Impacts of Applied Genetics: Micro-Organisms, Plants, and Animals

Home , Classical genetics, Microbial genetics, Molecular engineering, Plant genetics

April 1981

NTIS order #PB81-206609 Library of Congress Catalog Card Number 81-600046

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 Foreword

This report examines the application of classical and molecular genetic technologies to micro-organisms, plants, and animals. Congressional support for an assessment in the field of genetics dates back to 1976 when 30 Representatives requested a study of recombinant DNA technology. Letters of support for this broader study came from the then Senate Committee on Human Resources and the House Committee on Interstate and Foreign Commerce, Subcommittee on Health and the Environment. Current developments are especially rapid in the application of genetic technologies to micro-organisms; these were studied in three industries: pharmaceutical, chemical, and food. Classical genetics continue to play the major role in plant and animal breeding but new genetic techniques are of ever-increasing importance. This report identifies and discusses a number of issues and options for the Con- gress, such as: ● Federal Government support of R&D, ● methods of improving the germplasm of farm animal species, . risks of genetic engineering, patenting living organisms, and ● public involvement in decisionmaking. The Office of Technology Assessment was assisted by an advisory panel of scientists, industrialists, labor representatives, and scholars in the fields of law, economics, and those concerned with the relationships between science and society. Others contributed in two workshops held during the course of the assessment. The first was to investigate public perception of the issues in genetics; the second examined genetic applications to animals. Sixty reviewers drawn from universities, Government, industry, and the law provided helpful comments on draft reports. The Office expresses sincere appreciation to all those individuals. An abbreviated copy of the summary of this report (ch. 1) is available free of charge from the Office of Technology Assessment, U.S. Congress, Washington, D. C., 20510. In addition, the working papers on the use of genetic technology in human and in veterinary medicine are available as a separate volume from the National Technical Information Service.

Director Impacts of Applied Genetics Advisory

J. E. Legates, Chairman Dean, School of Agriculture and Life Sciences, North Carolina State University Ronald E. Cape Oliver E. Nelson, Jr. Cetus Corp. Laboratory of Genetics University of Wisconsin Nina V. Fedoroff Department of Embryology Carnegie Institution of Washington David Pimentel Department of Entomology June Goodfield Cornell University The Rockefeller University Harold P. Green Robert Weaver Fried, Frank, Harris, Shriver and Kampelman Department of Agricultural Economics Halsted R. Holman Pennsylvania State University Stanford University Medical School M. Sylvia Krekel James A. Wright Health and Safety Office Pioneer Hi-Bred International Oil, Chemical, and Atomic Workers Plant Breeding Division International Union Elizabeth Kutter Norton D. Zinder The Evergreen State College The Rockefeller University

iv Applied Genetics Assessment Staff

Joyce C. Lashof, Assistant Director, OTA Health and Life Sciences Division Gretchen Kolsrud, Program Manager Zsolt Harsanyi, Project Director

Project Staff

Marya Breznay, Administrative Assistant Lawrence Burton, Analyst Susan Clymer, Research Assistant Renee G. Ford, * Technical Editor Michael Gough, Senior Analyst Robert Grossman,* Analyst Richard Hutton, * Editor Geoffrey M. Karny, Legal Analyst

Major Contractors

Benjamin G. Brackett, University of Pennsylvania The Genex Corp. William P. O’Neill, Poly-Planning Services Plant Resources Institute Anthony J. Sinskey, Massachusetts Institute of Technology Aladar A. Szalay, Boyce-Thompson Institute Virginia Walbot, Washington University

OTA Publishing Staff

John C. Holmes, publishing Officer John Bergling* Kathie S. Boss Debra M. Datcher Patricia A. Dyson* Mary Harvey * Joe Henson

OTA contract personnel.

v Contents

Page . . . Glossary ...... viii Acronyms and Abbreviations ...... xii Chapter l. Summary: Issues and Options ...... 3 Chapter 2. Introduction ...... 00 29

Part I :Biotechnology Chapter 3. Genetic Engineering and the Fermentation Technologies ...... 49 Chapter 4. The Pharmaceutical Industry...... 59 Chapter 5. The Chemical Industry ...... 85 Chapter 6. The Food Processing Industry...... 107 Chapter 7. The Use of Genetically Engineered Micro-Organisms in the Environment...... 117

Part II: Agriculture Chapter 8. The Application of Genetics to Plants ...... 137 Chapter 9. Advances in Reproductive Biology and Their Effects on Animal Improvement ...... 167

Part III: Institutions and Society Chapter l0. The Question of Risk...... 199 Chapter ll. Regulation of Genetic Engineering...... 211 Chapter 12. Patenting Living Organisms...... 237 Chapter 13. Genetics and Society ...... 257

Appendixes I-A. A Case Study of Acetaminophen Production...... 269 I-B. A Timetable for the Commercial Production of Compounds Using Genetically Engineered Micro-Organisms in Biotechnology ...... 275 I-C. Chemical and Biological Processes...... 292 I-D. The Impact of Genetics on Ethanol-A Case Study ...... 293 II-A. A Case Study of Wheat...... 304 11-B. Genetics and the Forest Products Industry Case Study ...... 307 II-C. Animal Fertilization Technologies...... 309 III-A. History of the Recombinant DNA Debate ...... 315 III-B. Constitutional Constraints on Regulation...... 320 111-C. Information on International Guidelines for Recombinant DNA...... 322 IV. Planning Workshop Participants, Other Contractors and Contributors, and Acknowledgments...... 329

vii Glossary

Aerobic.—Growing only in the presence of oxygen. volve the use of genetically engineered micro- organisms. Anaerobic. —Growing only in the absence of oxygen. Blastocyst.— An early developmental stage of the embryo; the fertilized egg undergoes several cell Alkaloids.—A group of nitrogen-containing organic divisions and forms a hollow ball of cells called substances found in plants; many are pharmaco- the blastocyst. logically active—e.g., nicotine, caffeine, and cocaine. Callus.—The cluster of plant cells that results from tissue culturing a single plant cell. Allele.—Alternate forms of the same gene. For example, the genes responsible for eye color (blue, Carbohydrates.— The family of organic molecules brown, green, etc.) are alleles. consisting of simple sugars such as glucose and sucrose, and sugar chains (polysaccharides) such Amino acids.—The building blocks of proteins. as starch and cellulose. There are 20 common amino acids; they are’ joined together in a strictly ordered “string” Catalyst.—A substance that enables a chemical which determines the character of each protein. reaction to take place under milder than normal conditions (e.g., lower temperatures). Biological Antibody.—A protein component of the immune catalysts are enzymes; nonbiological catalysts in- system in mammals found in the blood. clude metallic complexes. Cell fusion.—The fusing together of two or more Antigen.—A large molecule, usually a protein or cells to become a single cell. carbohydrate, which when introduced in the body stimulates the production of an antibody Cell lysis.—Disruption of the cell membrane allow- that will react specifically with the antigen. ing the breakdown of the cell and exposure of its contents to the environment. Aromatic chemical.—An organic compound containing one or more six-membered rings. Cellulase.—An enzyme that degrades cellulose to glucose. Aromatic polymer.–Large molecules consisting of repeated structural units of aromatic chem- Cellulose.–A polysaccharide composed entirely of icals. several glucose units linked end to end; it constitutes the major part of cell walls in plants. Artificial insemination.—The manual placement of sperm into the uterus or oviduct. Chimera.—An individual composed of a mixture of genetically different cells. Bacteriophage (or phage).–A virus that multiplies in bacteria. Bacteriophage lambda is com- Chloroplast.-The structure in plant cells where monly used as a vector in recombinant DNA ex- photosynthesis occurs. periments. Chromosomes.—The thread-like components of a cell that are composed of DNA and protein. They Bioassay .–Determination of the relative strength contain most of the cell’s DNA. of a substance (such as a drug) by comparing its effect on a test organism with that of a standard Clone.—A group of genetically identical cells or preparation. organisms asexually descended from a common ancestor. All cells in the clone have the same ge- Biomass.—Plant and animal material. netic material and are exact copies of the original. Biome.—A community of living organisms in a ma- Conjugation.—The one-way transfer of DNA be- jor ecological region. tween bacteria in cellular contact. Biosynthesis. —The production of a chemical com- Crossing-over. —A genetic event that can occur pound by a living organism. during celluar replication, which involves the breakage and reunion of DNA molecules. Biotechnology .—The collection of industrial processes that involve the use of biological systems. Cultivar.—An organism developed and persistent For some of these industries, these processes in- under cultivat ion. Cytognetics.-A branch of biology that deals with Gene.–The hereditary unit; a segment of DNA the study of heredity and variation by the meth- coding for a specific protein. ods of both cytology (the study of cells) and Gene expression.—The manifestation of the ge- genetics. netic material of an organism as specific traits. Cytoplasm.—The protoplasm of a cell, external to Genetic drift.—Changes of gene frequency in small the cell’s nuclear membrane. population due to chance preservation or extinc- Diploid.—A cell with double the basic chromosome tion of particular genes. number. Genetic code.—The biochemical basis of heredity DNA (deoxyribonucleic acid).—The genetic ma- consisting of codons (base triplets along the DNA terial found in all living organisms. Every inher- sequence) that determine the specific amino acid ited characteristic has its origin somewhere in sequence in proteins and that are the same for all the code of each individual’s complement of DNA. forms of life studied so far. DNA vector.—A vehicle for transferring DNA from Genetic engineering.-A technology used at the one cell to another. laboratory level to alter the hereditary apparatus of a living cell so that the cell can produce more Dominant gene.—A characteristic whose expres- or different chemicals, or perform completely sion prevails over alternative characteristics for a new functions. These altered cells are then used given trait. in industrial production. Escherichia coli.-A bacterium that commonly in- Gene mapping-Determining the relative loca- habits the human intestine. It is a favorite orga- tions of different genes on a given chromosome. nism for many microbiological experiments. Genome.—The basic chromosome set of an Endotoxins.-Complex molecules (lipopolysaccha- organism—the sum total of its genes. rides) that compose an integral part of the cell wall, and are released only when the integrity of Genotype.—The genetic constitution of an individ- the cell is disturbed. ual or group. Embryo transfer.—Implantation of an embryo Germplasm.-The total genetic variability available into the oviduct or uterus. to an organism, represented by the pool of germ cells or seed. Enzyme.—A functional protein that catalyzes a chemical reaction. Enzymes control the rate of Germ cell.—The sex cell of an organism (sperm or metabolic processes in an organism; they are the egg, pollen or ovum). It differs from other cells in active agents in the fermentation process. that it contains only half the usual number of chromosomes. Germ cells fuse during fertiliza- Estrogens.—Female sex hormones. tion. Estrus (“heat").-The period in which the female Glycopeptides.—Chains of amino acids with at- will allow the male to mate her. tached carbohydrates. Eukaryote.—A higher, compartmentalized cell A conjugated protein in which the characterized by its extensive internal structure Glycoprotein.— nonprotein group is a carbohydrate. and the presence of a nucleus containing the DNA. All multicellular organisms are eukaryotic. Haploid .—A cell with only one set (half of the usual The simpler cells, the prokaryotes, have much number) of chromosomes. less compartmentalization and internal structure; bacteria are prokaryotes. Heterozygous.—When the two genes controlling a particular trait are different, the organism is Exotoxins.—Proteins produced by bacteria that are heterozygous for that trait. able to diffuse out of the cells; generally more potent and specific in their action than endotoxins. Homozygous.— When the two genes controlling a particular trait are identical for a pair of chro- Fermentation.—The biochemical process of con- mosomes, the organism is said to be homozygous v ti g a raw material such as glucose into a er n for that trait. product such as ethanol. Hormones.—The “messenger” molecules of the Fibroblast.—A cell that gives rise to connective body that help coordinate the actions of various tissues. tissues; they produce a specific effect on the ac- Gamete.—A mature reproductive cell. tivity of cells remote from their point of origin.

ix Hybrid.—A new variety of plant or animal that re- Nif genes.—The genes for nitrogen fixation present suits from cross-breeding two different existing in certain bacteria. varieties. Nucleic acid.—A polymer composed of DNA or Hydrocarbon. —All organic compounds that are RNA subunits. composed only of carbon and hydrogen. Nucleotides.—The fundamental units of nucleic Immunoproteins.— All the proteins that are part acids. They consist of one of the four bases— of the immune system (including antibodies, in- adenine, guanine, cytosine, and thymine (uracil terferon, and cytokines). in the case of RNA)—and its attached sugar-phosphate group. In vitro. -outside the living organism and in an artificial environment. Organic compounds.—Chemical compounds based on carbon chains or rings, which contain In vivo.—Within the living organism. hydrogen, and also may contain oxygen, nitro- Leukocytes.—The white cells of blood. gen, and various other elements. Lipids.–Water insoluble biomolecules, such as cel- Parthenogenesis.— Reproduction in animals with- lular fats and oils. out male fertilization of the egg. Lipopolysaccharides. —Complex substances com- Pathogen.—A specific causative agent of disease. posed of lipids and polysaccharides. Peptide. —Short chain of amino acids. Lymphoblastoid.–Referring to malignant white pH.–A measure of the acidity or basicity of a solu- blood cells. tion; on a scale of O (acidic) to 14 (basic): for exam- Lymphokines.–The biologically active soluble fac- pie, lemon juice has a pH of 2.2 (acidic), water has tor produced by white blood cells. a pH of 7.0 (neutral), and a solution of baking soda has a pH of 8.5 (basic). Maleic anhydride.—An important organic chemical used in the manufacture of synthetic resins, Phage.—(See bacteriophage.) in fungicides, in the dyeing of cotton textiles, and Phenotype. —The visible properties of an organism to prevent the oxidation of fats and oils during that are produced by the interaction of the geno- storage and rancidity. type and the environment. Messenger RNA.– Ribonucleic acid molecules that Plasmid.—Hereditary material that is not part of a serve as a guide for protein synthesis. chromosome. Plasmids are circular and self-repli- Metabolism.—The sum of the physical and chem- cating. Because they are generally small and rela- ical processes involved in the maintenance of life tively simple, they are used in recombinant DNA and by which energy is made available. experiments as acceptors of foreign DNA. Mitochondria.—Structures in higher cells that Plastid.–Any specialized organ of the plant cell serve as the “powerhouse” for the cell, producing other than the nucleus, such as the chloroplast. chemical energy. Ploidy. —Describes the number of sets of chromo- Monoclinal antibodies.—Antibodies derived somes present in the organism. For example, from a single source or clone of cells which humans are diploid, having two homologous sets recognize only one kind of antigen. of 23 chromosomes (one set from each parent) Mutants.–Organisms whose visible properties with for a total of 46 chromosomes; many plants are respect to some trait differ from the norm of the haploid, having only one copy of each chro- population due to mutations in its DNA. mosome. Mutation.—Any change that alters the sequence of Polymer.—A long-chain molecule formed from bases along the DNA, changing the genetic ma- smaller repeating structural units. terial. Polysaccharide. —A long-chain carbohydrate con- Myeloma.—A malignant disease in which tumor taining at least three molecules of simple sugars cells of the antibody producing system synthesize linked together; examples would include cellu- excessive amounts of specific proteins. lose and starch. n-alkanes.—Straight chain hydrocarbons–the Progestogens. —Hormones involved with ovula- main constituents of petroleum. tion.

x Prostaglandin.-Refers to a group of naturally oc- RNA, the reverse of the normal direction of proc- curring, chemically related long-chain fatty acids essing genetic information within the cell. that have certain physiological effects (stimulate contraction of uterine and other smooth muscles, RNA (ribonucleic acid).—In its three forms—mes- lower blood pressure, affect action of certain senger RNA, transfer RNA, and ribosomal RNA— hormones). it assists in translating the genetic message of DNA into the finished protein. Protein.—A linear polymer of amino acids; proteins are the products of gene expression and are the Somatic cell.—One of the cells composing parts of functional and structural components of cells. the body (e.g., tissues, organs) other than- a germ cell. Protoplasm.—A cell without a wall. in vitro method of propagat- Protoplasm fusion.—A means of achieving genetic Tissue culture.—An ing healthy cells from tissues, such as fibroblasts transformation by joining two protoplasts or join- from skin. ing a protoplasm with any of the components of another cell. Transduction.—The process by which foreign Recessive gene.—Any gene whose expression is DNA becomes incorporated into the genetic com- dependent on the absence of a dominant gene. plement of the host cell. Recombinant DNA.–The hybrid DNA produced Transformation.—The transfer of genetic infor- by joining pieces of DNA from different sources. mation by DNA separated from the cell. Restriction enzyme.—An enzyme within a bac- Vector.—A transmission agent; a DNA vector is a terium that recognizes and degrades DNA from self-replicating DNA molecule that transfers a foreign organisms, thereby preserving the genet- piece of DNA from one host to another. ic integrity of the bacterium. In recombinant DNA experiments, restriction enzymes are used Virus.–An infectious agent that requires a host cell as tiny biological scissors to cut up foreign DNA in order for it to replicate. It is composed of before it is recombined with a vector. either RNA or DNA wrapped in a protein coat. Reverse transcriptase.—An enzyme that can syn- Zygote.—A cell formed by the union of two mature thesize a single strand of DNA from a messenger reproductive cells.

xi Acronyms and Abbreviations

AA — amino acids IBCs — Institutional Biosafety Committees ACS — American Cancer Society ICI — Imperial Chemical Industries ACTH — adrenocorticotropic hormone IND — Investigational New Drug Application AI — artificial insemination (FDA) AIPL — Animal Improvement Programs kg – kilogram Laboratory 1 — liter APAP — acetaminophen lb – pound ASM — American Society for Microbiology mg — milligram bbl – barrel(s) 1% — microgram bbl/d – barrels per day pm — micrometer (formerly micron) BOD5 — 5-day biochemical oxygen demand MUA — Memorandum of Understanding and BBM — Biological Response Modifier Program Agreement bu – bushel NCI — National Cancer Institute CaMV — cauliflower mosaic virus NDA — new drug application (FDA) CCPA — The Court of Customs and Patent NDAB — National Diabetics Advisory Board Appeals NDCHIP — National Cooperative Dairy Herd CDC — Center for Disease Control Program CERB — Cambridge Experimentation Review NIAID — National Institute of Allergy and Board Infectious Diseases DHHS — Department of Health and Human NIAMDD — National Institute of Arthritis, Services (formerly Health, Education, Metabolism, and Digestive Diseases and Welfare) NIH – National Institutes of Health DHI — Dairy Herd Improvement NIOSH — National Institute of Occupational DNA – deoxyribonucleic acid Safety and Health DOD — Department of Commerce NSF — National Science Foundation DOD — Department of Defense OECD — The Organization for Economic DOE — Department of Energy Cooperation and Development DPAG — Dangerous Pathogens Advisory Group ORDA — Office of Recombinant DNA Activities EOR — enhanced oil recovery PD — predicted difference EPA — Environmental Protection Agency pH — unit of measure for acidity/ basicity FDA — Food and Drug Administration ppm – parts per million FMDV — foot-and-mouth disease virus R&D — research and development ftz — square foot RAC — Recombinant DNA Advisory Committee ft – foot rDNA — recombinant DNA FTC — Federal Trade Commission SCP — single-cell protein g – gram T-DNA — a smaller segment of the the plasmid gal – gallon Ti — tumor inducing GH – growth hormone TSCA — Toxic Substances Control Act ha — hectares HEW — Department of Health, Education, and Welfare hGH – human growth hormone HYV — high-yielding varieties chapter 1 Summary: Issues and Options Chapter 1

Page Page Biotechnology ...... 4 Findings ...... 17 The Pharmaceutical industry...... 4 Issue and Options-Animals ...... 17 Findings ...... 4 Institutions and Society...... 18 The Chemical Industry...... 7 Regulation of Genetic Engineering ...... 18 Findings ...... 7 Findings ...... 18 Food processing industry...... 8 Issue and Options—Regulation ...... 20 Findings ...... 8 Patenting Living Organisms ...... 22 The Use of Genetically Engineered Micro- Findings ...... 22 Organisms in the Environment ...... 8 Issue and Options—Patenting Living Organisms 23 Findings ...... 8 Genetics and Society ...... 24 Mineral Leaching and Recovery ...... 9 Issues and options—Genetics and Society. .. . 24 Enhanced Oil Recovery...... 9 Pollution Control ...... 9 Constraints in Using Genetic Engineering Technologies in Open Environments...... 10 Table Table No. Page Issue and Options—Biotechnology ...... 10 1. Containment Recommended by the National Agriculture ...... 11 Institutes of Health...... 19 The Applications of Genetics to Plants ...... 11 Findings ...... 11 New Genetic Technologies for Plant Breeding 12 Figures Constraints on Using Molecular Genetics Figure No. Page for Plant Improvements...... 13 1. Recombinant DNA: The Technique of Genetic Variability, CropVulnerability, and Recombining Genes From One Species With the Storage of Germplasm ...... 13 Those From Another...... 5 Issues and Options—Plants...... 14 2. The Product Development Process ...... 6 Advances in Reproductive Biology and Their 3. The Way the Reproductive Technologies Effects on Animal Improvement ...... 15 Interrelate ...... 16 Chapter 1 Summary: Issues and Options

The genetic alteration of plants, animals, and ronment. In addition specificsp subtopics are of micro-organisms has been an important part of interest to other committees, notably those hav- agriculture for centuries. It has also been an ining jurisdiction over science and technology and tegral part of the alcoholic beverage industry those concerned with patents. since the invention of beer and wine; and for the past century, a mainstay of segments of the This report describes the potentials and prob- pharmaceutical and chemical industries. lems of applying the new genetic technologies to a range of major industries. It emphasizes the However, only in the last 20 years have pow- present state of the art because that is what erful new genetic technologies been developed defines the basis for the future applications. It that greatly increase the ability to manipulate then makes some estimates of economic, envi- the inherited characteristics of plants, animals, ronmental, and institutional impacts—where, and micro-organisms. One consequence is the when, and how some technologies might be ap- increasing reliance the pharmaceutical and plied and what some of the results might be. chemical industries are placing on biotechnol- The report closes with the possible roles that ogy. Micro-organisms are being used to manu- Government, industry, and the public might facture substances that have previously been play in determining the future of applied extracted from natural sources. Animal and genetics. plant breeders are using the new techniques to applied genetics, help clarify basic questions about biological The term as used in this functions, and to improve the speed and effi- report, refers to two groups of technologies: ciency of the technologies they already use. ● Classical genetics—natural mating methods Other industries–from food processing and pol- for the selective breeding of organisms lution control to mining and oil recovery—are for desired characteristics–e. g., breeding considering the use of genetic engineering to in- cows for increased milk production. The crease productivity and cut costs. pool of genes available for selection is comprised of those that cause natural differ- Genetic technologies will have a broad impact ences among individuals in a population on the future. They may contribute to filling and those obtained by mutation. some of the most fundamental needs of mankind–from health care to supplies of food and ● Molecular genetics includes the technologies energy. At the same time, they arouse concerns of genetic engineering that involve the about their potential effects on the environment directed manipulation of the genetic mate- and the risks to health involved in basic and rial itself. These technologies—such as applied scientific research and development rDNA and the chemical synthesis of genes (R&D). Because genetic technologies are already —can increase the size of the gene pool for being applied, it is appropriate to begin con- any one organism by making available ge- sidering their potential consequences. netic traits from many different populations. Molecular genetics also includes Congressional concern with applied genetics technologies in which manipulation occurs dates back to 1976, when 30 Representatives re- at a level higher than that of the gene—at quested an assessment of recombinant DNA the cellular level, e.g., cell fusion and in (rDNA) technology. Support for the broader vitro fertilization. study reported here came in letters to the Office of Technology Assessment from the then Senate Significant applications of molecular genetics Committee on Human Resources and the House to micro-organisms, such as the efforts to man- Committee on Interstate and Foreign Com- ufacture human insulin, are already underway merce, Subcommittee on Health and the Envi- in several industries. Most of these applications

3 4 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals depend on fermentation—a technology in which plants and animals, which are biologically more substances produced by micro-organisms can complex and more difficult to manipulate suc- be obtained in large quantities. Applications to cessfully, will take longer to develop.

Biotechnology

Biotechnology-the use of living organisms or micro-organisms have been engineered to pro- their components in industrial processes—is duce human insulin, interferon, growth hor- possible because micro-organisms naturally pro- mone, urokinase (for the treatment of blood duce countless substances during their lives. clots), thymosin-a 1 (for controlling the immune Some of these substances have proved commer- response), and somatostatin (a brain hormone). cially valuable. A number of different industries (See figure 2.) have learned to use micro-organisms as natural The products most likely to be affected by factories, cultivating populations of the best genetic engineering in the next 10 to 20 years producers under conditions designed to en- are nonprotein compounds like most antibiotics, hance their abilities. and protein compounds such as enzymes and Applied genetics can play a major role in im- antibodies, and many hormones and vaccines. proving the speed, efficiency, and productivity Improvements can be made both in the prod- of these biological systems. It permits the ma- ucts and in the processes by which they are pro- nipulation, or engineering, of the micro-orga- duced. Process costs may be lowered and even nisms’ genetic material to produce the desired entirely new products developed. characteristics. Genetic engineering is not in The most advanced applications today are in itself an industry, but a technique used at the the field of hormones. While certain hormones laboratory level that allows the researcher to have already proved useful, the testing of modify the hereditary apparatus of the cell. The others has been hindered by their scarcity and population of altered identical cells that grows high cost. Of 48 human hormones that have from the first changed micro-organism is, in been identified so far as possible candidates for turn, used for various industrial processes. (See production by genetically engineered micro- figure 1.) organisms, only 10 are used in current medical The first major commercial effects of the ap- practice. The other 38 are not, partly because plication of genetic engineering will be in the they have been available in such limited quan- pharmaceutical, chemical, and food processing tities that tests of their therapeutic value have industries. Potential commercial applications of not been possible. value to the mining, oil recovery, and pollution Genetic technologies also open up new ap- control industries—which may desire to use ma- proaches for vaccine development for such in- nipulated micro-organisms in the open environ - tractable parasitic and viral diseases as amebic ment —are stll somewhat speculative. dysentery, trachoma, hepatitis, and malaria. At present, the vaccine most likely to be produced The pharmaceutical industry is for foot-and-mouth disease in animals. How- ever, should any one of the vaccines for human FINDINGS diseases become available, the social, economic, The pharmaceutical industry has been the and political consequences of a decrease in mor- first to take advantage of the potentials of ap- bidity and mortality would be significant. Many plied molecular genetics. Ultimately, it will of these diseases are particularly prevalent in probably benefit more than any other, with the less industrialized countries; the developments largest percentage of its products depending on of vaccines for them may profoundly affect the advances in genetic technologies. Already, lives of tens of millions of people. Ch. 1 Summary: Issues and Options ● 5

Figure 1.— Recombinant DNA: The Technique of Recombining Genes From One Species With Those From Another

Photo credits; Professor F. A. Eiserling, UCLA Molecular Biology Institute Electron micrograph of the DNA, which is the plasmid SPO1 from Bacillus subtilis. This plasm id which has been Electron micrograph of Bacillus subtilis in the process of sliced open is used for recombinant DNA research cell division. The twisted mass in the center of each in this bacterial host daughter cell is the genetic material, DNA

Donor DNA Restriction enzymes recognize certain sites along the DNA and can chemically cut the DNA at those sites. This makes it possible to remove selected genes from donor DNA molecules and insert them into plasmid DNA molecules to form the recombinant DNA. This recombinant DNA can then be cloned in its bacterial host and large amounts of a desired protein can be produced.

Expression produces amount of DNA protein

SOURCE: Office of Technology Assessment.

For some pharmaceutical products, biotech- will be available in ample amounts for clin- nology will compete with chemical synthesis ical testing. Interferon, for example, can be and extraction from human and animal organs. tested for its efficacy in cancer and viral Assessing the relative worth of each method therapy, and human growth hormone can must be done on a case-by-case basis. But for be evaluated for its ability to heal wounds. other products, genetic engineering offers the ● Other pharmacologically active substances only method known that can ensure a plentiful for which no apparent use now exists will supply; in some instances, it has no competition. be available in sufficient quantities and at low enough cost to enable researchers to By making a pharmaceutical available, genet- explore new uses. As a result, the potential ic engineering may have two types of effects: for totally new therapies exists. Regulatory ● Drugs that already have medical promise proteins, for example, which are an entire

76-565 0 - 81 - 2 6 . Impacts of Applied Genetics—Micro-Organ/ims, Plants, and Animals

Figure 2.-The Product Development Process

Micro-organisms such as E co/I

I 7. cutting 6. Plasmid 2. Tissues

14. Process development scale-up

16. Industrial

The development process begins by obtaining DNA either through organic synthesis (1) or derived from biological sources such as tissues (2). The DNA obtained from one or both sources is tailored to form the basic “gene” (3) which contains the genetic information to “code” for a desired product, such as human interferon or human insulin. Control signals (4) containing instructions are added to this gene (5). Circular DNA molecules called plasmids (6) are isolated from micro-organisms such as E. coIi; cut open (7) and spliced back(8) together with genes and control signals to form “recombinant DNA” molecules. These molecules are then introduced into a host cell (9). Each plasmid is copied many times in a cell (10). Each cell then translates the information contained in these plasmids into the desired product, a process called “expression” (11). Cells divide (12) and pass on to their offspring the same genetic information contained in the parent cell. Fermentation of large populations of genetically engineered micro-organisms is first done in shaker flasks (13), and then in small fermenters (14) to determine growth conditions, and eventually in larger fermentation tanks (15). Cellular extract obtained from the fermentation process is then separated, purified (16), and packaged (17) either for industrial use (18) or health care applications. Health care products are first tested in animal studies (19) to demonstrate a product’s pharmacological activity and safety, in the United States, an investigational new drug application (20) is submitted to begin human clinical trials to establish safety and efficacy. Following clinical testing (21), a new drug application (NDA) (22) is fried with the Food and Drug Administration (FDA). When the NDA has been reviewed and approved by the FDA the product maybe marketed in the United States (23).

SOURCE: Genentech, Inc. Ch. 1—Summary: Issues and Options ● 7

class of molecules that control gene activi- vantages over current chemical production ty, are present in the body in only minute techniques. quantities. Now, for the first time, they can The use of renewable resources: starches, be recognized, isolated, characterized, and sugars, cellulose, and other components of produced in quantity. biomass can serve as the raw material for The mere availability of a pharmacologically synthesizing organic chemicals. With prop- active substance does not ensure its adoption in er agricultural management, biomass can medical practice. Even if it is shown to have assure a continuous renewable supply for therapeutic usefulness, it may not succeed in the industry. the marketplace. The use of physically milder conditions: chemical processes often require high tem- The difficulty in predicting the economic im- peratures and extreme pressures. These pact is exemplified by interferon. If it is found to conditions are energy intensive and pose a be broadly effective against both viral diseases hazard in case of accidents. Biological proc- and cancers, sales would be in the tens of bil- esses operate under milder conditions, lions of dollars annually. If its clinical effec- which are compatible with living systems. tiveness is found to be only against one or two One-step production methods: micro-orga- viruses, sales would be significantly lower. nisms can carry out several steps in a syn- At the very least, even if there are no im- thetic process, eliminating the need for in- mediate medical uses for compounds produced termediate steps of separation and puri- by genetic engineering, their indirect impact on fication. medical research is assured. For the first time, Decreased pollution: because biological almost any biological phenomenon of medical processes are highly specific in the reac- interest can be explored at the cellular level. tions they catalyze, they offer control over These molecules are valuable tools for under- the products formed and decrease undesir- standing the anatomy and functions of cells. able side-products. As a result, they pro- The knowledge gained may lead to the develop- duce fewer pollutants that require management of new therapies or preventive measures ment and disposal, for diseases. The impact of this technology will cut across The chemical industry the entire spectrum of chemical groups: plastics and resin materials, flavors and perfumes mate- FINDINGS rials, synthetic rubber, medicinal chemicals, The chemical industry’s primary raw materi- pesticides, and the primary products from pe- al, petroleum, is now in limited supply. Coal is troleum that serve as the raw materials for the one appealing alternative; another is biomass, a synthesis of organic chemicals. Nevertheless, renewable resource composed of plant and ani- the specific products that will be affected in mal material. each group can only be chosen on a case-by-case Biomass has been transformed by fermenta- basis, with the applicability of genetics de- tion into organic chemicals like citric acid, etha- pending on a variety of factors. Crude estimates nol, and amino acids for decades. Other organic of the expected economic impacts are in the bil- chemicals such as acetone, butanol, and fumaric lions of dollars per year for dozens of chemicals within 20 years. acid were at one time made by fermentation until chemical production methods, combined INDUSTRY AND MANPOWER IMPACTS with cheap oil and gas, proved to be more eco- Although genetic engineering will develop nomical. In theory, most any industrial organic new techniques for synthesizing many sub- chemical can be produced by a biological proc- stances, the direct displacement of any current ess. industry seems doubtful. Genetic engineering Commercial fermentation using genetically should be considered simply another industrial engineered micro-organisms offers several ad- tool. Industries will probably use genetic 8 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals engineering to maintain their positions in their micro-organisms high in protein content respective markets. This is already illustrated (called single-cell protein or SCP). Its pres- by the variety of companies in the pharmaceu- ent cost of production in the United States tical, chemical, and energy industries that have is relatively high, and it must compete with invested in or contracted with genetic engineer- cheaper sources of protein such as soy- ing firms. Some large companies are already de- beans and fishmeal, among others. veloping inhouse genetic engineering research ● Isolated successes can be anticipated for capabilities. the production of such food additives as fructose (a sugar) and the synthetic sweet- Any predictions of the number of workers ener aspartame, and for improvements in that will be required in the production phase of SCP production. biotechnology will depend on the expected volume of chemicals that will be produced. At An industrywide impact is not expected in the present, this figure is unknown. An estimated near future because of several major conflicting $15 billion worth of chemicals maybe manufac- factors: tured by biological processes. This will employ ● The basic knowledge of the genetic charac- approximately 30,000 to 75,000 workers for su- teristics that could improve food has not pervision, services, and production. Whether been adequately developed. this will represent a net loss or gain in the num- ● The food processing industry is conserva- ber of jobs is difficult to predict since new jobs tive in its expenditures for R&D to improve in biotechnology will probably displace some of processes. Generally, only one-third to one- those in traditional chemical production. half as much is allocated for this purpose as in technologically intensive industries. Food processing industry ● Products made by new microbial sources must satisfy the Food and Drug Adminis- FINDINGS tration’s (FDA) safety regulations, which in- Genetics in the food processing industry can clude undergoing tests to prove lack of be used in two ways: to design micro-organisms harmful effects. It may be possible to that transform inedible biomass into food for reduce the amount of required testing by human consumption or into feed for animals; transferring the desired gene into micro- and to design organisms that aid in food proc- organisms that already meet FDA stand- essing, either by acting directly on the food ards. itself or by providing materials which can be added to food. The use of genetically engineered The use of genetics to design organisms with micro-organisms in the environment desired properties for food processing is an established practice. Fermented foods and FINDINGS beverages have been made by selected strains Genetically engineered micro-organisms are of mutant organisms (e.g., yeasts) for centuries. being designed now to perform in three areas Only recently, however, have molecular tech- (aside from agricultural uses) that require their nologies opened up new possibilities. In par- large-scale release into the environment: ticular, large-scale availability of enzymes will . mineral leaching and recovery, play an increasing role in food processing. . . enhanced oil recovery, and The applications of molecular genetics are ● pollution control. likely to appear in the food processing industry All of these are characterized by: in piecemeal fashion: ● the use of large volumes of micro-orga- ● Inedible biomass, human and animal nisms, wastes, and even various industrial efflu- decreased control over the behavior and ents are now being transformed into edible fate of the micro-organisms, Ch. 1—Summary: Issues and Options ● 9

the possibility of ecological disruption, and process to find micro-organisms that can func- less development in basic R&D (and more tion in an oil reservoir environment, and then to speculation-) than in the industries in which improve their characteristics genetically. micro-organisms are used in a controlled environment. The genetic alteration of micro-organisms to produce chemicals useful for enhanced oil re- MINERAL LEACHING AND RECOVERY covery has been more successful than the alter- Bacteria have been used to leach metals, such ation of micro-organisms that may be used in as uranium and copper, from low-grade ores. situ. However, rDNA technology has not been Although there is reason to believe leaching applied to either case. All attempts have em- ability is under genetic control in these orga- ployed artificially induced or naturally occur- nisms, practically nothing is known about the ring mutations. precise mechanisms involved. Therefore, the POLLUTION CONTROL application of genetic technologies in this area remains speculative. Progress has been slow in Many micro-organisms can consume various obtaining more information, partly because kinds of pollutants, changing them into relative- very little research has been conducted. ly harmless materials before they die. These micro-organisms always have had a role in In addition to leaching, micro-organisms can “natural” pollution control: nevertheless, cities be used to recover valuable metals or eliminate have resisted adding microbes to their sewerage polluting metals from dilute solutions such as in- systems. Although the Environmental Protec- dustrial waste streams. The process makes use tion Agency (EPA) has not recommended addi- of the ability of micro-organisms to bind metals tion of bacteria to municipal sewerage systems, to their surfaces and then concentrate them in- it suggests that they might be useful in smaller ternally. installations and for specific problems in large The economic competitiveness of biological systems. In major marine spills, the bacteria, methods is still unproved, but genetic modifica- yeast, and fungi already present in the water tions have been attempted only recently. The participate in degradation. The usefulness of cost of producing the micro-organisms has been added microbes has not been demonstrated. a major consideration. If it can be reduced, the Nevertheless, in 1978, the estimated market approach might be useful. of biological products for pollution control was ENHANCED OIL RECOVERY $2 million to $4 million per year, divided among Many methods have been tried in efforts to some 20 companies; the potential market was remove oil from the ground when natural estimated to be as much as $20 million per year. expulsive forces alone are no longer effective. To date, genetically engineered strains have Injecting chemicals into a reservoir has, in many not been applied to pollution problems. Restrict- cases, aided recovery by changing the oil’s flow ing factors include the problems of liability in characteristics. the event of health, economic, or environmental Micro-organisms can produce the necessary damage; the contention that added organisms chemicals that help to increase flow. Theoreti- are not likely to be a significant improvement; cally, they can also be grown in the wells and the assumption that selling microbes rather themselves, producing those same chemicals in than products or processes is not likely to be situ. The currently favored chemical, xanthan, profitable. is far from ideal for increasing flow. Genetic Convincing evidence that microbes could re- engineering should be able to produce chem- move or degrade an intractable pollutant would icals with more useful characteristics. encourage their application. In the meantime, The current research approach, funded by however, these restrictions have acted to inhibit the Department of Energy (DOE) and independ- the research necessary to produce marked im- ently by various oil companies, is a two-phase provements. 10 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

CONSTRAINTS IN USING GENETIC ENGINEERING systems on a scale large enough to exploit their TECHNOLOGIES IN OPEN ENVIRONMENTS biological activity. This will necessitate a con- The genetic data base for the potentially use- tinual dialog among microbial geneticists, geolo- ful micro-organisms is lacking. Only the sim- gists, chemists, and engineers; an interdisci- plest methods of mutation and selection for de- plinary approach is required that recognizes the sirable properties have been used thus far. needs and limitations of each discipline. These are the only avenues for improvement The second obstacle is ecological. Introducing until more is learned about the genetic mech- large numbers of genetically engineered micro- anisms. organisms into the environment might lead to Even when the scientific knowledge is avail- ecological disruption or detrimental effects on able, two other obstacles to the use of geneti- human health, and raise questions of legal— lia- cally engineered micro-organisms will remain. bility. The first is the need to develop engineered

Issue and Options—Biotechnology ISSUE: How can the Federal Govern- OPTIONS: ment promote advances in bio- A. Congress could allocate funds specifically for technology and genetic engi- genetic engineering and biotechnology R&Din neering? the budget of appropriate agencies. The United States is a leader in applying genetic engineering and biotechnology to in- Congress could promote two types of programs in biotechnology: those with long-range dustry. One reason is the long-standing commitment by the Federal Government to the funding payoffs (basic research), and those that industry of basic biological research; several decades of is not willing to undertake but that might be in the national interest. support for some of the most esoteric basic research has unexpectedly provided the foun- B. Congress could establish a separate Institute dation for a highly useful technology. A second of Biotechnology as a funding agency. is the availability of venture capital, which has allowed the formation of small, innovative com- The merits of a separate institute lie in the panies that can build on the basic research. possibility of coordinating a wide range of efforts, all related to biotechnology. On the other The chief argument for Government subsidi- hand, biotechnology and genetic engineering zation for R&D in biotechnology and genetic cover such a broad range of disciplines that a engineering is that Federal help is needed in new funding agency would overlap the man- areas such as general (generic) research or high- dates of existing agencies. Furthermore, the ly speculative investigations not now being de- creation of yet another agency carries with it all veloped by industry. The argument against the the disadvantages of increased bureaucracy and need for this support is that industry will devel- competition for funds at the agency level. op everything of commercial value on its own. C. Congress could establish research centers in A look at what industry is now attempting in- universities to foster interdisciplinary ap- dicates that sufficient investment capital is proaches to biotechnology. In addition, a pro- available to pursue specific manufacturing ob- gram of grants could be offered to train scien- jectives. Some high-risk areas, however, that tists in biological engineering. might be of interest to society, such as pollution control, may justify promotion by the Govern- The successful use of biological techniques in ment, while other, such as enhanced oil recov- industry depends on a multidisciplinary ap- ery might might not be profitable soon enough proach involving biochemists, geneticists, mi- to attract investment by industry. crobiologists, process engineers, and chemists. Ch. 1—Summary: lssues and Options ● 11

Little is now being done publicly or privately to gress, small businesses and universities may re- develop the expertise necessary. tain title to inventions developed under federal- D. Congress could use tax incentives to stimulate ly funded research. Currently, some Federal biotechnology. agencies award contractors these exclusive rights, while others insist on the nonexclusive The tax laws could be used to stimulate bio- licensing of inventions. technology by expanding the supply of capital for small, high-risk firms, which are generally F. Congress could mandate support for specific considered more innovative than established research tasks such as pollution control using firms because of their willingness to undertake microbes. the risks of innovation. In addition to focusing on the supply of capital, tax policy could at- Microbes may be useful in degrading intrac- tempt to directly increase the profitability of table wastes and pollutants. Current research, potential growth companies. however, is limited to isolating organisms from natural sources or from mutated cultures. More A tax incentive could also be directed’ at in- elaborate efforts, involving rDNA techniques or creasing R&D expenditures. It has been sug- other forms of microbial genetic exchange, will gested that companies be permitted to take tax require additional funding. credits: 1) on a certain percentage of their R&D expenses; and 2) on contributions to universities G. Most efforts could be left to industry and each for research. Government agency allowed to develop pro- E. Congress could improve the conditions under grams in the fields of genetic engineering and which U.S. companies collaborate with aca- biotechnology as it sees fit. demic scientists and make use of the technol- Generic research will probably not be under- ogy developed in universities, which has been taken by any one company. Leaving all R&D in wholly or partly supported by tax funds. industry’s hands would still produce major com- Developments in genetic engineering have mercial successes, but does not ensure the de- kindled interest in this option. Under legislation velopment of needed basic general knowledge that has recently passed both Houses of Con- or the undertaking of high-risk projects.

Agriculture

The complexity of plants and animals pre- ment, pest control, and cropping techniques sents a greater challenge to advances in applied using herbicides, irrigation, and fertilizers. genetics than that posed by micro-organisms. Nevertheless, the impacts of breeding technol- Nevertheless, the successful genetic manipula- ogies have been extensive. tion of microbes has encouraged researchers in the agricultural sciences. The new tools will be The plant breeder’s approach is determined for the most part by the particular biological used to complement, but not replace, the well- factors of the crop being bred. The new genetic established practices of plant and animal technologies potentially offer additional tools to breeding. allow development of new varieties and even species of plants by circumventing current bio- The applications of genetics to plants logical barriers to the exchange of genetic FINDINGS material. It is impossible to exactly determine the ex- Technologies developed for classical plant tent to which applied genetics has directly con- breeding and those of the new genetics should tributed to increases in plant yield because of not be viewed as being competitive; they are simultaneous improvements in farm manage- both tools for effectively manipulating genetic 12 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals information. One new technology -e.g., proto- often quality for quantity, such as increased plasm fusion, or the artificial fusion of two cells– protein content but decreased yield. allows breeders to overcome incompatibility NEW GENETIC TECHNOLOGIES FOR between plants. But the plant that may result PLANT BREEDING still must be selected, regenerated, and eval- The new technologies fall into two categories: uated under field conditions to ensure that the those involving genetic transformations genetic change is stable and that the attributes through cell fusion and those involving the in- of the new variety meet commercial require- sertion or modification of genetic information ments. through the cloning of DNA and its vectors. In theory, the new technologies will expand Techniques are available for manipulating the capability of breeders to exchange genetic organs, tissues, cells, or protoplasts in culture; information by overcoming natural breeding for regenerating plants; and for testing the barriers. To date, however, they have not had a genetic basis of novel traits. So far these tech- widespread impact on the agricultural industry. niques are routine only in a few species. The approach to exploiting molecular biology As a note of caution, it must be emphasized for plant breeding is similar in some respects to that no plant can possess every desirable trait. the genetic manipulation of micro-organisms. There will always have to be some tradeoff; However, there is one major conceptual dif-

Photo credits: Weyerhaeuser Co. A plantlet of Ioblolly pine grown in Weyerhaeuser Co.’s tissue culture laboratory. The next step in this procedure A young Douglas fir tree propagated 4 years ago from a is to transfer the plantlet from its sterile and humid small piece of seedling leaf tissue. Three years ago this was environment to the soil at the test-tube stage seen in the Ioblolly pine photograph Ch. 1—Summary: Issues and Options . 13 ference. In micro-organisms, the changes made plant sciences from NSF is approximately $25 on the cellular level are the goals of the million, only $1 million of which is specifically manipulation. With crops, changes made on the designated for plant genetics. cellular level are meaningless unless they can be The shortage of a trained workforce is a reproduced in the entire plant as well. There- significant constraint. Only a few universities fore, unless single cells in culture can be have expertise in both plants and molecular bi- selected and grown into mature plants and the ology. In addition, there are only a few people desired traits expressed in the mature plant– who have the ability to work with modern mo- procedures which at this time have had limited lecular techniques related to whole plant prob- success—the benefits of genetic engineering will lems. As a result, a business firm could easily not be widely felt in plant breeding. develop a capability in this area exceeding that Moderate success has been achieved for at any individual U.S. university. However, the growing cells in tissue culture into mature building of industrial laboratories and subse- plants. Tissue culture programs of commercial quent hiring from the universities could easily significance in the United States include the deplete the expertise at the university level. asparagus, citrus fruits, pineapples, and straw- With the recent investment activity by many berries. Breeders have had little success, how- bioengineering firms, this trend has already ever, in regenerating mature plants of wide begun; in the long-run it could have serious con- agronomic importance) such as corn and wheat. sequences for the quality and quantity of university research. Some success can be claimed for engineering changes to alter genetic makeup. Both the stable GENETIC VARIABILITY, CROP VULNERABILITY, integration of genetic material into a cell and AND THE STORAGE OF GERMPLASM the fusion of genetically different cells are still Successful plant breeding is based on the largely experimental techniques. Technical availability of genetically diverse plants for the breakthroughs have come on a species-by- insertion of new genes into plants. The number species basis, but key discoveries are not often of these plants has been diminishing for a varie- applicable to all plants. Initial results suggest ty of reasons. However, the rate and extent of that agronomically important traits, such as this trend is unknown; the data simply do not disease resistance, can be transferred from one exist. Therefore, it is essential to have an ade- species to another. Limited success has also quate scientific understanding of how much ge- been shown in attempts to create totally new netic loss has taken place and how much germ- species by fusing cells from different genera. plasm (the total genetic variability available to a Attempts to find both suitable vectors and genes species) is needed. Neither of these questions for transferring one plant genes to another are can be answered completely at this time. only now beginning to show promise. Even if-genetic needs can be adequately iden- CONSTRAINTS ON USING MOLECULAR GENETICS FOR PLANT IMPROVEMENTS tified, there is disagreement about the quantity of germplasm to collect. Furthermore, the ex- Molecular engineering has been impeded by a tent to which the new genetic technologies will lack of answers to basic questions in molecular affect genetic variability, vulnerability, or the biology and plant physiology owing to insuffi- storage technologies of germplasm has not been cient research. Federal funding for plant molec- determined. As a result, it is currently difficult, ular genetics in agriculture has come primarily if not impossible, to state how much effort from the U.S. Department of Agriculture should be expended by the National Germplasm (USDA) and the National Science Foundation System to collect, maintain, and test new gene (NSF). In USDA, research support is channeled resources (in this case as seed). primarily through the flexible competitive grants program (fiscal year 1980 budget of $15 Finally, even if an adequate level of genetic million) for the support of new research direc- variability can be assessed, the real problem of tions in plant biology. The total support for the vulnerability—the practice of planting only a 14 ● Impacts Of Applied Genetics—MicrO.Organ/SrnS, Plants, and Animals single variety—must be dealt with at an institu- nologies existed, farmers would still select only tional or social level. Even if no genetic tech- one or a few “best” varieties for planting.

Issues and Options—Plants ISSUE: Should an assessment be con- ISSUE: What are the most appropriate ducted to determine how much approaches in overcoming the diversity in plant germplasm various technical constraints needs to be maintained? that limit the success of molecular genetics for plant improve- An understanding of how much germplasm ment? should be protected and maintained would make the management of genetic resources Although genetic information has been trans- simpler. ferred by vectors and protoplasm fusion, DNA transformations of commercial value have not yet been performed. Molecular engineering has OPTIONS: been impeded by the lack of vectors that can A. Congress could commission a study of how transfer novel genetic material into plants, much genetic variability is necessary or desir- by insufficient knowledge about which genes able to meet present and future needs. would be useful for breeding purposes, and by a lack of understanding of the incompatibility of A comprehensive evaluation of the National chromosomes from diverse sources. Another Germplasm System’s requirements for collec- impediment has been the lack of researchers ting, evaluating, maintaining, and distributing from a variety of disciplines. genetic resources for plant breeding and research could serve as a baseline for a further OPTIONS: assessment. A. The level of funding could be increased for B. Congress could commission a study on the plant molecular genetics research supported need for international cooperation to manage by NSF and USDA. and preserve genetic resources both in natural B. Research units devoted to plant molecular ge- ecosystems and in repositories. netics could be established under the auspices This investigation could include an evaluation of the National Institutes of Health (NIH), with of the rate at which genetic diversity is being emphasis on potential pharmaceuticals derived from plants. lost from natural and agricultural systems along with an estimate of the effects this loss will c. An institute for plant molecular genetics could have. be established under the Science and Educa- tion Administration at USDA that would in- C. Congress could commission a study on how to clude multidisciplinary teams to consider both develop an early warning system to recognize basic research questions and direct applica- the potential vulnerability of crops. tions of the technology to commercial needs Where high genetic uniformity still exists, and practices. proposals could be suggested to reduce any The discoveries of molecular plant genetics risks due to uniformity. Alternatively, the will be used in conjunction with traditional avenues by which private seed companies could breeding programs. Hence, each of the three be encouraged to increase the levels of genetic options could require additional appropriations diversity could be investigated. for agricultural research. Ch. 1—Summary: Issues adoptions ● 15

Advances in reproductive biology and hundreds of millions of dollars to producers and their effects on animal improvement consumers. Coupled with a technology for estrus-cycle FINDINGS regulation, the use of AI could be expanded for Much improvement can be made in the germ- both dairy and beef breeding. Embryo transfer plasm of all major farm animal species using ex- technology, already well-developed but still isting technology. The expanded use of artificial costly, can be used to produce valuable breed- insemination (AI) with stored frozen sperm, es- ing stock. Sexing technology, which is not yet pecially in beef cattle, would benefit both pro- perfected, would be of enormous benefit to the ducers and consumers. New techniques for syn- beef industry because bulls grow faster than chronizing estrus should encourage the wider heifers. use of AI. Various manipulations of embryos will find limited use in producing breeding In the case of animals other than cows: stocks, and sex selection and twinning tech- ● Expanded use of AI for swine production niques should be available for limited applica- will be encouraged by the strong trend to tions within the next 10 to 20 years. confinement housing, although the poor The most important technology in reproduc- ability of boar sperm to withstand freezing tive physiology will continue to be AI. Due in will continue to be a handicap. part to genetic improvement, the average milk ● The benefits of applied genetics have not yield of cows in the United States has more than been realized in sheep production because doubled in the past 30 years, while the total neither AI nor performance testing has number of milk cows has been reduced by more been used. As long as the use of AI con- than half. AI along with improved management tinues to be limited by the inability to and the availability and use of accurate progeny freeze semen and by a lack of agents on the records on breeding stock have caused this market for synchronizing estrus, no rapid great increase. (See figure 3.) major gains can be expected. ● Increasing interest in goats in the United The improvement lags behind what is theo- States and the demand for goat products retically possible. In practice, the observed in- throughout the world, should encourage crease is about 100 lb of milk per cow per year, attention to the genetic gains that the use while a hypothetical breeding program using AI of AI and other technologies make possible. would result in a yearly gain of 220 lb of milk ● Poultry breeders will continue to concen- per cow. The biological limits to this rate of gain trate on improved egg production, growth are not known. rate, feed efficiency, and reduced body fat In comparison with dairy cattle, the beef cat- and diseases. The use of frozen semen tle industry has not applied AI technology wide- should increase as will the use of AI and ly. Only 3 to 5 percent of U.S. beef is artificially dwarf broiler breeders. inseminated, compared to 60 percent of the ● Genetics applied to production of fish, dairy herd. This low rate for beef cattle can be mollusks, and crustaceans in either natural explained by several factors, including manage- environments or manmade culture systems ment techniques (range v. confined housing) is only at the rudimentary stage. and the conflicting objectives of individual Breeders must have reliable information breeders, ranchers, breed associations, and about the genetic value of the germplasm they commercial farmers. are considering introducing. Since farmers do The national calf crop—calves alive at wean- not have the resources to collect and process ing as a fraction of the total number of cows ex- data on the performance of animals other than posed to breeding each year—is only 65 to 81 those in their own herds, they must turn to out- percent. An improvement of only a few percent- side sources. The National Cooperative Dairy age points through AI would result in savings of Herd Improvement Program (NCDHIP) is a mod- 16 . The Impacts of Genetics:Applicatjions to Micro-Organisms, Plants, and Animals

Figure 3.-The Way the Reproductive Technologies Interrelate

Sperm Artificial insemination

Recovered embryos Bull Superovulated cow Sexed?

Sexed?

Frozen?

Calves

Recipient Herd: Synchronized estrus Embryo transfer Each get two for twinning

Photo Credit: Science These 10 calves from Colorado State University were the result of superovulation, in vitro culture, and transfer to the surrogate mother cows on the left. The genetic mother of all 10 calves is at upper right SOURCE: Office of Technology Assessment. Ch. 1—Summary: Issues and Options . 17 el information system and could be adapted to for other species, although pork production in other species. the United States would greatly benefit from a national swine testing program. Selection—deciding which animals to mate —is the breeder’s most basic tool. When going The more esoteric methods of genetic manip- outside his herd to purchase new germplasm, ulation will probably have little impact on the the breeder needs impartial information about production of animals or animal products with- the quality of the available germplasm. NCDHIP in the next 10 years. other in vitro manipula- had recorded 2.8 million of the 10.8 million U.S. tions, such as cloning, cell fusion, the produc- dairy cattle in 1979. In 1978, cows enrolled in tion of chimeras, and the use of rDNA tech- the official plans of NCDHIP outproduced cows niques, will continue to be of intense interest, not enrolled by 5,000 lb of milk per cow, repre- especially for research purposes. It is less likely, senting 52 percent more milk per lactation. however, that they will have widespread prac- No comparable information system exists for tical effects on farm production in this century. other types of livestock. Beef bulls, for example, continue to be sold to a large extent on the basis Each technique requires more research and of pedigrees, but with relatively little objective refinement. Until specific genes of farm animals information on their genetic merit. Data on can be identified and located, no direct gene dairy goats in the United States became avail- manipulation will be practicable. In addition able through NCDHIP for the first time in late this will be difficult because most traits of im- 1980. No nationwide information systems exist portance are due to multiple genes.

Issue and Options—Animals ISSUE: How can the Federal Govern- sentatives for data collecting, can provide the ment improve the germplasm of individual farmer or breeder with accurate in- major farm animal species? formation on the available germplasm so that he can make his own breeding decisions. OPTIONS: A. Programs like the NCDHIP could have in- This option implies that the Federal Govern- creased governmental participation and fund- ment would play such a role in new programs, ing. The efforts of the Beef Cattle Improve- and expand its role in existing ones. ment Federation to standardize procedures could receive active support, and a similar in- B. Federal funding could be increased for basic formation system for swine could be estab- research in total animal improvement. lished. This option, in contrast to option A, assumes The fastest and least expensive way to up- that it is necessary to maintain or expand basic grade breeding stock in the United States is R&D to generate new knowledge that can be through effective use of information. Computer applied to the production of improved animals technology, along with a network of local repre- and animal products. * * * The wide variety of applications for genetic Biological materials can also be converted to engineering is summarized in figure 4. Genetics useful products. In this process, genetic engi- can be used to improve or increase the quality neering can be used to develop micro-organisms and output of plants and animals for direct use that will carry out the conversions. Therefore, by man. Alternatively, materials can be ex- genetic manipulation cannot only provide more tracted from plants and animals for use in food, or better biological raw materials but can also chemical, and pharmaceutical industries. aid in their conversion to useful products. 18 . The Impacts of Genetics: Applications to Micro-Organisms, Plants, and Animals

Figure 4.—AppIications of Genetics

AGRICULTURAL I INDUSTRY I

Plants

Animals

(lncrease/Improve, Output)

SOURCE: Office of Technology Assessment.

Institutions and society

Regulation of genetic engineering relevant to rDNA, and scientific meetings and workshops. FINDINGS No evidence exists that any unexpectedly A program of risk assessment was established harmful genetically engineered organism has at NIH in 1979 to conduct experiments and col- been created. Yet few experts believe that mo- late relevant information. It assesses one form lecular genetic techniques are totally without of genetic engineering, rDNA. On the basis of risk to health and the environment. Information these data, conjectured, inadvertent risk is that has proved useful in assessing the risks generally regarded as less likely today than from these techniques has come from three originally suspected. Risk due to the manipula- sources: experiments designed specifically to tion of genes from organisms known to be haz- test the consequences of working with rDNA, ardous is considered to be more realistic. There- experiments designed for other purposes but fore, microbiological safety precautions that are Ch. 1—Summary: lssues and Options . 19 appropriate to the use of the micro-organisms Table I.—Containment Recommended by the serving as the source of DNA are required. Nev- National institutes of Health ertheless, it has not been demonstrated that Biological—Any combination of vector and host must be combining those genes in the form of rDNA is chosen to minimize both the survival of the system any more hazardous than the original source of outside of the laboratory and the transmission of the vector to nonlaboratory hosts. There are three levels the DNA. of biological containment: Perceptions of the nature, magnitude, and ac- HVl– Requires the use of Escherichia coli K12 or other weakened strains of micro-organisms that ceptability of the risk differ. In addition, public are less able to live outside the laboratory. concern has been expressed about possible HV2– Requires the use of specially engineered strains long-range impacts of genetic engineering. In that are especially sensitive to ultraviolet light, detergents, and the absence of certain this context, the problem facing the policy- uncommon chemical compounds. maker is how to address the risk in a way that HV3– No organism has yet been developed that can accommodates the perceptions and values of qualify as HV3. those who bear it. Physical—Special laboratories (P1-P4) Pl— Good laboratory procedures, trained personnel, The NIH Guidelines for Research Involving wastes decontaminated Recombinant DNA Molecules and existing Fed- P2– Biohazards sign, no public access, autoclave in building, hand-washing facility eral laws appear adequate in most cases to deal P3– Negative pressure, filters in vacuum line, class II with the risks to health and the environment safety cabinets presented by genetic engineering. However, the P4– Monolithic construction, air locks, all air decontaminated, autoclave in room, all Guidelines are not legally binding on industry, experiments in class Ill safety cabinets (glove and no single statute or combination will clearly box), shower room cover all foreseeable commercial applications of SOURCE: Office of Technology Assessment. genetic engineering. The Guidelines are a flexible evolving over- ment programs, certifying new host-vector sys- sight mechanism that combines technical exper- tems, serving as an information clearinghouse, tise with public participation. They cover the and coordinating Federal and local activities. most widely used and possibly risky molecular Limitations in NIH’s oversight are that: it lacks genetic technique–rDNA–prohibiting experi- legal authority over industry; its procedures for ments using dangerous toxins or pathogens and advising industry on large-scale projects have setting containment standards for other poten- not incorporated sufficient expertise on large- tially hazardous experiments. Although compli- scale fermentation technology; its monitoring ance is mandatory only for those receiving NIH for either compliance or consistent application funds, other Federal agencies follow them, and of the Guidelines by individuals or institutions is industry has proclaimed voluntary compliance. virtually nonexistent; and it has not systemati- Rare cases of noncompliance have occurred in cally evaluated other techniques, such as cell fu- universities but have not posed risks to health sion, that might present risks. or the environment. As scientists have learned Federal laws on health and environment will more about rDNA and molecular genetics, the cover most commercial applications of genetic restrictions have been progressively and sub- engineering. Products such as drugs, chemicals, stantially relaxed to the point where 85 percent and foods can be regulated by existing laws. of the experiments can now be done at the However, uncertainty exists about the regula- lowest containment levels, and virtually all tion of either production methods using engi- monitoring for compliance now rests with ap- neered micro-organisms or their intentional proximately 200 local self-regulatory commit- release into the environment, when the risk has tees called institutional biosafety committees not been clearly demonstrated. While a broad (IRCs). (See table 1.) interpretation of certain statutes, such as the Under the Guidelines, NIH serves an impor- Occupational Safety and Health Act and the tant oversight role by sponsoring risk assess- Toxic Substances Control Act, might cover these 20 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals situations, regulatory actions based on such in- substantial regulatory authority over commer- terpretations could be challenged in court. In cial genetic engineering have not yet officially any evident those agencies that could have acted to assert that authority.

Issue and Options—Regulation

ISSUE: How could Congress address the C. Congress could require that all recombinant risks presented by genetic engi- DNA activity be monitored for a limited num- neering? ber of years. OPTIONS: This represents a “wait and see” position by A. Congress could maintain the status quo by let- Congress and the middle ground between the ting NIH and the regulatory agencies set the status quo and full regulation. It recognizes and Federal policy. balances the following factors: 1) the absence of demonstrated harm to human health or the en- Congress might determine that legislation to vironment from genetic engineering; 2) the con- remedy the limitations in current Federal over- tinuing concern that genetic engineering pre- sight would result in unnecessary and burden- sents risks; 3) the lack of sufficient knowledge some regulation. No known harm to health or and experience from which to make a final judg- the environment has occurred under current ment; 4) the existence of an oversight mech- regulation. Also the agencies generally have the anism that seems to be working well, but that legal authority and expertise to adapt to most has clear limitations with respect to commercial new problems posed by genetic engineering. activities; 5) the virtual abolition of Federal The disadvantages are the lack of a central- monitoring of rDNA activities by recent amend- ized, uniform Federal response to the problem, ments to the Guidelines; and 6) the expected in- and the possibility that risks associated with crease in commercial genetic engineering. commercial applications will not be adequately This option would provide a data base that addressed. Conflicting or redundant regulations could be used for: 1) determining the effec- of different agencies would result in unneces- tiveness of voluntary compliance with the sary burdens on those regulated. Guidelines by industry, and mandatory compli- B. Congress could require that the Federal Inter- ance by Federal grantees; 2) determining the agency Advisory Committee on Recombinant quality and consistency of the local self-regu- DNA Research prepare a comprehensive re- latory actions; 3) continuing a formal risk assess- port on its members’ collective authority to ment program; 4) identifying vague or conflict- regulate rDNA and on their regulatory inten- ing provisions of the Guidelines for revision; 5) tions. identifying emerging trends or problems; and 6) The Industrial Practices Subcommittee of this tracing any long-term adverse impacts on health Committee has been studying agency authority or the environment to their sources. over commercial rDNA activities. Presently, there is little official guidance on regulatory re- The obvious disadvantage of this option quirements for companies that may soon mar- would be the required paperwork and effort by ket products made by rDNA methods. A con- scientists, universities, corporations, and the gressionally mandated report would ensure full Federal Government. consideration of these issues by the agencies D. Congress could make the NIH Guidelines ap- and expedite the process. On the other hand, plicable to all rDNA work done in the United the agencies are studying the situation, which States. must be done before they can act. Also, it is often easier and more efficient to act on each This option would eliminate any concern case as it arises, rather than on a hypothetical about the effectiveness of voluntary compliance basis before the fact. with the Guidelines, and it has the advantage of Ch. 1—Summary: Issues adoptions ● 21

using an existing oversight mechanism. The ma- This option would deal comprehensively and jor changes that would have to be made in the directly with the risks of novel molecular area of enforcement. Present penalties for non- genetic techniques. A specific statute would compliance—suspension or termination of re- eliminate the uncertainties over the extent to search funds-are obviously inapplicable to in- which present law covers particular applica- dustry. In addition, procedures for monitoring tions of genetic engineering and any concerns compliance would have to be strengthened. about the effectiveness of voluntary compliance with the Guidelines. Alternatively, the legisla- The main disadvantage of this option is that tion could take the form of amending existing NIH is not a regulatory agency. Since NIH has laws to clarify their applicability to genetic traditionally viewed its mission as promoting engineering. biomedical research, it would have a conflict of interest between regulation and promotion. Other molecular genetic techniques, while One of the regulatory agencies could be given not as widely used and effective as rDNA, raise the authority to enforce the Guidelines. similar concerns. Of the current techniques, cell fusion is the prime candidate for being treated E. Congress could require an environmental im- like rDNA in any regulatory framework. No risk pact statement and agency approval before assessment of this technique has been done, and any genetically engineered organism is inten- no Federal oversight exists. tionally released into the environment. The principal argument against this option is There have been numerous cases where an that the current system appears to be working animal or plant species has been introduced into fairly well, and the limited risks of the tech- a new environment and has spread in an uncon- niques may not warrant the significantly in- trolled and undesirable fashion. Yet in pollution creased regulatory burden that would result control, mineral leaching, and enhanced oil from such legislation. recovery, it might be desirable to release large G. Congress could require NIH to rescind the numbers of engineered micro-organisms into Guidelines. the environment. Deregulation would have the advantage of al- The Guidelines currently prohibit deliberate lowing money and personnel currently involved release of any organism containing rDNA within implementing the Guidelines at the Federal out approval of NIH. One disadvantage of this and local levels to be used for other purposes. prohibition is that it lacks the force of law. There are several reasons for retaining the Another is that approval may be granted on a Guidelines. Sufficient scientific concern exists finding that the release would present “no sig- for the Guidelines to prohibit certain experi- nificant risk to health or the environment;” a ments and to require containment for others. tougher or more specific standard may be de- Most experiments can be done at the lowest, sirable. least burdensome containment levels. NIH is A required study of the possible conse- serving an important role as a centralized over- quences of releasing a genetically engineered sight and information coordinating body, and organism would be an important step in ensur- the system has been flexible enough in the past ing safety. An impact statement could be filed to liberalize the restrictions as evidence in- before permission is granted to release the dicated lower risk than originally thought. organism. However, companies and individuals H. Congress could consider the need for regulat- might be discouraged from developing useful ing work with all hazardous micro-organisms organisms if this process became too burden- and viruses, whether or not they are genet- some and costly. ically engineered. F. Congress could pass legislation regulating all It was not within the scope of this study to ex- types and phases of genetic engineering from amine this issue, but it is an emerging one that research through commercial production. Congress may wish to consider.

76-565 0 - 81 - 3 22 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

Patenting living organisms nisms as trade secrets; 2) patenting microbiological processes and their products; and 3) pat- On June 16, 1980, in a 5-to-4 decision, the Su- enting inanimate components of micro-orga- preme Court ruled that a human-made micro- nisms, such as genetically engineered plasmids. organism was patentable under Federal patent statutes. The decision while hailed by some as 3. Impact on the Patent Law and the Patent and assuring this country’s technological future was Trademark Office.— Because of the complexity, at the same time denounced by others as creat- reproducibility, and mutability of living orga- ing Aldous Huxley’s Brave New World. It will do nisms, the decision may cause some problems neither. for a body of law designed more for inanimate objects than for living organisms. It raises ques- FINDINGS tions about the proper interpretation and appli- 1. Meaning and Scope of the Decision.—The cation of the patent law requirements of novel- decision held that a patent could not be denied ty, nonobviousness, and enablement. In addi- on a genetically engineered micro-organism that tion, it raises questions about how broad the otherwise met the legal requirements for pat- scope of patent coverage on important micro- entability solely because it was alive. It was organisms should be, and about the continuing based on the Court’s interpretation of a provi- need for two statutes, the Plant Patent Act of sion of the patent law which states that a patent 1930 and the Plant Variety Protection Act of may be granted on “. . . any new and useful . . . 1970. These uncertainties could result in in- manufacture, or composition of matter. . . .“ (35 creased litigation, making it more difficult and U.S.c. $101) costly for owners of patents on living organisms to enforce their rights. It is uncertain whether the case will serve as a legal precedent for patenting more complex The impact on the Patent and Trademark Of- organisms. Such organisms, however, will prob- fice is not expected to be significant in the next ably not meet other legal prerequisites to paten- few years. Although the number of patent ap- tability that were not at issue here. In any event, plications on micro-organisms have almost fears that the case would be legal precedent doubled during 1980, the approximately 200 sometime in the distant future for patenting hu- pending applications represent less than 0.2 man beings are unfounded because the 13th percent of those processed each year by the Of- amendment to the Constitution absolutely pro- fice. While the number of such applications is hibits ownership of humans. expected to increase in the next few years because of of the decision and developments in 2. Impact on the Biotechnology Industry.—The the field, the Office should be able to ac- decision is not crucial to the development of the commodate the increase. A few additional ex- industry. It will stimulate innovation by encour- aminers may be needed. , aging the dissemination of technical information that otherwise would have been main- 4. Impact on Academic Research.—Because the tained as trade secrets because patents are pub- decision may encourage academic scientists to lic documents that fully describe the inventions. commercialize the results of their research, it In addition, the ability to patent genetically engi- may inhibit the free exchange of information, neered micro-organisms will reduce the risks but only if scientists rely on trade secrecy and uncertainties facing individual companies rather than patents to protect their inventions in the commercial development of those orga- from competitors in the marketplace. In this re- nisms and their products, but only to a limited spect, it is not clear how molecular biology dif- degree because reasonably effective alterna- fers from other research fields with commercial tives exist. These are: 1) maintaining the orga- potential. Ch. 1—Summary: Issues and Options ● 23

Issue and Options—Patenting Living Organisms ISSUE: To what extent could Congress acts that grant ownership rights to plant provide for or prohibit the pat- breeders who develop new and distinct ent ing of living organisms? varieties of plants.

C Congress could mandate a study of the Plant OPTIONS: . Patent Act of 1930 and the Plant Variety Pro- The Supreme Court stated that it was under- tection Act of 1970. taking only the narrow task of determining These Acts could serve as models for studying whether or not Congress, in enacting the patent statutes, had intended a manmade micro-orga- the broader, long-term potential impacts of patenting living organisms. Such a study would nism to be excluded from patentability solely be timely not only because of the Court’s deci- because it was alive. Moreover, the opinion sion, but also because of allegations that the specifically invited Congress to overrule the decision if it disagreed with the Court’s inter- Acts have encouraged the planting of uniform varieties, loss of genetic diversity, and increased pretation. Congress can act to resolve the questions left unanswered by the Court, overrule concentration in the plant breeding industry. the decision, or develop a comprehensive statu- D. Congress could prohibit patents either on any tory approach. Most importantly, Congress can living organism or on organisms other than draw lines; it can decide which organisms, if those already subject to the plant protection any, should be patentable. Acts. A. Congress could maintain the status quo. By prohibiting patents on any living organisms, Congress would be accepting the Congress could choose not to address the arguments of those who consider ownership issue of patentability and allow the law to be rights in living organisms to be immoral, or who developed by the courts. The advantage of this are concerned about other potentially adverse option is that issues will be addressed as they impacts of such patents, A total prohibition arise, in the context of a tangible, nonhypo- would slow but not stop the development of thetical case. molecular genetic techniques and the biotech- There are two disadvantages to this option: a nology industry because there are severa] good uniform body of law may take time to develop; alternatives for maintaining exclusive control of and the Federal judiciary is not designed to take biological inventions. Development would be sufficient account of the broader political and slowed primarily because information that social interests involved. might otherwise become public would be withheld as trade secrets. A major consequence B. Congress could pass legislation dealing with would be that desirable products would take the specific legal issues raised by the Court's longer to reach the market. decision. Alternatively, Congress could overrule the Many of the legal questions are so broad and Supreme Court’s decision by amending the pat- varied that they do not readily lend themselves ent law to prohibit patents on organisms other to statutory resolution, The precise meaning of than the plants covered by the two statutes the requirements for novelty, nonobviousness, mentioned in option C. This would demonstrate and enablement as applied to biological inven- congressional intent that living organisms could tions will be most readily developed on a case- be patented only by specific statute. by-case basis by the Patent Office and the E. Congress could pass a comprehensive law cov- Federal courts. On the other hand, some ques- ering any or all organisms (except humans). tions are fairly narrow and well-defined; thus, they could be better resolved by statute. The This option recognizes that Congress can most important question is whether there is a draw lines where it sees fit in this area. It could continuing need for the two plant protection specifically limit patenting to micro-organisms, 24 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

or it could encourage the breeding of agricul- characteristics of organisms and the potential turally important animals by granting patent for altering inheritance in a directed fashion rights to breeders of new and distinct breeds. In raise again questions about the relationship of the interest of comprehensiveness and uniform- humans to each other and to other living things. ity, one statute could cover plants and all other People respond in different ways to this poten- organisms that Congress desires to be patent- tial; some see it (like many predecessor develop- able. ments in science) as a challenging opportunity, others as a threat, and still others respond with Genetics and society vague unease. Although many people cannot articulate fully the basis for their concern, ethical, FINDINGS moral, and religious reasons are often cited. Continued advances in science and technol- The public’s increasing concern about the ad- ogy are beginning to provide choices that strain vance of science and impacts of technology has human value systems in areas where previously led to demands for greater participation in deci- no choice was possible. Existing ethical and sions concerned with scientific and technologi- moral systems do not provide clear guidelines cal issues, not only in the United States but and directions for those choices. New programs, throughout the world. The demands imply new both in public institutions and in the popular challenges to systems of representative govern- media, have been established to explore the ment. In every Western country, new mecha- relationships among science, technology, socie- nisms have been devised for increasing citizen ty, and value systems, but more work needs to participation. be done. The public has already become involved in decisionmaking with regard to genetics. As the Genetics-and other areas of the biological science develops, additional issues in which the sciences—have in common a much closer rela- public will demand involvement can be antici- tionship to certain ethical questions than do pated for the years ahead. The question then be- most advances in the physical sciences or comes one of how best to involve the public in engineering. The increasing control over the decisionmaking.

Issues and Options—Genetics and Society Issue: How should the public be ining the public to participate only when it volved in determining policy re- decides to do so on its own initiative. lated to new applications of genetics? If option A were followed, there would be no Because public demands for involvement are cause for claiming that public involvement was unlikely to diminish, ways to accommodate inadequate (as occurred after the first set of these demands must be considered. Guidelines for Recombinant DNA Research was promulgated). Option A poses certain problems: OPTIONS: How to identify a major policy and at what stage A. Congress could specify that public opinion public involvement would be required. Should must be sought in formulating all major pol- it take place only when technological develop- icies concerning new applications of genetics, ment and application are imminent, or at the including decisions on the funding of specific basic research stage? research projects. A “Public Participation Statement” could be mandated for all such decisions. Option B would be less cumbersome to effect. It would permit the establishment of ad hoc B. Congress could maintain the status quo, allow- mechanisms when necessary. —

Ch. 1—Summary: Issues adoptions . 25

ISSUE: How can the level of public tion. On the other hand, this option could have knowledge concerning genetics significant economic consequences to the copy- and its potential be raised? right owner, whose market is often limited to educational institutions. There are some educators who believe that too little time is spent on genetics within the ISSUE: Should Congress begin prepar- traditional educational system. outside the traditional school system, a number of sources ing now to resolve issues that may contribute to increased public understand- have not yet aroused much pub- ing of science and the relationship between lic debate but which may in the science and society. future? Efforts to increase public understanding As scientific understanding of genetics and should, of course, be combined with carefully the ability to manipulate inherited character- designed evaluation programs so that the effec- istics develops, society may face some difficult tiveness of a program can be assessed. questions that could involve tradeoffs between individual freedom and the needs of society. OPTIONS: This will be increasingly the case as genetic technologies are applied to humans. Develop- A. Programs could be developed to increase ments are occurring rapidly. Recombinant DNA public understanding of science and the rela- technology was developed in the 1970’s. In the tionship between science, technology, and spring of 1980, investigators succeeded in the society. first gene replacement in mammals; in the fall Public understanding of science in today's of 1980, the first gene substitution in humans world is essential, and there is concern about was attempted. the adequacy of the public's knwledge. Although this study was restricted to nonhu- B. programs could be established to monitor the man applications, many people assume from level of public understanding of genetics and these and other examples that what can be done of science in general, and to determine wheth- with lower animals can be done with humans er public concern with decisionmaking in and will be. Therefore, some action might be science and technology is increasing. taken to better prepare society for decisions on Selecting this option would indicate that the application of genetic technologies to there is need for additional information, and humans. that Congress is interested in involving the public indeveloping science policy. OPTIONS: C. The copyright laws could be amended to per- A. A commission could be established to identify mit schools to videotape television programs central issues, the probable time frame for ap- for educational purposes. plication of various genetic technologies to Under current copyright law, videotaping tel- humans, and the probable effects on society, evision programs as they are being broadcast and to suggest courses of action. The commission might also consider the related area of may infringe on the rights of the program’s owner, generally its producer. The legal status how participatory democracy might be combined with representative democracy in deci- of such tapes is presently the subject of litiga- sionmaking. tion. In favor of this option, it should be noted that B. The life of the President Commission could many of the programs are made at least in part be extended for the study of Ethical problems with public funds. Removing the copyright con- in Medicine and Biomedical and Behavioral Re- straint on schools would make these programs search, for the purpose of addressing these more available for another public good, educa- issues. 26 . Impacts of Applied Genetics—Micro-Organisms, plants, and Animals

This 1 l-member ‘Commission was established issues already exist, and more are likely to arise in November 1978 and terminates on December in the years ahead. Yet there are also other 31, 1982. It could be asked to broaden its cover- issues in medicine and biomedical and behav- age to additional areas. This would require that ioral research not associated with genetic engi- the life span of the commission be extended and neering that also need review. Whether all additional funds be appropriated. these issues can be addressed by one commission should be considered. Comments from the A potential disadvantage to using the existing existing commission would assist in deciding the commission to address societal issues associated most appropriate course of action. with genetic engineering is that a number of Chapter 2 Introduction Chapter 2

Page The Origins of Genetics...... , . . 29 7. The Griffith Experiment ...... 34 (Genetics in the 20th Century...... 33 8. The Structure of DNA ...... 36 The Riddle of the Gene , ...... , . . . . . , . . . . . 33 9. Replication of DNA ...... , . . . . 37 The Genetic Code ...... , .37 10. The Genetic Code ...... , 38 Developing Genetic “technologies ...... , . . . . 39 11. The Expression of Genetic Information in The Basic Issues ...... 43 the Cell ...... , 39 How Will Applied Genetics Be Used? ...... 43 12. Transduction: The Transfer of Genetic What Are the Dangers? ...... , . . 43 Material in Bacteria by Means of Viruses . . . . 39 13. Conjugation: The Transfer of Genetic Material in Bacteria by Mating...... 40 14. Recombinant DNA: The Technique of Figures Recombining Genre From One Species With Those From Another , ! ...... , ...... 41 Figure No. Page 15. An Example of How the Recombinant DNA 5. The Inheritance Pattern of Pea Color. . . . . , , 30 Technique May Be Used To Insert New 6. Chromosomes . . . . . , ...... 32 Genes Into Bacterial Cells...... , . . . . . 42 Chapter 2 Introduction

Humankind is gaining an increasing under- equally as important: the dwindling supplies of standing of heredity and variation among living natural resources, the risks involved in basic things—the science of genetics. This report ex- and applied scientific research and develop- amines both the critical issues arising from the ment, and the nature of innovation itself. science and technologies that spring from ge- As always, some decisions concerning the use netics, and the potential impacts of these ad- of the new technologies will be made by the vances on society. They are the most rapidly marketplace, while others will be made by var- progressing areas of human knowledge in the ious institutions, both public and private. In the world today. coming years, the public and its representatives Genetic technologies exist only within the in Congress and other governmental bodies will larger context of a maturing science. The key to be called on to make difficult decisions because planning for their potential is understanding of society’s knowledge about genetics and its not simply a particular technology, or breeding capabilities. program, or new opportunity for investment, This report does not make recommendations but how the field of genetics works and how it nor does it attempt to resolve conflicts. Rather, interacts with society as a whole. it clarifies the bases for making judgments by The technologies that this report assesses can defining the likely impacts of a group of technol- be expected to have pervasive effects on life in ogies and tracing their economic, societal, legal, the future. They touch on the most fundamen- and ethical implications. The new genetics will tal and intimate needs of mankind: health care, be influential for a long time to come. Although supplies of food and energy, and reproduction. it will continue to change, it is not too early to At the same time, they trigger concerns in areas begin to monitor its course.

The origins of genetics

For the past 10,000 years, a period encom- perfectly good grain during one season in the passing less than one-half of 1 percent of man’s hope of growing a new crop several months time on Earth, the human race has developed later–faith not only that the seed would indeed under the impetus of applied genetics. As tech- return, but that it would do so in the form of the niques for planning, cultivating, and storing same grain-producing crop from which it had crops replaced subsistence hunting and forag- sprung. This permanence of form from one ing, the character of humanity changed as well. generation to the next has been scientifically From the domestication of animals to the devel- understood only within the past century, but opment of permanent settlements, from the rise the understanding has transformed vague be- of modern science to the dawn of biotech- liefs in the inheritance of traits into the science nology, the genetic changes that mankind has of genetics, and rule-of-thumb animal and plant directed have, in turn, affected the nature of his breeding into the modern manipulations of society. genetic engineering. Applied genetics depends on a fundamental principle–that organisms both resemble and The major conceptual boost for the science differ from their parents. It must have required of genetics required a shift in perspective, great faith on the part of Neolithic man to bury from the simple observation that characteristics

29 30 . lmpacts of Applied Genetics—Micro-Organisms, Plants, and Animals passed from parents to offspring, to a study of the green-seed trait—and it occurred at a rate the underlying agent by which this transmission consistent with the random selection of one of is accomplished. That shift began in the garden two genes from each parent for passage to the of Gregor Mendel, an obscure monk in mid-19th new generation. (See figure 5.) century Austria. By analyzing generations of Genes were real—Mendel’s work made that controlled crosses between sweet pea plants, clear. But where were they located, and what Mendel was able to identify the rudimentary were they? The answer, lay within the nucleus characteristics of what was later termed the of the cell. Unfortunately, most of the contents gene. of the nucleus were unobtainable by biologists Mendel reasoned that genes were the vehicle in Mendel’s time, so his published findings were and repository of the hereditary mechanism, ignored. Only during the last decades of the and that each inherited trait or function of an 19th century did improved microscopes and organism had a specific gene directing its devel- new dyes permit cells to be observed with an opment and appearance. An organism’s observ- acuity never before possible. And only by the able characteristics, functions, and measurable properties taken together had to be based some- how on the total assemblage of its genes. Figure 5.-The Inheritance Pattern of Pea Color Mendel’s analysis showed that the genes of Y = yellow gene = green gene his pea plants remained constant from one gen- g eration to the next, but more importantly, he found that genes and observable traits were not simply matched one-for-one. There were, in fact, two genes involved in each trait, with a Each parent contributes only one seed-color gene to the off- single gene contributed by each parent. When spring. When the two YY and gg homozygotes are crossed, the genetic composition of all offspring is Yg: the genes controlling a particular trait are identical, the organism is homozygous for that trait; if they are not, it is heterozygous. In the Mendelian crosses, homozygous plants always retained the expected characteristics. But heterozygous plants did not simply display a mixture of their different genes; one of the two Offspring tended to predominate. Thus, when homozy - All Yg offspring are heterozygous, and all have yellow gous yellow-seed peas were crossed with homo- seeds, indicating that the Y yellow gene is dominant over zygous green-seed plants, all the offspring were the g green gene. now heterozygous for seed color, possessing a When these Yg heterozygotes are crossed with each other: “green” gene from one parent and a “yellow” from the other. Yet all of them turned out to be indistinguishable from the yellow-seed parent: Yellow-seed color in peas was dominant to green. But even though the offspring resembled their dominant parent, they could be shown to contain a genetic difference. For when the heterozygotes were now crossed with each other, a certain number of recessive green-seed plant again appeared among the offspring. This oc- Thus, 3/4 of these offspring will have yellow seeds, but their curred whenever an offspring was endowed individual genetic composition, YY of Yg, maybe different. with a pair of genes that was homozygous for SOURCE: Office of Technology Assessment. Ch. 2—Introduction ● 37 beginning of the 20th century did scientists traits that Mendel had prescribed for genes rediscover Mendel’s work and begin to appre- almost three decades earlier. By the beginning ciate fully the significance of the cell nucleus of the 20th century, it was clear that chromo- and its contents. somes were of central importance to the life history of the cell, acting in some unspecified man- Even in the earliest microscopic studies, ner as the vehicle for the Mendelian gene. however, certain cellular components stood out; they were deeply stained by added dye. As If this conclusion was strongly implied by the a result, they were dubbed ‘(colored bodies, ” or events of cell division, it became obvious when chromosomes. Chromosomes were seen rela- reproduction in whole organisms was analyzed. tively rarely in cells, with most cells showing It had been established by the latter part of the just a central dark nucleus surrounded by an 19th century that the germ cells of plants and extensive light grainy cytoplasm. But periodi- animals—pollen and ovum, sperm and egg—ac- cally the nucleus seemed to disappear, leaving tually fuse in the process of fertilization. Germ in its place long thready material that con- cells differ from other body cells in one impor- solidated to form the chromosomal bodies. (See tant respect—they contain only half the usual figure 6a.) Once formed, the chromosomes number of chromosomes. This chromosome assembled along the middle of the cell, copied halving within the cell was apparently done themselves, and then moved apart while the cell very precisely, for every sperm and egg con- pinched itself in half, trapping one set of tained exactly one representative from each chromosomes in each of the two halves. Then chromosome pair. When the two germ cells the chromosomes themselves seemed to dis- then fused during fertilization, the offspring solve as two new nuclei appeared, one in each were supplied with a fully reconstituted chro- of the two newly formed cells. (See figure 6b. ) mosome complement, half from each parent. Thus, the same number of chromosomes ap- Clearly, chromosomes were the material link from one generation to the next. Somewhere peared in precisely the same form in every cell of an organism except the germ, or sex, cells. locked within them was the substance of both heredity—the fidelity of traits between genera- Furthermore, the chromosomes not only remained constant in form and number from one tions; and diversity—the potential for genetic generation to the next, but were inherited in variation and change. pairs. They were, in short, manifesting all the 32 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Figure 6.— Chromosomes

Photo credit: Professor Judith Lengyel, Molecular Biology institute, UCLA

Optical micrograph of chromosomal material from the salivary gland of the larva of the common fruit fly, Drosophila melanogaster

6a. An example of a chromosome body from a higher organism. Step 5 Step 6

Step 3 Step 4 Step 1

6b. In Step 1, the chromosome bodies are still uncondensed.

In Steps 2 and 3, the chromosomes condense into thread-like bodies and align themselves near the center of the cell. In Steps 4 and 5, the chromosomes begin to separate and are pulled to the opposite poles of the cell. In Step 6, the chromosomes return to an uncondensed state and the cell begins to constrict about the middle to form two new cells.

SOURCE: Office of Technology Assessment. Ch. 2—introduction . 33

(kinetics in the 20th century

During the first few decades of the 20th cen- because linkage itself was not permanent, tury, scientists searched for progressively linked genes sometimes separated. For instance, simpler experimental organisms to clarify pro- while yellow bodies, ruby eyes, and forked bris- gressively more complex genetic concepts. First tles were all linked traits, the first two stayed was Thomas Hunt Morgan’s Drosophila—gnat- together far more frequently than either did sized fruit flies with bulbous eyes. These insects with the third. have a simple array of four easily distinguish- The degree of linkage between two genes was able chromosome pairs per cell. They repro- hypothesized to be directly proportional to the duce rapidly and in large numbers under the distance between them on the chromosome, simplest of laboratory conditions, supplying a mainly because of a unique event that occurs new generation every month or so. Thus, re- during the development of germ cells. Before searchers could carry out an enormous number the normal chromosome number is halved, the of crosses employing a whole catalog of dif- chromosomes crowd together in the center of ferent fruit fly traits in a relatively brief time. the cell, coiling tightly around each other, prac- It became obvious from the extensive Dros- tically fusing along their entire length. It is in ophila data that certain traits were more likely this state that crossing-over (or natural recombi- to be inherited together than others. Yellow nation)—the actual physical exchange of parts bodies and ruby eyes, for instance, almost al- between chromosomes—occurs. No chromo- ways went together, with both in turn, appear- some emerges from the exchange in the same ing more frequently than expected with the condition as before; the lengths of chromo- trait known as “forked bristles. ” All three traits, somes are reshuffled before being transferred however, showed up only randomly with to the next generation. curved wings. Certain genes thus seemed to be The idea of linkage meant that Mendel’s for- linked to one another. The entire Drosophila mulations had to be modified. Clearly, genes genome, in fact, fell into four distinct linkage were not completely independent units. Further groups. The physical basis for these groups, not work with Drosophila in the 1920’s showed that surprisingly, consisted of the four fruit fly genes were also not permanent and could chromosomes. Linked genes behaved as they change over time. Although natural mutations did because they were located on the same occurred at a very slow rate, exposing fruit flies chromosome. to X-rays accelerated their frequency enor- Soon, scientists learned that they could not mously. Exposure of a parental fly population only assign particular genes to particular Droso- led to an array of new traits among their off- phila chromosomes but could identify the rela- spring—traits which, if they were neither lethal tive locations of different genes on a given nor sterilizing, could be passed from one gen- chromosome. This gene mapping was possible eration to the next.

The riddle of the gene _

With all this research, nobody yet knew what triguing observations made a decade earlier by the gene was made of. The first evidence that a British physician, Fred Griffith. He had it consisted of deoxyribonucleic acid (DNA) worked with two types of pneumococcus (the emerged from the work of Oswald Avery, Colin bacteria responsible for pneumonia) and with MacLeod, and Maclyn McCarty at the Rockefel- two different bacteria within each type. One ler Institute in New York in the early 1940’s. bacterium in each type was coated in a polysac- Avery’s group took as its starting point some in- charide capsule; the other was bare. Bare bac- 34 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals teria gave rise only to bare progeny, while those Figure 7.—The Griffith Experiment with capsules produced only encapsulated forms. Only the encapsulated forms of both 7a. There are two types of pneumococcus, each of which can exist in two forms: types II and III could cause disease; bare bacteria were benign. (See figure 7a.) But when Griffith took some encapsulated type III bacteria that had been killed and rendered harmless and where R represents the rough, nonencapsulated, benign mixed them with bare bacteria of type II, the form; and presumably safe mixture became virulent: Mice S represents the smooth, encapsulated, virulent injected with it died of a massive pneumonia inform. fection, Bacteria recovered from these animals 7b. The experiment consists of four steps: were found to be of type 11—the only living bacteria the mice had received—now wrapped in type III capsules. (See figure 7b.) Avery’s group recognized Griffith’s finding as a genetic phenomenon; the dead type 111 bacte- Virulent strain (1) ria must have delivered the gene for making capsules into the genetic complement of the living type 11 recipients. By meticulous research, Avery’s group found that the substance which caused the genetic transformation was DNA. It had been in 1868, just 3 years after Mendel had published his findings, that DNA was dis- Mice injected with nonvirulent R do not become infected. covered by Friedrich Miescher. It is an extreme- ll ly simple molecule composed of a small sugar molecule, a phosphate group (a phosphorous atom surrounded by four oxygen atoms), and four kinds of simple organic chemicals known as nitrogenous (nitrogen-containing) bases. To- Virulent gether, one sugar, one phosphate, and one base strain, form a nucleotide—the basic structural unit of heat-killed the large DNA molecule. Because it is so simple, (3) DNA had appeared to be little more than a The virulent SIII is heat-killed. Mice injected with it do not monotonous conglomeration of simple nucleo- die. tides to scientists in the early 20th century. It seemed unlikely that such a prosaic molecule could direct the appearance of genetic traits while faithfully reproducing itself so that information could be transferred between generations. Although Avery’s results seemed clear enough, many were reluctant to accept them. Those doubts were finally laid to rest in a brief report published in 1953 by James Watson heat-killed II and Francis Crick. By using X-ray crystallo- graphic techniques and building complex mod- (4) els—and without ever having actually seen the When mice are injected with the nonvirulent RII and the molecule itself—Watson and Crick reported that heat-killed SIII, they die. Type II bacteria wrapped in type Ill they had discovered a consistent scientifically capsules are recovered from these mice. sound structure for DNA. SOURCE: Office of Technology Assessment. Ch. 2—introduction . 35

The structure that Crick and Watson uncov- If the actual order of the bases on one of the ered solved part of the genetic puzzle. Accord- pair of chains were given, one could write down ing to them, the phosphates and sugars formed the exact order of the bases on the other one, two long chains, or backbones, with one nitrog- because of the specific pairing. Thus one chain enous base attached to each sugar. The two is, as it were, the complement of the other, and it is this feature which suggests how the desoxy- backbones were held together like the supports ribonucleic acid molecule might duplicate of a ladder by weak attractions between the itself. ’ bases protruding from the sugar molecules. Of the four different nitrogenous bases—adenine, When a double-stranded DNA molecule is un- thymine, guanine, and cytosine—attractions ex- zipped, it consists of two separate nucleotide isted only between adenine(A) and thymine(T), chains, each with a long stretch of unpaired and between guanine(G) and cytosine(C). (See bases. In the presence of a mixture of nucleo- figure 8a) Thus, if a stretch of nucleotides on tides, each base attracts its complementary one backbone ran: match in accordance with the inherent affinities of adenine for thymine, thymine for adenine, guanine for cytosine, and cytosine for guanine. the other backbone had to contain the directly The result of this replication is two DNA mole- opposite complementary sequence: cules, both precisely identical to each other and to the original molecule—which explains the T-A-G-A-A-T-T. . . faithful duplication of the gene for passage from The complementary pairing between bases run- one generation to the next. (See figure 9.) ning down the center of the long molecule was responsible for holding together the two other- Crick and Watson’s work solved a major rid- wise independent chains. (See figure 8b. ) Thus, dle in genetic research. Because George Beadle the DNA molecule was rather like a zipper, with and Edward Tat urn had recently discovered that genes control the appearance of specific the bases as the teeth and the sugar-phosphate chains as the strands of cloth to which each zip- proteins, and that one gene is responsible for per half was sewn. Crick and Watson also found producing one specific protein, scientists now that in the presence of water, the two poly - knew what the genetic material was, how it rep- nucleotide chains did not stretch out to full licated, and what it produced. But they had yet length, but twisted around each other, forming to determine how genes expressed themselves what has undoubtedly become the most glori- and produced proteins. fied structure in the history of biology-the double helix. (See figure 8c.) The structure was scientifically elegant. But it was received enthusiastically also because it im- ‘James D. Watson and Francis Crick, %enetical Implications of plied how DNA worked. As Crick and Watson the Structures of Deoxyribose Nucleic Acid, ” Nature 171, 1953. pp. themselves noted: 737-8. 36 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Figure 8.—The Structure of DNA

A o -o”

5 0. -o”

Adenine 8a. The pairing of the four nitrogenous bases of DNA: Adenine (A) pairs with Thymidine (T) Guanine (G) pairs with Cytosine (C) -o”

8b. The four bases form the four letters in the alphabet of the genetic code. The sequence of the bases along the sugar-phosphate backbone encodes the genetic information.

A schematic diagram of the DNA double helix. A three-dimensional representation of the DNA double helix.

&. The DNA molecule is a double helix composed of two chains. The sugar-phosphate backbones twist around the outside, with the paired bases on the inside serving to hold the chains together. SOURCE: Office of Technology Assessment. Ch. 2—introduction . 37

Figure 9.—Replication of DNA

Old Old

Old New New Old When DNA replicates, the original strands unwind and serve as templates for the building of new complementary strands. The daughter molecules are exact copies of the parent, with each having one of the parent strands.

SOURCE: Office of Technology Assessment.

The genetic code

Proteins are the basic materials of cells. Some Ironically, proteins are far more complex and proteins are enzymes, which catalyze reactions diverse than the four nucleotides that help within a cell. In general, for every chemical re- create them. Proteins, too, are long chains made action in a living organism, a specific enzyme is up of small units strung together. In this case, required to trigger the process. Other proteins however, the units are amino acids rather than are structural, comprising most of the raw ma- nucleotides—and there are 20 different kinds of terial that forms cells. amino acids. Since an average protein is a few

76-565 0 - 81 - 4 38 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals hundred amino acids in length, and since any manufactured, how their synthesis was regu- one of 20 amino acids can fill each slot, the num- lated, and the role of DNA in both processes her of possible proteins is enormous. Neverthe- were understood in considerable detail. The less, each protein requires the strictest ordering process of transcribing DNA’s message-carry- of amino acids in its structure. Changing a ing the message to the cell’s miniature protein single amino acid in the entire sequence can factories and building proteins-took place drastically change the protein’s character. through a complex set of reactions. Each amino acid in the protein chain was represented by It was now possible for scientists to move three nucleotides from the DNA. That three- nearer to an appreciation of how genes func- base unit acted as a word in a DNA sentence tioned. First had come the recognition that DNA that spelled out each protein-the genetic code. determined protein; now it was evident that the (See figure 10.) sequence of nucleotides in DNA determined a linear sequence of amino acids in proteins. Through the genetic code, an entire gene—a By the early 1980’s, the way proteins were linear assemblage of nucleotides-could now be

Figure 10.—The Genetic Code

THIRD BASE THIRD BASE Amino acid symbol alanine ala arginine arg asparagine asn aspartic acid asp asn and/or asp asx cysteine Cys glutamine gln glutamic acid glu gin and/or glu glx glycine gly histidine his isoleucine ileu Ieucine Ieu Iysine Iys methionine met SECOND THIRD BASE THIRD BASE phenylaianine phe proline pro serine ser threonine thr tryptophan trp tyrosine tyr valine val

Each amino acid is determined by a three letter code (A, G, T, or C) along the DNA. If the first letter in the code is A, the second is T, and third is A, the amino acid will be tyrosine (or tyr) in the complete protein molecule. For Ieucine (or Ieu), the code is GAT, and so forth. The dictionary above gives ● och (ochre); amb (ambert, and end are step signals for translation, i.e., the entire code, signals the end of synthesis of the protein chain.

SOURCE: Office of Technology Assessment. Ch. 2—introduction ● 39

read like a book. By the 1970’s, researchers had Figure 11.—The Expression of Genetic Information learned to read the code of certain proteins, in the Cell synthesize their DNA, and insert the DNA into bacteria so that the protein could be produced. (See figure 11.) Meanwhile, other scientists were studying the genetics of viruses and bacteria. The combination of these studies with those investigating the genetic code led to the innovations of genetic engineering. SOURCE: Office of Technology Assessment.

Developing genetic technologies

In the early 1960’s, scientists discovered ex- DNA along with it. Thus, when the new virus actly how genes move from one bacterium to particles now infect other bacteria, they bring another. One such mechanism uses bacterio- along several genes from their previous host. phages-viruses that infect bacteria-as inter- This viral transduction-the transfer of genes mediaries. Phages act like hypodermic needles, by an intermediate viral vector or vehicle- injecting their DNA into bacterial hosts, where could be used to confer new genetic traits on it resides before being passed along from one recipient bacteria. (See figure 12. ) generation to the next as part of the bacterium’s own DNA. Sometimes, however, the injected Bacteria also transfer genes directly in a proc- DNA enters an active phase and produces a crop ess called ’conjugation, in which one bacterium of new virus particles that can then burst out of attaches small projections to the surface of a their host. Often during this process, the viral nearby bacterium. DNA from the donor bacte- DNA inadvertently takes a piece of the bacterial rium is then passed to the recipient through the

Figure 12.—Transduction: The Transfer of Genetic Material in Bacteria by Means of Viruses

Bacterial Empty chromosome Bacterium fragments viral coat . New virus

bacterium

In step 1 of viral transduction, the infecting virus injects its DNA into the cell. In step 2 when the new viral particles are formed, some of the bacterial chromosomal fragments, such as gene A, may be accidently incorporated into these progeny viruses instead of the viral DNA. In step 3 when these particles infect a new cell, the genetic elements incorporated from the first bacterium can recombine with homologous segments in the second, thus exchanging gene A for gene a.

SOURCE: Office of Technology Assessment 40 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals projections. The ability to form projections and lated, for instance, would cut DNA only when it donate genes to neighbors is a genetically con- located the sequence: trolled trait. The genes controlling this trait, (G-A-A-) however, are not located on the bacterial chromosomes. Instead, they are located on separate (C-T-T) genetic elements called plasmids-relatively If the sequence occurred once in a circular plas- small molecules of double-stranded DNA, ar- mid, the effect would simply be to open the ranged as closed circles and existing autono- circle. If the sequence were repeated several mously within the bacterial cytoplasm. (See times along a length of DNA, the DNA would be figure 13.) chopped into several small pieces. Plasmids and phages are two vehicles—or By the late 1970’s, scores of different restric- vectors-for carrying genes into bacteria. As tion enzymes had been isolated from a variety such, they became tools of genetic engineering; of bacteria, with each enzyme having a unique for if a specifically selected DNA could be intro- specificity for one specific nucleotide sequence. duced into these vectors, it would then be pos- These enzymes were another key to genetic en- sible to transfer into bacteria the blueprints for gineering: they not only allowed plasmids to be proteins—the building blocks of genetic charac- opened up so that new DNA could be inserted, teristics. but offered a way of obtaining manageable pieces of new DNA as well. (See figure 14.) But bacteria had been confronting the inva- Using restriction enzymes, almost any DNA sion of foreign DNA for millennia, and they had molecule could be snipped, shaped, and evolved protective mechanisms that preserved trimmed with precision. their own DNA while destroying the DNA that did not belong. Bacteria survive by producing Cloning DNA—that is obtaining a large quanti- restriction enzymes. These cut DNA molecules ty of exact copies of any chosen DNA molecule in places where specific sequences of nucleo- by inserting it into a host bacterium-became tides occur—snipping the foreign DNA, yet leav- technically almost simple. The piece in question ing the bacteria’s own genetic complement was merely snipped from the original molecule, alone. The first restriction enzyme that was iso- inserted into the vector DNA, and provided with

Figure 13.—Conjugation: The Transfer of Genetic Material in Bacteria by Mating

Plasmid

In conjugation, a plasmid inhabiting a bacterium can transfer the bacterial chromosome to a second cell where homologous segments of DNA can recombine, thus exchanging gene B from the first bacterium for gene b from the second.

SOURCE: Office of Technology Assessment. Ch. 2—introduction ● 41

Figure 14.—Recombinant DNA: The Technique of into an appropriate vector. This was the Recombining Genes From One Species procedure used to obtain the gene for hu- With Those From Another man insulin in 1979. (See figure 15. ) ● The gene can also be synthesized, or created, directly, since the nucleotide sequence of the gene can be deduced from New DNA the amino acid sequence of its protein product. This procedure has worked well for small proteins—like the growth regulatory hormone somatostatin—which have relatively short stretches of DNA coding. But somatostatin is a tiny protein, only 14 amino acids long. With three nucleotides coding for each amino acid, scientists had to synthesize a DNA chain 42 nucleotides long to produce the complete hormone. For larger proteins, the gene-synthesis ap- amount of DNA protein proach rapidly becomes highly impractical. ● The third method is also the most controversial. In this “shotgun” approach, the Restriction enzymes recognize certain sites along the DNA entire genetic complement of a cell is and can chemically cut the DNA at those sites. This makes chopped up by restriction enzymes. Each it possible to remove selected genes from donor DNA mole- of the DNA fragments is attached next to cules and insert them into plasmid DNA molecules to form the recombinant DNA. This recombinant DNA can then be vectors and transferred into a bacterium; cloned in its bacterial host and large amounts of a desired the bacteria are then screened to find those protein can be produced. making the desired product. Screening thousands of bacterial cultures was part of SOURCE: Office of Technology Assessment. the technique that enabled the isolation of a bacterial host as a suitable environment for the human interferon gene. * replication. The desired piece of DNA could be At present, these techniques of recombina- recombined with a plasmid vector, a procedure tion work mainly with simple micro-organisms. that gave rise to recombinant DNA (rDNA), aIso Scientists have only recently learned how to in- known as gene splicing. Since bacteria can be troduce novel genetic material into cells of grown in vast quantities, this process could higher plants and animals. These higher cells result in large-scale production of otherwise are being ‘engineered’ in totally different ways, scarce and expensive proteins. by growing plant or animal cells in ‘tissue cul- Although placing genes inside of bacteria is ture’ systems, in vitro. now a relatively straightforward procedure, ob- Tissue culture systems work with isolated taining precisely the right gene can be difficult. cells, with entire pieces of tissue, and to a far Three techniques are currently available: more limited extent, with whole organs or even early embryos. The techniques make it possible ● Ribonucleic acid—RNA—is the vehicle through which the message of DNA is read to manipulate cells experimentally and under and transcribed to form proteins. The RNA controlled conditions. Several techniques are available. For example, in one set of experi- that carries the message for the desired protein is first isolated. An enzyme, called ments, complete plants have been grown from ‘reverse transcriptase, is then added to the single cells—a breakthrough that may permit RNA. The enzyme triggers the formation of “Strictly speaking, RNA was transcribed using the shotgun DNA—reversing the normal process of pro- approach into DNA, which was then cloned into bacteria and tein production. The DNA is then inserted screened. 42 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Figure 15.-An Example of How the Recombinant DNA Technique May Be Used To Insert New Genes Into Bacterial Cells

The first part of the technique involves the manipula- 11. A bacterial plasmid, which is a small piece of circular tions necessary to isolate and reconstruct the desired DNA, serves as the vehicle for introducing the new gene gene from the donor: (obtained. in part. I above), into the bacterium: ‘a) The RNA that carries the message (mRNA) for the a) The circular plasmid is cleaved by the appropriate desired protein product is isolated. restriction enzyme. b) The double-stranded DNA is reconstructed from the b) The enzyme terminal transerase extends the DNA mRNA. strands of the broken circle with identical bases c) in the final step of this sequence, the enzyme ter- (four cytosines in this case, to allow complemen- minal transferase acts to extend the ends of the tary base pairing with the guanines added to the DNA strands with short sequences of identical gene obtained in part i). bases (in this case four guanines). II I Messenger RNA Bacterial plasmid DNA from animal cell a) Enzymatic reconstruction

Double-strand DNA t b) I

Terminal transferase a)

Ill. The final product, a bacterial plasmid containing the new gene, is obtained. This plasmid can then be inserted into a bacterium where it can be replicated and produce the desired protein product: a) The gene obtained in part i and the plasmid DNA from part ii are mixed together and anneai because of the complementary base-pairing between them. b) Bacterial enzymes fill in any gaps in the circle, sealing the connection between the plasmid DNA and the inserted DNA to generate an intact circular plasmid now containing a new gene.

Uptake by cell; Ill repair by enzymes

SOURCE: Office of Technology Assessment. Ch. 2—introduction ● 43 hundreds of plants to be grown asexually from fusion* can be used to direct efficient, fast ge- a small sample of plant material. Just as with netic changes in plants. (See ch. 8.) bacteria, the cells can be induced to take up Cell culture techniques, while not strictly ge- pieces of DNA in a process call transformation. netic manipulation, form a major aspect of mod- They can also be exposed to mutation-causing ern biotechnology. Combined with genetic ap- agents so that they produce mutants with de- proaches, their potential is only on the verge of sired properties. in another set of experiments, being realized. two different cells have been fused to form a “hybrid” that contains the ● A related technique is protoplasm fusion, or the fusion of cells new, single-cell whose walls have been removed to leave only membrane-bound genetic complements of both antecedents. In cells. The cells of bacteria, fungi, and plants must all be freed of both cases, the success of tissue culture and cell their walls before they can be fused.

The basic issues

Applied genetics is like no other technology. What are the dangers? By itself, it may enable tremendous advances in conquering diseases, increasing food produc- Even before scientists recognized the poten- tion, producing new and cheaper industrial sub- tial power of applied genetics, some questioned stances, cleaning up pollution, and understand- its consequences; for with its benefits, appeared ing the fundamental processes of life. Because hypothetical risks. Although most experts today the technology is so powerful, and because it in- agree that the immediate hazards of the basic volves the basic roots of life itself, it carries with research itself appear to be minimal, nobody it potential hazards, some of which might arise can be certain about all the consequences of from basic research, others of which may stem placing genetic characteristics in micro-orga- from its applications. nisms, plants, and animals that have never carried them before. There are at least three sepa- As the impacts of genetic technologies are dis- rate areas of concern: cussed, two fundamental questions must be kept in mind: First, genetically engineered micro-organisms might have potentially deleterious effects on hu- How will applied genetics be used? man health, other living organisms, or the environment in general. Unlike toxic chemicals, or- Interest in the industrial use of biological ganisms may reproduce and spread of their processes stems from a merging of two paths: own accord; if they are released into the envi - the revolution in scientific understanding of the ronment, they may be impossible to control. nature of genetics; and the accelerated search for a sustainable society in which most indus- Second, some observers question whether trial processes are based on the use of renew- sufficient knowledge exists to allow the extinc- able resources. The new genetic technologies tion of diverse species of “genetically inferior” will spur that search in three ways: they will plants and animals in favor of a few strains of provide a means of doing something biolog- “superior” ones. Evolution thus far has de- ically—with renewable raw materials—that pre- pended, in part, on genetic diversity; replacing viously required chemical processes using non- in nature diverse inferior strains by genetically renewable resources; they will offer more ef- engineered superior strains may increase the ficient, more economical, less polluting ways for susceptibility of living things to disease and en- producing both old and new products; and they vironmental insults. will increase the yield of the plant and animal resources that are responsible for providing the Finally, this new knowledge affects the un- world’s supplies of food, fibers, and some fuels. derstanding of life itself. It is tied to the ultimate 44 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals questions of how humans view themselves and locating and monitoring its benefits and bur- what they legitimately control in the world. dens. That process requires knowledge. The following sections of the report describe the im- Because of the significant and wide-ranging pacts of applied genetics on specific industries, scope of applied genetics, society as whole must and assess many of their consequences. begin to debate the issues with a view toward al- Part I Biotechnology

Chapter 3-Genetic Engineering in the Fermentation Technologies ...... , ...... 49 Chapter 4-The Pharmaceutical Industry ...... 59 Chapter 5-The Chemical Industry...... , ...... , ...... 85 Chapter 6-The Food Processing Industry ...... 107 (C hapter 7- The Use of Genetically Engineered Micro-Organisms in the Environment. . 117 Chapter 3 Genetic Engineering and the Fermentation Technologies Chapter 3

Page Figures Biotechnology—An Introduction...... 49 Fermentation ...... 49 Figure No. 16. Diagram of Products Available From Cells . . . 51 Fermentation Industries ...... , ...... 50 17. Features of a Standard Fermenter ...... 52 Fermentation Using Whole Living Cells ...... 51 18. Immobilized Cell System ...... 52 The Process of Enzyme Technology...... 53 19. Diagram of Conversion of Raw Material Comparative Advantages of Fermentations to Product...... Using whole Cells and Isolated Enzymes. . . . 54 The Relationship of Genetics to Fermentation . 54 Fermentation and Industry ...... , . 55

Table Table No. Page 2. Enzyme Products ...... 54 Chapter 3 Genetic Engineering and the Fermentation Technologies

Biotechnology—an introduction

Biotechnology involves the use in industry of some of the secondary impacts that the technol- living organisms or their components (such as ogies might have. Because the chemical and enzymes). It includes the introduction of geneti- food industries will feel the major impact of bio- cally engineered micro-organisms into a variety technology later, specific impacts are less cer- of industrial processes. tain and particular products are less identifiable. The mining, oil recovery, and pollution The pharmaceutical, chemical, and food proc- control industries are also candidates for the essing industries, in that order, are most likely use of genetic technologies. However, because to take advantage of advances in molecular ge- of technical, scientific, legal, and economic un- netics. Others that might also be affected, al- certainties, the success of applications in these though not as immediately, are the mining, industries is more speculative. crude oil recovery, and pollution control industries. The generalizations made with respect to each of the industries should be viewed as just Because nearly all the products of biotechnol- that—generalizations. Because a wide array of ogy are manufactured by micro-organisms, fer- products can be made biologically, and because mentation is an indispensable element of bio- different factors influence each instance of pro- technology’s support system. The pharmaceuti- duction, isolated examples of success may ap- cal industry, the earliest beneficiary of the new pear throughout the industries at approximate- knowledge, is already producing pharmaceu- ly the same time. In almost every case, specific ticals derived from genetically engineered predictions can only be made on a product-by- micro-organisms. The chemical industry will product basis; for while it may be true that bio- take longer to make use of biotechnology, but technology’s overall impact will be profound, the ultimate impact may be enormous. The food identifying many of the products most likely to processing industry will probably be affected be affected remains speculative. last. This report examines many of the pharmaceutical industry’s products in detail, as well as

Fermentation

There are several ways that DNA can be cut, success of biotechnology has been around for spliced, or otherwise altered. But engineered centuries. It is fermentation, essentially the DNA by itself is a static molecule. To be any- process used to make wine and beer. It can also thing more than the end of a laboratory exer- produce organic chemical compounds using cise, the molecule must be integrated into a sys- micro-organisms or their enzymes. tem of production; to have an impact on society Over the years, the scope and efficiency of at large, it must become a component of an in- the fermentation process has been gradually im- dustrial or otherwise useful process. proved and refined. Two processes now exist, The process that is central to the economic both of which will benefit from genetic engi-

49 50 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals neering. In fermentation technology, living or- ● structural components, such as collagen, ganisms serve as miniature factories, convert- used in skin transplants following burn ing raw materials into end products. In enzyme trauma; technology, biological catalysts extracted from ● certain hormones, such as insulin and those living organisms are used to make the human growth hormone; products. ● substances in the immune system, such as antibodies and interferon; and Fermentation industries ● specialized functional components, such as hemoglobin. The food processing, chemical, and pharmaceutical industries are the three major users of Fermentation technologies are so useful for pro- fermentation today. The food industry was the ducing proteins partly because these are the first to exploit micro-organisms to produce direct products of genes. But proteins (as en- alcoholic beverages and fermented foods. Mid- zymes) can also be used in thousands of addi- 16th century records describe highly sophisti- tional conversions to produce practically any cated methods of fermentation technology. Heat organic chemical and many inorganic ones as processing techniques, for example, anticipated well: (See figure 16. ) pasteurization by several centuries.

[n the early 20th century, the chemical in- Figure 16.-Diagram of Products Available dustry began to use the technology to produce From Cells organic solvents like ethanol, and enzymes like amylase, used at the time to treat textiles. The chemical industry’s interest in fermentation arose as the field of biochemistry took shape around the turn of the century. But it was not until World War I that wartime needs for the organic solvent acetone—to produce the cor- dite used in explosives–substantially increased research into the potential of fermentation. Thirty years later after World War 11, the phar- isolated maceutical industry followed the chemical industry’s lead, applying fermentation to the production of vitamins and new antibiotics. Today, approximately 200 companies in the United States and over 500 worldwide use fermentation technologies to produce a wide variety of products. Most use them as part of B production processes, usually in food processing. But others manufacture either proteins, which can be considered primary products, or a host of secondary products, which these proteins help produce. For genes can make en- Raw material zymes, which are proteins; and the enzymes Product can help make alcohol, methane, antibiotics, and many other substances. In (A) DNA directs the formation of a protein, such as insulin, which is itself the desired product. In (B), DNA directs Proteins, the primary products, function as: the formation of an enzyme which, in turn, converts some raw material, such as sugar, to a product, such as ethanol. enzymes such as asparaginase which are used in the treatment of leukemia; SOURCE: Office of Technology Assessment. Ch. 3—Genetic Engineering and the Fermentation Technologies . 51

carbohydrates, such as fructose sweeten- from citrus fruits—are now made by fer- ers; mentation. As a result of more efficient

lipids, such as vitamins A, E, and K; technology, products from vitamin B12 to alcohols, such as ethanol; steroids have come into wider use. other organic compounds, such as acetone; 3. Discovery of a new product.—The discovery and that antibiotics were produced by micro- inorganic chemicals, such as ammonia, for organisms sparked searches for an entirely use in fertilizers. new group of products. Several thousand antibiotics have been discovered to date, of Fermentation is not the only way to manufac- which over a hundred have proved to be ture or isolate these products. Some are tradi- clinically useful. tionally produced by other methods. If a change 4. Environmental concerns. —The problems of from one process to another is to occur, both sewage treatment and the need for new economic and societal pressures will help deter- sources of energy have triggered a search mine whether an innovative approach will be for methods to convert sewage and munici- used to produce a particular product. Alan Bull pal wastes to methane, the principal com- has identified four stimuli for change and in- 1 ponent of natural gas. Because micro-orga- novation: nisms play a major role in the natural cy- 1. abundance of a potentially useful raw cling of organic compounds, fermentation material; has been one method used for the conver- 2. scarcity of an established product; sion. 3. discovery of a new product; and 5. Scarcity of a currently used raw materi- 4. environmental concerns. a/.—Because the Earth’s supplies of fossil fuels are rapidly dwindling, there is intense And conditions existing today have added a fifth interest in finding methods for converting stimulus: other raw materials to fuel. Fermentation 5. scarcity of a currently used raw material. offers a major approach to such conversions. Each of these factors has tended to accelerate the application of fermentation. Fermentation technologies can be effective in each of these situations because of their out- 1. Abundance of a potentially useful raw material.—The use of a raw material can be standing versatility and relative simplicity. The processes of fermentation are basically identi- the driving force in developing a process. cal, no matter what organism is selected, what When straight chain hydrocarbons (n-al- medium used, or what product formed. The kanes) were produced on a large scale as same apparatus, with minor modifications, can petroleum refinery byproducts, fermenta- be used to produce a drug, an agricultural prod- tion processes were developed to convert uct, a chemical, or an animal feed supplement. them to single-cell proteins for use in animal feed. 2. Scarcity of an established product.—The Fermentation using whole living cells new-found potential for producing human Originally, fermentation used some of the hormones through fermentation technol- most primitive forms of plant life as cell fac- ogy is a major impetus to the industry to- tories. Bacteria were used to make yogurt and day. Similarly, many organic compounds antibiotics, yeasts to ferment wine, and the once obtained by other processes—like filamentous fungi or molds to produce organic citric acid, which was extracted directly acids. More recently, fermentation technology has begun to use cells derived from higher ‘/\. ‘I”. lhJll, 11. [:. FllWrCM)Ci, and (:. Rat ledge, kficrobia/ Techndo,gv: plants and animals under growth conditions (~urrent Sfafe, Future Prospects, Z$lth Symposium ot’ the Society for (kllf>l’iil hlicrut)iub~y al 1 llll)f~rsitv uf (:iilllt)l’itl~f~, April 1979 known as cell or tissue culture. In all cases, ((:illllt)l’idg(’, l’;ll~lillld: (kmt)ridge [~l;ilwrsit}~ Press, 1 979), pp. 4-8. large quantities of cells with uniform character- 52 . Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

istics are grown under defined, controlled con- raw materials and other nutrients and an at- ditions. tendant output of fermented material. The most recent approaches use micro-organisms that In its simplest form, fermentation consists of have been immobilized in a supporting struc- mixing a micro-organism with a liquid broth ture. (See figure 18.) As the solution containing and allowing the components to react. More so- the raw material passes over the cells, the phisticated large-scale processes require control micro-organisms process the material and re- of the entire environment so that fermentation lease the products into the solution flowing out proceeds efficiently and, more importantly, so of the fermenter. that it can be repeated exactly, with the same amounts of raw materials, broth, and micro- In general, products obtained by fermenta- organisms producing the same amount of prod- tion also can be produced by chemical synthe- uct. Strict control is maintained of such vari- sis, and to a lesser extent can be isolated by ex- ables as pH (acidity/alkalinity), temperature, and traction from whole organs or organisms. A oxygen supply. (See figure l?. ) The newest mod- fermentation process is usually most competi- els are regulated by sensors that are monitored tive when the chemical process requires several by computers. The capacity of industrial-sized fermenters can reach 50,000 gal or more. The one-shot system of fermentation is called batch Figure 18.-immobilized Cell System fermentation—i.e., fermentation in which a single batch of material is processed from start Solution with product out to finish. . In continuous fermentation, an improvement on the batch process, fermentation goes on without interruption, with a constant input of

Figure 17.- Features of a Standard Fermenter

Exhaust to atmosphere unfiltered

harvest & fill Compressed air inlet

sampler

anisms Cooling jacket immobilized in inert u material Agitator

Raw material solution in Air sparger Typically, a solution of raw materials is pumped through a bed of immobilized micro-organisms which convert the materials to the desired product.

SOURCE: Eli Lilly & Co. SOURCE: Office of Technology Assessment. Ch. 3—Genetic Engineering and the Fermentation Technologies ● 53 individual steps to complete the conversion. In a try and even from region to region within a chemical synthesis, the raw material (shown in country; the economics of the production proc- figure 19 as a) might have to be transformed to ess varies accordingly. an intermediate b, which, in turn, might have to The cost of the raw material can contribute be converted to intermediates c and d before significantly to the cost of production. Usually, final conversion to the product e–each step the most useful micro-organisms are those that necessitating the recovery of its products before consume readily available inexpensive raw ma- the next conversion. In fermentation technol- terials. For large volume, low-priced products ogy, all steps take place within those miniature chemical factories, the micro-organisms; the (such as commodity chemicals), the relationship between the cost of the raw material and the microbial chemist merely adds the raw material cost of the end product is significant. For low a and recovers the product e. volume, high-priced products (such as certain A wide variety of carbohydrate raw materials pharmaceuticals), the relationship is negligible. can be used in fermentation. These can be pure substances (sucrose or table sugar, glucose, or The process of enzyme technology fructose) or complex mixtures still in their original form (cornstalks, potato mash, sugar- Although live yeast had been used for several cane, sugar beets, orcellulose). They can be of thousand years in the production of fermented recent biological origin (biomass) or derived foods and beverages, it was not until 1878 that from fossil fuels (methane or oil). The availabili- the active agents of the fermentation process ty of raw materials varies from country to coun- were given the name “enzymes” (from the Greek, meaning “in yeast”). The inanimate nature of enzymes was demonstrated less than two decades later when it was shown that extracts from yeast cells could effect the conversion of glucose to ethanol. Finally, their actual chemical nature was established in 1926 with the purification and crystallization of the enzyme urease. Fermentation carried out by live cells provided the conceptual basis for designing fermentation processes based on isolated enzymes. A single enzyme situated within a living cell is needed to convert a raw material into a product. A lactose-fermenting organism, e.g., can be used to convert the sugar lactose, which is found in milk, to glucose (and galactose). But if the actual enzyme responsible for the conversion is identified, it can be extracted from the cell and used in place of a living cell. The purified enzyme carries out the same conver- 3) In the Chemical Conversion of raw material a to final product e, intermediates b, c, and d must be synthe- sion as the cell, breaking down the raw material sized. Each intermediate must be recovered and purified in the absence of any viable micro-organism. An before it can be used in the next step of the conversion. enzyme that acts inside a cell to convert a raw )) A cell can perform the same conversion of a to e, but material to a product can also do this outside of with the advantage that the chemist does not have to the cell. deal with the intermediates: the raw material a k simply added and the final product e, recovered. Both batch and continuous methods are used in enzyme technology. However, in the batch OURCE: Office of Technology Assessment. method, the enzymes cannot be recovered eco- —

54 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals .

nomically, and new enzymes must be added for however, will be partly determined by which each production cycle. Furthermore, the en- method is chosen. With isolated enzymes, ge- zymes are difficult to separate from the end netic manipulation can readily increase the sup- product and constitute a potential contaminant. ply of enzymes, while with whole organisms, a Because enzymes used in the continuous meth- wide variety of manipulations is possible in con- od are reusable and tend not to be found in the structing more productive strains. product, the continuous method is the method of choice for most processes. Depending on the desired conversion, the immobilized micro- The relationship of genetics organisms of figure 18 could be replaced by an to fermentation appropriate immobilized enzyme. Applied genetics is intimately tied to fermen- Although more than 2,000 enzymes have tation technology, since finding a suitable spe- been discovered, fewer than 50 are currently of cies of micro-organism is usually the first step in industrial importance. Nevertheless, two major developing a fermentation technique. Until re- features of enzymes make them so desirable: cently, geneticists have had to search for an their specificity and their ability to operate organism that already produced the needed under relatively mild conditions of temperature product. However, through genetic manipula- and pressure. (The most frequently used. en- tion a totally new capability can be engineered; zymes are listed in table 2.) micro-organisms can be made to produce substances beyond their natural capacities. The Comparative advantages of most striking successes have been in the phar- fermentations using whole cells maceutical industry, where human genes have been transferred to bacteria to produce insulin, and isolated enzymes growth hormone, interferon, thymosin a-1, and At present, it is still uncertain whether the somatostatin, (See ch. 4.) use of whole cells or isolated enzymes will be more useful in the long run. There are advan- In general, once a species is found, conven- tages and disadvantages to each. The role of ge- tional methods have been used to induce muta- netic engineering in the future of the industry, tions that can produce even more of the desired compound. The geneticist searches from among hundreds of mutants for the one micro-orga- Table 2.—Enzyme Products nism that produces most efficiently. Most of the many methods at the microbiologist’s disposal Commercially Current available before: production involve trial-and-error. Newer genetic tech. Source/name 1900 1950 1980 tons/yr nologies, such as the use of recombinant DNA Animal (rDNA), allow approaches in which useful genet Rennet...... X 2 ic traits can be inserted directly into the micro Trypsin...... x 15 Pepsin ...... x 5 organism. Plant Malt amylase...... X 10,000 The current industrial approach to fermenta Papain ...... x 100 tion technologies therefore considers two prob Microbial lems: First, whether a biological process car Koji...... X produce a particular product; and second, wha Fungal protease ...... X 10 Bacillus protease ...... x 500 micro-organism has the greatest potential for Amyloglucosidase . . . . . x 300 production and how the desired characteristic; Fungal amylase...... X 10 can be engineered for it. Finding the desired Bacterial amylase...... x 300 Pectinase...... x 10 micro-organism and improving its capability i Glucose isomerase. . . . . x 50 so fundamental to the fermentation industry Microbial rennet ...... x 10 that geneticists have become important mem SOURCE: Office of Technology Assessment. bers of fermentation research teams. Ch. 3—Genetic Engineering and the Fermentation Technologies ● 55

Genetic engineering can increase an orga- case with many traits of agronomic impor- nism’s productive capability (a change that can tance, such as plant height. make a process economically competitive); but it Even if the genes are identified and suc- can also be used to construct strains with char- cessfully transferred, methods must be de- acteristics other than higher productivity. Prop- veloped to recognize the bacteria that re- erties such as objectionable color, odor, or slime ceived them. Therefore, the need to devel- can be removed. The formation of spores that op appropriate selection methods has im- could lead to airborne spread of the micro- peded the application of molecular ge- organism can be suppressed. The formation of netics. harmful byproducts can be eliminated or re- As a consequence of these limitations, genetic duced. Other properties, such as resistance to engineering will be applied to the development bacterial viruses and increased genetic stability, of capabilities that require the transfer of only can be given to micro-organisms that lack them. one or a few identified genes. Applying recent genetic engineering techniques to the production of industrially valuable Fermentation and industry enzymes may also prove useful in the future. For example, a strain of micro-organism that Genetic engineering is not in itself an indus- carries the genes for a desired enzyme may be try, but a technology used at the laboratory pathogenic. If the genes that express (produce) level. It allows the researcher to alter the hered- the enzyme can be transferred to an innocuous itary apparatus of a living cell so that the cell micro-organism, the enzyme can be produced can produce more or different chemicals, or safely. perform completely new functions. The altered cell, or more appropriately the population of CURRENT TECHNICAL LIMITS ON altered identical cells is, in turn, used in indus- GENETIC ENGINEERING trial production. It is within this framework that Despite the many genetic manipulations that the impacts of applied genetics in the various in- are theoretically possible, there are several dustries is examined. notable technical limitations: Regardless of the industry, the same three ● Genetic maps—the identification of the lo- criteria must be met before genetic technologies cation of desired genes on various chromo- can become commercially feasible. These cri- somes have not been constructed for most teria represent major constraints that industry industrially useful micro-organisms. must overcome before genetic engineering can ● Genetic systems for industrially useful play a part in bringing a product to market. micro-organisms, such as the availability of They include the need for: useful vectors, are at an early stage of 1. a useful biochemical product; development. 2. a useful biological fermentation approach ● Physiological pathways—the sequence of to commercial production; and enzymatic steps leading from a raw material to the desired product, are not known 3. a useful genetic approach to increase the efficiency of production. for many chemicals. Much basic research will be necessary to identify all the steps. The three criteria interrelate and can be met The number of genes necessary for the con- in any order; the demonstration of usefulness version is a major limitation. Currently, can begin with any of the three. Insulin, e.g., rDNA is most useful when only a single was first found to have value in therapy; easily identifiable gene is needed. It is more fermentation was then shown to be useful in difficult to use when several genes must be its production; and, now genetic engineering transferred. Finally, the problems are for- promises to make the fermentation process eco- midable, if not impossible, when the genes nomically competitive. In contrast, the value of have not yet been identified. This is the thymosin a-1, has not yet been proved, although . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals the usefulness of genetic engineering and fer- fulness of genetic technologies must be proved. mentation in its production have been demon- In others, the genetic technologies make pro- strated. duction at the industrial level possible, but their market has not yet been established. In still As these examples indicate, the limits on a others, the feasibility of fermentation is the ma- product’s commercial potential vary with the jor problem. product. In some cases, the usefulness of the product has already been shown, and the use- Chapter 4 The Pharmaceutical Industry Chapter 4

Page Table No. Page Background ...... 59 4. Naturally Occurring Small Peptides of Past Uses of Genetics...... 59 Potential Medical Interest ...... 64 Potential Uses of Molecular Genetic Technologies 61 5, Summary of Potential Methods for Hormones ...... 61 Interferon Production...... 70 Insulin ...... 65 6. Immunoassays ...... 73 Growth Hormone ...... 67 7. Diseases Amenable to Drugs Produced by Other Hormones...... 67 Genetic Engineering in the Pharmaceutical Immunoproteins ...... 68 Industry ...... 78 Antigens (Vaccines)...... 68 8. Major Diseases for Which Vaccines Need To Interferon ...... $ ...... 70 Be Developed ...... 78 Lymphokines and Cytokines ...... 71 Antibodies ...... 71 Enzymes and Other Proteins ...... 72 Enzymes ...... 72 Other Proteins ...... 74 Antibiotics ...... 75 Nonprotein Pharmaceuticals ...... 76 Figures Impacts ...... 77 .Figure No. Page Technical Notes ...... 80 20. The Development of a High Penicillin- Producing Strain via Genetic Manipulation . . 60 21. The Product Development Process for Tables Genetically Engineered Pharmaceuticals . . . . 63 Table No. Page 22. The Amino Acid Sequence of Proinsulin . . . . 65 3. Large Human Polypeptides Potentially 23. Recombinant DNA Strategy for Making Attractive for Biosynthesis ...... 61 Foot-and-Mouth Disease Vaccine ...... 69 Chapter 4 The Pharmaceutical Industry

, Background

The domestic sales of prescription drugs by Past uses of genetics U.S. pharmaceutical companies exceeded $7.5 billion in 1979. Of these, approximately 20 per- Genetic manipulation of biological systems cent were products for which fermentation for the production of pharmaceuticals has two processes played a significant role. They in- general goals: cluded anti-infective agents, vitamins, and bio- 1. to increase the level or efficiency of the logical, such as vaccines and hormones. Genet- production of pharmaceuticals with prov- ics is expected to be particularly useful in the en or potential value; and production of these pharmaceuticals and bio- 2. to produce totally new pharmaceuticals logical) which can only be obtained by extrac- and compounds not found in nature. tion from human or animal tissues and fluids. The first goal has had the strongest influence Although the pharmaceutical industry was on the industry. It has been almost axiomatic the last to adopt traditional fermentation tech- that if a naturally occurring organism can pro- nologies, it has been the first industry to make duce a pharmacologically valuable substance, widespread use of such advanced genetic tech- genetic manipulation can increase the output. nologies as recombinant DNA (rDNA) and cell The following are three classic examples. fusion. Two major factors triggered the use of ● genetics in the pharmaceutical industry: The genetic improvement of penicillin production is an example of the elaborate long- ● The biological sources of many pharmaco- term efforts that can lead to dramatic logically active products are micro-orga- increases. The original strains of Penicilli- nisms, which are readily amenable to ge- um chrysogenum, NRRL-1951) were treated netic engineering. with chemicals and irradiation through ● The major advances in molecular genetic successive stages, as shown in figure 20, engineering have been made under an in- until the strain E-15.1 was developed. This stitutional structure that allocates funds strain had a 55-fold improvement in pro- largely to biomedical research. Hence, the ductivity over the fungus in which penicil- Federal support system has tended to fos- lin was originally recognized–the Fleming ter studies that have as their ostensible goal strain. the improvement of health. ● Chemically induced mutations improved a Two factors, however, have tended to dis- strain of Escherichia coli to the point where courage the application of genetics in the chem- it produced over 100 times more L-asparag- cal and food industries. In the former, econom- inase (which is used to fight leukemia) than c considerations have not allowed biological the original strain. This increase made the production systems to be competitive with the task of isolating and purifying the pharma- existing forms of chemical conversion, with ceutical much easier, and resulted in low- rare exceptions. And in the latter, social and in- ering the cost of a course of therapy from stitutional considerations have not favored the nearly $15,000 to approximately $300. development of foods to which genetic engi- ● Genetic manipulation sufficiently improved neering might make a contribution. the production of the antibiotic, gentami-

59 60 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Figure 20.—The Development of a High Penicillin= cin, so that Schering-Plough, its producer, Producing Strain via Genetic Manipulation did not have to build a scheduled manufacturing plant, thereby saving $50 million. Most industry analysts agree that, overall, genetic manipulation has been highly significant First [ in increasing the availability of many pharmaceuticals or in reducing their production costs. mutant — strain The second major goal of genetic manipulation, the production of new compounds, has

Wis. Q-176 been achieved to a lesser degree. A recent new antibiotic, deoxygentamicin, was obtained by mutation and will soon be clinically tested in man. Earlier, an important new antibiotic, amikacin, was produced through classical molecular genetic techniques. And before that, the well-known antibiotic, tetracycline, which is normally not found in nature, was produced by a strain of the bacterium, Streptomyces, after appropriate genetic changes had been carried out in that bacterium.

Legend Methods used to create each subsequent mutant in line.

S: natural selection UV: ultraviolet radiation X: X-ray radiation NM: nitrogen mustard [methyl bis (&chlorethyarnine] SA: sarcolyeine (2-chloroethyl) amlnophenylalanine]

An illustration of the extensive use of genetics to increase the yield of a commercially valuable substance. A variety of laboratories and methods were responsible for the successful outcome.

SOURCE: Adapted by Office of Technology Assessment from R. P. Elander in Genetics of Industrial Microorganisms, O. K. Sebek and A. 1. Laekin (eds.) (Washington, D.C.: American Society for Microbiology, 1979), P. 23. Ch. 4—The Pharmaceutical Industry ● 61

Potential uses of molecular genetic technologies —

Polypeptides—proteins—are the first abun- they are taken orally, a process that curtails dant end products of genes. They include pep- their usefulness and causes side-effects. tide hormones, enzymes, antibodies, and cer- There are four technologies for producing tain vaccines. Producing them is the goal of polypeptide hormones and polypeptides: most current efforts to harness genetically directed processes. However, it is just a matter . extraction from human or animal organs, of time and the evolution of technology before serum, or urine; complex nonproteins like antibiotics can also be ● chemical synthesis; manufactured through rDNA techniques. ● production by cells in tissue culture; and ● production by microbial fermentation after Hormones genetic engineering. The most advanced applications of genetics One major factor in deciding which technol- today, in terms of technological sophistication ogy is best for which hormone is the length of and commercial development, are in the field of the hormone’s amino acid chains. (See table 3.) hormones, the potent messenger molecules that Modern methods of chemical synthesis have help the body coordinate the actions of various made the preparation of low-molecular weight tissues. (See Tech. Note 1, p. 80.) The capacity to polypeptides a fairly straightforward task, and synthesize proteins through genetic engineer- chemically synthesized hormones up to at least ing has stemmed in large part from attempts to 32 amino acids (AA) in length—like calcitonin prepare human peptide hormones (like insulin Table 3.—Large Human Polypeptides Potentially and growth hormone). The diseases caused by Attractive for Biosynthesis their deficiencies are presently treated with extracts made from animal or human glands. Amino acid Molecular residues weight The merits of engineering other peptide hor- Proactin ...... 198 Placental Iactogen ...... 192 mones depend on understanding their actions ● Growth hormone...... 191 22,005 and those of their derivatives and analogs. Nerve growth factor ...... 118 13,000 Evidence that they might be used to improve Parathyroid hormone (PTH)...... 84 9,562 bovine Prokm.din ...... 82 the treatment of diabetes, to promote wound Insulin-like growth factors healing, or to stimulate the regrowth of nerves (IGF-I & IGF-2) ...... 70,67 7,649,7471 will stimulate new scientific investigations. Epidermal growth factor ...... 6,100 ● insulin ...... 51 5,734 Other relatively small polypeptides that influ- Thymopoietin ...... 49 ence the sensation of pain, appetite suppression, Gastric inhibitory polypeptide and cognition and memory enhancement are (GIP) ...... 43 5,104 porcine ● Corticotropin (ACTH) ...... 39 4,567 porcine also being tested. If they prove useful, they will Cholecystokinin (CCK-39) ...... 39 unquestionably be evaluated for production via Big gastrin (BG)...... 34 fermentation. Active fragment of PTH ...... 34 4,109 bovine Cholecystokinin (CCK-33) ...... 33 3,918 porcine While certain hormones have already at- ● Calcitonin ...... 32 3,421 human 3,435 salmon tained a place in pharmacology, their testing Endorphins ...... 31 3,465 and use has been hindered to some extent by ● Glucagon ...... 29 3,483 porcine their scarcity and high cost. Until recently, Thymosin-a1 ...... 28 3,108 Vasoactive intestinal peptide (VIP) 28 3,326 porcine animal glands, human-cadaver glands, and ● Secretin...... 27 urine were the only sources from which they ● Active fragment of ACTH...... 24 could be drawn. Their use is also limited Motilin ...... 22 2,698 because polypeptide hormones must be ad- ● Currently used in medical practice. ministered by injection. They are digested if SOURCE: Office of Technology Assessment. 62 . Impacts o/Applied Genetics—Micro-Organisms, Plants, and Animals

–have become competitive with those derived . The cost and suitability of comparable from current biological sources. Since frag- materials gathered from organs or fluids ments of peptide hormones often express activ- obtained from animals or people. ities comparable or sometimes superior to the intact hormone, a significant advantage of In the past decade, some simpler hormones have been chemically synthesized and a few are chemical synthesis for research purposes is that analogs having slight pharmacological differ- being marketed. However, synthesizing glycoproteins—proteins bound to carbohydrates—is ences from natural hormones can be prepared still beyond the capabilities of chemists. Data by incorporating different amino acids into obtained from companies directly involved in their structures. In principle however, genetically engineered biosynthetic schemes can be the production of peptides by chemical synthesis indicate that the cost of chemically preparing devised for most desirable peptide hormones and their analogs, although the practicality of polypeptides of up to 50 AA in length is ex- doing so must be assessed on a case-by-case tremely sensitive to volume (see Tech. Note 2, p. basis. Ultimately, the principal factors bearing 80,); although the costs are high, the production on the practicality of the competing alternatives of large quantities by chemical synthesis offers are: a competitive production method. ● The cost of raw materials. For genetically Nevertheless, rDNA production, also known engineered biosynthesis, this includes the as molecular cloning, has already been used to cost of the nutrient broth plus some amor- produce low-molecular weight polypeptides. In tization of the cost of developing the syn- 1977, researchers at Genentech, Inc., a small thetic organism. In the case of chemical biotechnology company in California, inserted a synthesis, it includes the cost of the pure totally synthetic DNA sequence into an E. coli amino acid subunits plus the chemicals plasmid and demonstrated that it led to the pro- used as activating, protecting, coupling, lib- duction of the 14 AA polypeptide sequence cor- erating, and supporting agents in the proc- responding to somatostatin, a hormone found in ess. the brain. The knowledge of somatostatin’s ● The different costs of separating the de- amino acid sequence made the experiment pos- sired product from the cellular debris and sible, and the existence of sensitive assays al- the culture medium in biological produc- lowed the hormone’s expression to be detected. tion, and from the supporting resin, by- Although the primary motive for using this par- products, and excess reagents in chemical ticular hormone for the first demonstration was synthesis. simply to show that it could be done, Genentech ● The cost of purification and freedom from has announced that it plans to market its toxic contaminants. The process is more genetically engineered molecule for research expensive for biologically produced materi- purposes. (See figure 21.) al than for materials produced by conven- Somatostatin is one of about 20 recognized tional chemistry, although hormones from small human polypeptides that can be made any source can be contaminated. without difficulty by chemical synthesis. (See ● Differences in the costs of labor and equip- table 4.) Unless a sizable market is found for one ment. Chemical synthesis involves a se- of them, it is unlikely that fermentation meth quence of similar (but different) operations ods will be developed in the foreseeable future during a time period roughly proportional Some small peptides that may justify the devel to the length of the amino acid chain (three opment of a biosynthetic process of production AA per day) in an apparatus large enough are: to produce 100 grams (g) to 1 kilogram (kg) per batch; biological fermentations use vats ● The seven AA sequence known as MSH —with capacities of several thousand gal- ACTH 4-10, which is reputed to influence lons–for a few days, regardless of the memory, concentration, and other psyche length of the amino acid chain. logical-behavioral effects: should such Ch. 4—The Pharmaceutical Industry ● 63

Figure 21.—The Product Development Process for Genetically Engineered Pharmaceuticals

Micro-organisms such as E. coli

The development process begins by obtaining DNA either through organic synthesis (1) or derived from biological sources such as tissues (2). The DNA obtained from one or both sources is tailored to form the basic “gene” (3) which contains the genetic information to “code” for a desired product, such as human interferon or human insulin. Control signals(4) containing plasmids (6) are isolated from micro-organisms such as E. coli; cut open (7) and spliced back (8) together with genes and control signals to form “recombinant DNA” molecules. These molecules are then introduced into a host cell (9). Each plasm id is copied many times in a cell (10). Each cell then translates the information contained in these plasmids into the desired product, a process called “expression” (11). Cells divide (12) and pass on to their offspring the same genetic information contained in the parent cell. Fermentation of large populations of genetically engineered micro-organisms is first done in shaker flasks (13), and then in small fermenters (14) to determine growth conditions, and eventually in larger fermentation tanks (15). Cellular extract obtained from the fermentation process is then separated, purified (16), and packaged (17) for health care applications. Health care products are first tested in animal studies (18) to demonstrate a product’s pharmacological activity and safety, In the United States, an investigational new drug application (19) is submitted to begin human clinical trials to establish safety and efficacy. Following clinical testing (20), a new drug application (N DA) (21) is filed with the Food and Drug Administration (FDA). When the NDA has been reviewed and approved by the FDA the product maybe marketed in the United States (22).

SOURCE: Genentech, Inc. 64 . Impacts of Applied Genetics—Micro-0rganisms, Plants, and Animals

Table 4.-Naturally Occurring Small Peptides of up to 3 percent of the population over 40 Potential Medical interest years of age, in WesternEurope. Number of Molecular . Adrenocorticotropic hormone (ACTH) (39 amino acids weight AA), which promotes and maintains the Dynorphin ...... 17 normal growth and development of the Little gastrin (LG) ...... 17 2,178 adrenalglands and stimulates the secretion Somatostatin...... 14 1,639 Bombesin ...... 14 1,620 of other hormones: in the United States, Melanocyte stimulating hormone. 13 1,655 ACTH is used primarily as a diagnostic Active dynorphin fragment ...... 13 agent for adrenal insufficiency, but in Neurotensin ...... 13 Mini-gastrin (G13) ...... 13 principle, ACTH might be used for at least Substance? ...... 11 l,347bovine one-third of the medical indications—like Luteinizing hormone-releasing rheumatic disorders, allergic states, and hormone(LNRH)...... 10 Active fragment of CCK...... 10 eye inflammation—for which about 5 mil- Angiotensin l ...... 10 1,297 lion Americans annually receive corticos- Caerulein ...... 10 l,252porcine teroids. Bradykinin ...... 9 1,060 ● Vasopressin(ADH) ...... 9 Within the last 5 years, other small polypep- ● Oxytocin ...... 9 1,007 Facteur thymique serique(FTH).. 9 tides have been identified in many tissues and Substance P(4-11)octapeptide. . . 8 have been linked to a variety of activities. Some Angiotensin lI ...... 8 1,046 certainly bind to the same receptor sites as the Angiotensin Ill...... 7 931 MSH/ACTH4-10 ...... 7 pain-relieving opiates related to the morphine Enkephalins...... 5 575 family. These peptides are called endogenous Active fragment of thymopoietin opiates: the smaller (5 AA) peptides are called (TP5)...... 5 ● Thyrotropin releasing hormone enkephalins and the larger (3 AA), endorphins. (TRY) ...... 3 362 Certain enkephalins produce brief analgesia .Currently used in medical practice. when injected directly into the brains of mice. SOURCE: Office of Technology Assessment Synthetic analogs that are less susceptible to enzymatic inactivation produce longer analgesia agents prove of value in wider testing, they even if they are injected intravenously, as does have an enormous potential for use. the larger endorphin molecule. Very recently, Both cholecystokinin (33 AA) and bombesin a 17 AA polypeptide, dynorphin, was reported (10 AA), which have been shown to sup- to be the most potent pain killer yet found—it is press appetite, presumably as a satiety 1,200 times more powerful than morphine. signal from stomach to brain: there is a The preparation of new analgesic agents ap- large market for antiobesity agents–appears a likely outcome of the new research, but proximately $85 million per year at the problems similar to those associated with clas- manufacturer’s level. sical opiates must be overcome. Consequently, Several hormones, such as somatostatin, unnatural analogs—including some made with which are released by nerves in the hypo- amino acids not found in micro-organisms— thalamus of the brain to stimulate or in- might prove more useful. The value of microbi- hibit release of hormones by the pituitary al biosynthesis for these substances is ques- gland: hormones produced by these glands tionable at this time. However, the importance are crucial in human fertility; analogs of of genetic technologies in clarifying the some are being investigated as possible underlying mechanisms should not be under- contraceptives. estimated. Calcitonin (32 AA), which is currently the largest polypeptide produced by chemical Higher molecular weight polypeptides cannot synthesis for commercial pharmaceutical be made practically by chemical synthesis, and use: it is useful for pathologic bone dis- must be extracted from human or animal tis- orders, such as Paget’s disease, that affect sues or produced in cells growing in culture. Ch. 4—The Pharmaceutical Industry ● 65

Now they can also be manufactured by fermen- proceeded quickly. A year after one group re- tation using genetically designed bacteria, as ported that the insulin gene had been incorpo- has been demonstrated by the production of in- rated into E. coli without expression, a second sulin and human growth hormone. group managed to grow colonies of E. coli that actually excreted rat proinsulin. Then, within a INSULIN couple of months, workers at Genentech, in col- Insulin, is composed of two chains—A and B— laboration with a group at City of Hope Medical of amino acids. It is initially produced as a Center, announced the separate synthesis of the single, long chain called pre-proinsulin, which is A (21 AA) and B (30 AA) chains of human insulin. cut into a shorter chain, proinsulin. Proinsulin, The synthesis of the DNA sequences depended in turn, is cut into the A and B chains when a on advances in organic chemistry as well as in piece is cleaved from the middle. (See figure 22.) genetics. Six months were required simply to Work on the genetic engineering of insulin has synthesize the necessary building blocks.

Figure 22.—The Amino Acid Sequence of Proinsulin

Connecting peptide

+ NH 3

B chain Proinsulin is composed of 84 amino acid residues. When the connecting peptide is removed, the retaining A and B chains form the insulin molecule. The A chain contains 21 amino acids; the B chain contains 30 amino acids.

SOURCE: Office of Technology Assessment. 66 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

A comparison with the traditional source of For insulin, therefore, the limitation on bring- animal insulin is interesting. If 0.5 milligram ing the fruits of genetic engineering to the (mg) of pure insulin can be obtained from a liter marketplace is not technological but institu- of fermentation brew, 2,000 liters (1) (roughly tional. The drug must first be approved by the 500 gal) would yield 1 g of purified insulin—the Food and Drug Administration (FDA) and then amount produced by about 16 lb of animal pan- marketed as a product as good as or better than creas. If, on the other hand, the efficiency of the insulin extracted by conventional means. production could be increased to that achieved Lilly has stated that it anticipates a 6-month for asparaginase (which is produced commer- testing period in humans. Undoubtedly, FDA cially by the same organism, E. coli), 2,000 will examine the evidence presented in the in- would yield 100 g of purified insulin—the vestigational new drug application (INDA) with amount extracted from 1,600 lb of pancreas. special care. Its review will establish criteria (The average diabetic uses the equivalent of that may influence the review of subsequent ap- about 2 mg of animal insulin per day.) plications in at least the following requirements: The extent of the actual demand for insulin is evidence that the amino acid sequence of a controversial issue. Eli Lilly & Co. estimates the material is identical to that of the nor- that there are 60 million diabetics in the world mal human hormone; (35 million in underdeveloped countries, where freedom from bacterial endotoxins that few are diagnosed or treated). Of the 25 million may cause fever at extremely low concen- in the developed countries, perhaps 15 million trations—an inherent hazard associated have been diagnosed; according to Lilly’s esti- with any process using E. coli; and mate, 5 million are treated with insulin. Only freedom from byproducts, including sub- one-fourth of those diabetics treated with instances of very similar structure that may sulin live in the United States, but they use 40 to give rise to rare acute or chronic reactions 50 percent of the insulin consumed in the of the immune system. world. A number of studies indicate that while Furthermore, as development continues, FDA the emphasis on diet (alone) and oral antidia- might require strict assurances that the mole- betic drugs varies, approximately 40 percent of cules produced from batch to batch are not sub- American patients in large diabetes clinics or ject to subtle variations resulting from their practices take insulin injections. In the United genetic origin. States, diabetes ranks as the fifth most common cause of death and second most common cause If the insulin obtained from rDNA techniques of blindness. Roughly 2 million persons require manages to pass FDA requirements, it must daily injections of insulin. overcome a second obstacle—competition in the marketplace. The clinical rationale for using Today, at least, there is no real shortage of human rather than animal insulin rests on the glands from slaughter houses for the produc- differences in structure among insulins pro- tion of animal (principally bovine and porcine) duced by different species. Human and porcine insulin. A study conducted by the National Dia- insulins for example, differ in a single amino betes Advisory Board (NDAB) concluded that a acid, while human and cattle insulins differ maximum demand and a minimum supply with respect to three. As far as is known, these would lead to shortages in the 1990’s. Eli Lilly’s variations do not impair the effectiveness of the projection, presented in that report, also antici- insulin, but no one has ever been in a position to pates these shortages. But, Novo Industri, a ma- conduct a significant test of the use of human jor world supplier of insulin, told the NDAB that insulin in a diabetic population. Many conse- it estimates that the 1976 free-world consump- quences of the disease, such as retinopathy (ret- tion of insulin of 51 X 109 units constituted only inal disease) and nephropathy (kidney disease), 23 percent of the potential supply, and the are not prevented by routine injection of animal 87X 109 units projected for 1996 would only insulin. Patients also occasionally respond ad- equal 40 percent of the supply, assuming that versely or produce antibodies to animal insulin, the animal population stays constant. with subsequent allergic or resistant reaction. Ch. 4—The Pharmaceutical Industry . 67

It remains to be seen how many patients will 1,600 patients, each of whom receives therapy be better off with human insulin. The proof that for several years), and for research. it improves therapy will take years. Progress on the etiology of the disease—especially in identi- While the National Pituitary Agency feels that fying it in those at risk or in improving the it can satisfy the current demand for hGH (see dosage form and administration of insulin–may Tech. Note 3, p. 80.), it welcomes the promise of have far more significant effects than new de- additional hGH at relatively low cost to satisfy velopments in insulin production. Nevertheless, areas of research that are handicapped more by as long as private enterprise sees fit to invest in a scarcity of funds than by a scarcity of the hor- such developments, and as long as the cost of mone. However, if hGH is shown to be thera- treating diabetics who respond properly to ani- peutically valuable in these areas, widespread mal insulin is not increased, biological produc- use could severely strain the present supply. At tion of human insulin may become a kind of in- present, the potential seems greatest for pa- surance for diabetics within the next few tients with: decades. ● senile osteoporosis (bone decalcification); GROWTH HORMONE ● other nonpituitary growth deficiencies such The second polypeptide hormone currently a as Turner’s syndrome (1 in 3,000 live candidate for FDA approval is growth hormone female births); ● intrauterine growth retardation; (GH). It is one of a family of closely related, rel- ● atively large pituitary peptide hormones—sin- bleeding ulcers that cannot be controlled gle-chain polypeptides 191- to 198-AA in length. by other means; and ● It is best known for the growth it induces in burn, wound, and bone-fracture healing many soft tissues, cartilage, and bone, and it is a Two groups have already announced the requirement for postnatal growth in man. preparation of micro-organisms with the capaci- The growth of an organism is a highly com- ty for synthesizing GH. (See Tech. Note 4, p. 80.) plex process that depends on the correct bal- In December 1979, one of these groups—Genen- ance of many variables: The action of GH in the tech —requested and received permission from body for example, depends on the presence of the National Institutes of Health (NIH), on the insulin, whose secretion is stimulated by GH. recommendation of the Recombinant DNA Ad- Under some circumstances, one or more inter- visory Committee (RAC), to scale-up its process. mediary polypeptides produced under the in- Its formation of a joint-venture with Kabi Gen fluence of GH by the liver (and possibly the AB is typical of the kind of alliance that develops kidneys) may actually be the proximate causes as a result of the different expertise of groups in of some of the effects attributed to GH. In any the multidisciplinary biomedical field. Kabi has case, the biological significance of GH is most been granted a New Drug Application (NDA) clearly illustrated by the growth retardation under which to market pituitary GH imported that characterizes its absence before puberty, from abroad. and by the benefits of replacement therapy. OTHER HORMONES In the United States, most of the demand for Additional polypeptide hormones targeted human growth hormone (hGH) is met by the Na- for molecular cloning (rDNA production) in- tional Pituitary Agency, which was created in clude: the early 1960’s by the College of Pathologists and the National Institute of Arthritis, Metab- ● Parathyroid hormone (84 AA), which may olism, and Digestive Diseases (NIAMDD) to col- be useful alone or in combination with cal- lect pituitary glands from coroners and private citonin for bone disorders such as osteo- donors. Under the programs of the NIAMDD, porosis. hGH is provided without charge to treat chil- ● Nerve growth factor (118 AA), which influ- dren with hypopituitarism, or dwarfism (about ences the development, maintenance, and 68 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

repair of nerve cells and thus could be sig- of conventional vaccines and change the meth- nificant for nerve restoration in surgery. ods and the dosages in which vaccines are ● Erythropoietin, a glycopeptide that is large- administered. ly responsible for the regulation of blood cell development. Its therapeutic applica- Some vaccines are directed against toxic pro- tions may range from hemorrhages and teins (like the diphtheria toxin produced by burns to anemias and other hematologic some organisms), preparing the body to neutral- conditions. (See Tech. Note 5, p. 80.) ize them. Molecular cloning might make it possible to produce inactivated toxins, or better Immunoproteins nonvirulent fragments of toxins, by means of micro-organisms that are incapable of serving Immunoproteins include all the proteins that as disease-causing organisms. are part of the immune system—antigens, interferon, cytokines, and antibodies. Since poly- Immunity conferred by live vaccines invari- peptides, the primary products of every molec- ably exceeds that conferred by nonliving anti- ular cloning scheme, are at the heart of immu- genic material–possibly because a living micro- nology, developments made possible by recent organism creates more antigen over a longer breakthroughs will presumably affect the entire period of time, providing continuous “booster field. There is little doubt that applied genetics shots.” Engineered micro-organisms might be- will play a critical role in developing a pharma- come productive sources of high-potency anti- cology for controlling immunologic functions, gen, offering far larger, more sustained, doses since it provides the only apparent means of of vaccine without the side-effects from the con- synthesizing many of the agents that will com- taminants found in those vaccines that consist prise immunopharmacology. of killed micro-organisms.

ANTIGENS (VACCINES) However, it is clear that formidable Federal One early dramatic benefit should be in the regulatory requirements would have to be met area of vaccination, where genetic technologies before permission is granted for a novel living may lead to the production of harmless sub- organism to be injected into human subjects. stances capable of eliciting specific defenses Because of problems encountered with live vac- against various stubborn infectious diseases. cines, the most likely application will lie in the Vaccination provides effective immunity by area of killed vaccines (often using only parts of introducing relatively harmless antigens into micro-organisms). the immune system thereby allowing the body to establish, in advance, adequate levels of anti- It is impossible in the scope of this report to discuss the pros, cons, and consequences of de- body and a primed population of cells that can veloping a vaccine for each viral disease. How- grow when the antigen reappears in its virulent ever, the most commercially important are the form. Obviously, however, the vaccination itself should not be dangerous. As a result, several influenza vaccines, with an average of 20.8 mil- methods have been developed over the past two lion doses given per year from 1973 to 1975–a centuries to modify the virulence of micro-orga- smaller number than the 25.0 million doses per nisms used in vaccines without destroying their year of polio vaccine, but more profitable. ability to trigger the production of antibodies. Influenza is caused by a virus that has re- (See Tech. Note 6, p. 80.) mained uncontrolled largely because of the fre- Novel pure vaccines based on antigens syn- quency with which it can mutate and change its thesized by rDNA have been proposed to fight antigenic structures. It has been suggested that communicable diseases like malaria, which have antigenic protein genes for influenza could be resisted classical preventive efforts. Pure vac- kept in a “gene bank” and used when needed. In cines have always been scarce; if they were addition, the genetic code for several antigens available, they might reduce the adverse effects could be introduced into an organism such as E. Ch. 4—The Pharmaceutical Industry ● 69 coli, so that a vaccine with several antigens Figure 23.-Recombinant DNA Strategy for Making might be produced in one fermentation. 1 Foot-and-Mouth Disease Vaccine Two more viral diseases deserve at least brief comment. Approximately 800 million doses of foot-and-mouth disease virus (FMDV) vaccine are annually used worldwide, making it the largest volume vaccine produced. This vaccine must be given frequently to livestock in areas where the disease is endemic, which includes I most of the world outside of North America. The present methods of producing the vaccine require that enormous quantities of hazardous virus be contained. Many outbreaks are attributed to incompletely inactivated vaccine or to the escape of the virus from factories. (See figure 23.) Molecular cloning of the antigen could produce a stable vaccine at considerably less expense, without the risk of the virus escaping. On the basis of that potential, RAC has approved a joint program between the U.S. Department of Agriculture (USDA) and Genentech to clone pieces of the FMDV genome to produce pure antigen. The RAC decision marked the first exception to the NIH prohibition against cloning DNA that is derived from a virulent pathogen.2 FMDV. vaccine made by molecular cloning will probably be distributed commercially by 1985, although not in the United States. It will be the first vaccine to achieve that status, and illus- Growing E. co/i bacteria may produce VP3 for use as vaccine trates the potential veterinary uses of genetic for foot-and-mouth disease. No virus or infectious RNA is technologies. produced by the harmless bacteria strain. Hepatitis has also received significant atten- ● VP3 is the protein from the shell of the virus, which can act tion. Vaccines against viral hepatitis, which afas a vaccine for immunizing livestock against foot-and- fects some 300,000 Americans each year, may mouth disease. The idea outlined above is to make this VP3 be produced by molecular cloning. This disease protein without making any virus or infectious RNA. is second only to tuberculosis as a cause of SOURCE: Office of Technology Assessment. death among reportable infectious diseases. R is extremely difficult to cultivate the causative agents. Hepatitis A has a good chance of being is in the testing stage, but cloning is being in- the first human viral disease for which the investigated as a better source of an appropriate itial preparation of experimental vaccine will in- antigen. The causative agent for a third form of volve molecular cloning. A vaccine against hepa- hepatitis has not even been identified. Since at titis B, made from the blood of chronic carriers, least 16 million U.S. citizens are estimated to be at high risk of contracting hepatitis, there is I For other aspects of vaccine production see: Office of Technol- keen interest in the development of vaccines ogy Assessment, U.S. Congress, working Papers, The impacts of 3 Generics, vol. 2. (Springfield, Va.: National Technical Information among academic and industrial researchers. Service, 1981). ‘Ibid. 31bid.

76-565 0 - 81 - 6 70 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

More hypothetically, molecular cloning may Genentech, for example, has been shown to pro- lead to three other uses of antigens as well: vac- tect squirrel monkeys from infection by the le- cination against parasites, such as malaria and thal myocarditis virus. Once interferon is avail- hookworm (see Tech. Note 7, p. 80.); immuniza- able in quantity, large-scale tests on human pop- tion in connection with cancer treatment; and ulations can be conducted to confirm its ef- counteracting abnormal antibodies, which are ficacy in man. made against normal tissues in the so-called Several production techniques are being ex- “autoimmune diseases, ” such as multiple sclero- plored. (See Tech. Note 10, p. 81.) Extraction of sis. (See Tech. Note 8, p. 81.) interferon from leukocytes (white blood cells), INTERFERON the current method of choice, may have to com- Interferon are glycoproteins normally made pete with tissue culture production as well as rDNA. (See table 5.) by a variety of cells in response to viral infection. All interferon (see Tech. Note 9, p. 81) can Recombinant DNA is widely regarded as the induce an antiviral state in susceptible cells. In key to mass production of interferon, and addition, interferon has been found to have at important initial successes have already been least 15 other biochemical effects, most of achieved. Each of the four major biotechnology which involve other elements of the immune companies is working on improved production system. methods, and all have reported some success. promising preliminary studies have sup- An enormous amount remains to be learned ported the use of interferon in the treatment of about the interferon system. It now appears such viral diseases as rabies, hepatitis, varicella- that the interferon are simply one of many zoster (shingles), and various herpes infections. families of molecules involved in physiological To date, the effect of interferon has been far regulation of response to disease. Only now more impressive as a prophylactic than as a have molecular biology and genetics made their therapeutic agent. The interferon produced by study—and perhaps their use—possible.

Table 5.—Summary of Potential Methods for Interferon Production

Types of Potential interferon for Present projected Potential for Means of Production produced scale-up ($/10 6 units) Problems improvement “Buffy coat” leukocytes leukocyte, 95% No 50 – —lack of scale up —minimal fibroblast, 5% —pathogen contamination Lymphoblastoid cells leukocyte, 80% Yes — =25 —poor yields —improved yields fibroblast, 20% —cells derived from tumor —expression of fibroblast interferon Fibroblasts fibroblast Yes 43-200 = 1-10 —cell culture —improved yields —economic competition —improved cell- with recombinant DNA culture technology —expression of leukocyte-type interferon Recombinant DNA leukocyte or Yes — z 1-10 —does not produce —improved yields fibroblast interferon —in vitro drug stability —poor yields —modified interferon —drug approval —possible economic competition with fibroblast cell production

SOURCE: Office of Technology Assessment. Ch. 4—The Pharmaceutical Industry . 71

The interferon are presently receiving atten- fects similar to lymphokines, include several tion largely because studies in Sweden and the compounds associated with the thymus gland, United States stimulated the appropriation of referred to as thymic hormones.4 $5.4 million by the American Cancer Society In 1979, the BRM subcommittee concluded (ACS) for expanded clinical trials in the treat- that several of these agents probably have great ment of cancer. That commitment by the non- potential for cancer treatment. Nevertheless, profit ACS–the greatest by far in its history– adequate quantities for laboratory and clinical was followed by a boost in NIH funding for in- testing of many of them will probably not be terferon research from $7.7 million to $19.9 available until the problems of producing glyco- million for fiscal year 1980. Much of the cost of proteins by molecular cloning are overcome. No interferon research is allotted to procuring the system is currently available for the industrial glycopeptide. Initially, the ACS bought 40 billion production of glycoproteins, although yeasts units of leukocyte interferon from the Finnish may prove to be the most useful micro-orga- Red Cross for $50 per million units. In March nisms. 1980, Warner-Lambert was awarded a contract to supply the National Cancer Institute (NCI) ANTIBODIES with 50 billion units of leukocyte interferon Antibodies are the best known and most ex- within the next 2 years at an average price of ploited protein components of the immune sys- $18 per million units. NCI is also planning to tem. Until recently, all antibodies were obtained purchase 50 billion units each of fibroblast and from the blood of humans or animals; and they lymphoblastoid interferon. were often impure. Within the past 5 years, The bulk of the NIH funding is included in however, it has become possible to produce an- NCI’s new Biological Response Modifier (BRM) tibodies from cells in culture, and to achieve program—interferon accounts for $13.9 million levels of purity previously unattainable. As with of the $34.1 million allocated for BRM work in previous advances in antibody technology, re- fiscal year 1980. (NCI expenditures on inter- searchers are examining ways to put this new feron in 1979 were $2.6 million, 19 percent of level of purity to use. There have been hun- the amount budgeted for 1980.) Other impor- dreds, if not thousands, of examples of new tant elements of that BRM program concern diagnostic and research methods, new methods immunoproteins known as lymphokines and of purification, and new therapies published thymic hormones, for which molecular genetics within the first 3 years that the technique has has major implications. The program is aimed at been available. (See Tech, Note 11, p. 81.) identifying and testing molecules that control This high level of purity was attained by the the activities of different cell types. development of monoclinal antibodies. These antibodies that recognize only one kind of anti- LYMPHOKINES AND CYTOKINES gen were the unanticipated fruit of fundamen- Lymphokines and cytokines are regulatory tal immunological research conducted by Drs. molecules that have begun to emerge from the Caesar Milstein and Georges Kohler at the Med- obscure fringes of immunology in the past 10 ical Research Council in England in 1975. They years. (Interferon is generally considered a lym- fused two types of cells–myeloma and plasma- phokine that has been characterized sufficiently spleen cells—to form hybridomas that produce to deserve independent status.) the monoclinal antibodies. (See Tech. Note 12, Lymphokines are biologically active soluble p. 81.) Not only are the antibodies specific, but factors produced by white blood cells. Studied because the hybridomas can be grown in mass in depth only within the last 15 years, they are culture, a virtually limitless supply is available. being implicated at virtually every stage in the The most immediate medical application for complex series of events that make up the im- monoclinal antibodies lies in diagnostic testing. 4 mune response. They now include about 100 For 40 of the best characterized cytokines, see footnote 1, p. different compounds. Cytokines, which have ef- 69. 72 . Impacts of Applied Genetics—Micro-Organ/SmS, Plants, and Animals

Over the past 20 years, large segments of the Enzymes and other proteins diagnostic and clinical laboratory industries have sprung up to detect and quantify particu- ENZYMES lar substances in specimens. Because monoclon- Enzymes are involved in virtually every bio- al antibodies are so specific, hybridomas seem logical process and are well-understood. Never- certain to replace animals as the source of anti- theless, despite their potency, versatility, and bodies for virtually all diagnosis and monitor- diversity, they play a small role in the practice ing. Their use will not only improve the accu- of medicine today. Therapeutic enzymes ac- racy of tests and decrease development costs, counted for American sales of about $70 million but should result in a more uniform product. (wholesale) in 1978, but one-half of those sales involved the blood-plasma-derived coagulation Today, such assays are used to: factors used to treat hemophilia. Although the determine hormone levels in order to figure is difficult to estimate, the total number assess the proper functioning of an endo- of patients receiving any type of enzyme ther- crine gland or the inappropriate produc- apy in 1980 probably does not exceed 50,000. tion of a hormone by a tumor; Enzymes cannot be synthesized by conven- detect certain proteins, the presence of tional chemistry. Almost all those presently which has been found to correlate with a employed in medicine are extracted from tumor or with a specific prenatal condition; human blood, urine, or organs, or are produced detect the presence of illicit drugs in a per- by micro-organisms. Already the possibility of son’s blood, or monitor the blood or tissue using rDNA clones as the source of enzymes— level of a drug to ensure that the dosage primarily to reduce the cost of production—is achieves a therapeutic level without ex- being explored. ceeding the limits that could cause toxic effects; and However, problems associated with the use of identify microbial pathogens. nonhuman enzymes (such as immune and feb- rile responses) and the scarcity of human en- The extent of the use of antibodies and the zymes, have hindered research, development, biochemical properties that they can identify is and clinical exploitation of enzymes for ther- suggested by table 6. No one assay constitutes a apeutic purposes. Today, the experimental ge- major market, and short product lifetime has netic technologies of rDNA and somatic cell fu- been characteristic of this business. sion and culture open the only conceivable Other applications of monoclinal antibodies routes to relatively inexpensive production of include: compatible human enzymes. The genetic engineering of enzymes is prob- the improvement of the acceptance of kid- ably the best example of a dilemma that ham- ney (and other organ) transplants by injec- pers the exploitation of rDNA: Without a clinical tion of the recipient with antibodies against need large enough to justify the investment, certain antigens; there is no incentive to produce a product; yet passive immunization against an antigen in- without adequate supplies, the therapeutic pos- volved in reproduction, as a reversible im- sibilities cannot be investigated. The substances munological approach to contraception. that break this cycle will probably be those that localizing tumors with tumor-specific anti- are already produced in quantity from natural bodies (see Tech. Note 13, p. 81); and tissue. targeting cancer cells with antibodies that have anticancer chemicals attached to The only enzymes administered today are them. given to hemophiliacs–and they are actually Ch. 4—The Pharmaceutical Industry ● 73

Table 6-Immunoassays .

Analgesics and narcotics Hallucinogenic drugs Methyl digoxin Steroid hormones Anileridine Mescaline Ouabain Skeletal muscle relaxants Antipyrine Tetrahydrocannabi nol Proscillaridin d-Tubocurarine Codeine Hypoglycemic agents Dihydroergotamine Synthetic peptides Etorphine Butylbiguanide Propranolol DDAVP Fentanyl Glibenclamid Quinidine Saralasin Meperidine Insecticides CNS stimulants Synthetic steroids Methadone Aldrin Amphetamine Anabolic steroids Morphine DDT Benzoyl ecgonine Trienbolone acetate Pentazocine Dieldrin (cocaine metabolize) Androgens Antibiotics Malathion Methamphetamine Fluoxymesterone Amikacin Narcotic antagonists Pimozide Estrogens Chloramphenicol Cyclazocine Diuretics Diethylstilbestrol Clindamycin Naloxone Bumetanide Ethinylestradiol Gentamicin Peptide hormones Hallucinogenic drugs Mestranol Isoniazid Angiotensin Bile acid conjugates Glucocorticoids Penicillin Anterior pituitary Cholylglycine Dexamethasone Sisomycin Bradykinin Cholyltaurine Methyl prednisolone Tobramycin Gastric Catecholamines Prednisolone Anticonvulsants Hypothalamic Epinephrine Prednisone Clonazepam Intestinal Norepinephrine Metyrapone Phenytoin Pancreatic Tyramine Progestins Primidone Parathyroid Fibrinopeptides Medroxyprogesterone Anti-inflammatory agents Posterior pituitary Fibrinopeptide A acetate Colchicine Thyroid (calcitonin) Fibrinopeptide B Norethindrone Indomethacin Plant hormones Indolealkylamines Norethisterone Phenyibutazone lndole-3-acetic acid Melatonin Norgestrel Antineoplastic agents Gibberelilic acid Serotonin Toxins Adriamycin Polyamides Insect hormones Aflatoxin B, Bleomycin Spermine Ecdysone Genistein Daunomycin Prostaglandins Nucleosides and Nicotine and metabolizes Methotrexate Sedatives and nucleotides Ochratoxin A Bronchodilators tranquilizers Cyclic AMP Paralytic shellfish poison Theophylline Barbiturates Cyclic GMP Thyroid hormones Cardiovascular drugs Barbital N2-Dimethylguanosine Thyroxine Cardiac glycosides Pentobarbital 7-Methylguanosine Triodothyronine Acetylstrophanthidin Phenobarbital Pseudouridine Vitamins Cedilanid Chlordiazepoxide Thymidine Vitamin B12 Deslanoside Chlorpromazine Glutethimide Vitamin D Digitoxin Desmethylimipramine Methaqualone Digoxin Diazepam and Gitoxin N-desmethyldiazepam

SOURCE: ‘“lmmunoassays of Drugs–Comprehensive Immunology,” hrrrnunal Pharmacology, Hadde~ Caffey (cd.) (New York: Plenum Press, 1977), P. 325.

proenzymes, which are converted to active en- use of human plasma-derived products is ex- zymes in the body when needed. The most com- tremely high. One recent study found chronic mon agents are called Factor VIII and Factor IX, hepatitis in a significant percentage of asymp- which are found in serum albumin and are cur- tomatic patients treated with Factor VIII and rently extracted from human blood plasma. Factor IX. Hemophilia A and Hemophilia B—accounting for The plasma fractionation industry, which over 90 percent of all major bleeding disor- produces the proenzymes, is currently faced ders—are characterized by a deficiency of these with excess capacity, intense competition, high 5 factors. Supplies of the proenzymes will exceed plasma costs, and tight profit margins. The cost demand well beyond 1980 if the harvesting and and availability of any one plasma protein is

processing of plasma continues as it has. Never- ‘For details of the factors governing the industry. see footnote 1, theless, the risk of hepatitis associated with the p. 69. 74 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

coupled to the production of the others. Hence, complexity exceed the synthetic and analytic the industry would still have to orchestrate the capabilities that will be available in the next few production of the other proteins even if just one years; on the other, either their use in medicine of them, such as Factor VIII, becomes a target has yet to be established or material derived for biological production. from animals appears adequate, as is the case with collagen, for which uses are emerging. Another enzyme, urokinase, has been targeted for use in removing unwanted blood clots, Plasma, the fluid portion of the blood, con- which lead to strokes, myocardial infarctions, tains about 10 percent solids, most of which are and pulmonary emboli. Currently, the drug is proteins. During World War II, a simple pro- either isolated from urine or produced in tissue cedure was developed to separate the various culture. (See Tech. Note 14, p. 81.) components. It is still used today. Urokinase is thus far the only commercial Serum albumin is the smallest of the main therapeutic product derived from mammalian plasma proteins but it constitutes about half of cell culture. Nevertheless, some calculations plasma’s total mass. Its major therapeutic use is suggest that production by E. coli fermentation to reverse the effects of shock. p It is a reason- would have economic advantages. The costs im- able candidate for molecular cloning, although plicit in having to grow cells for 30 days on fetal its relatively high molecular weight complicates calf serum (or its equivalent) or in having to col- purification, and its commercial value is rela- lect and fractionate urine–as reflected in uroki- tively low. The market value of normal serum nase’s market price ($150/mg at the manufac- albumin is approximately $3/g, but the volume turer’s level) —should be enough incentive to en- is such that domestic sales exceed $150 million. courage research into its production. In fact, in Including exports, annual production is in the April 1980, Abbott Laboratories disclosed that range of 100,000 kg. E. coli had been induced to produce urokinase Normal serum albumin for treating shock is through plasmid-borne DNA. already regarded as too expensive compared The availability of urokinase might be guar- with alternative treatments, to expand its use anteed by the new genetic technologies, but its would require a lower price. On the other hand, use is not. For a variety of reasons, the Amer- the Federal Government—and especially the De- ican medical community has not accepted the partment of Defense—might disregard the im- drug as readily as have the European and Japa- mediate economic prospects and conclude that nese communities. Studies to establish the use having a source of human serum albumin that of urokinase for deep vein thrombosis, for ex- does not depend on payments to blood donors ample, are now being conducted almost exclu- might be in the national interest. Since many na- sively in Europe.6 tions import serum albumin, products derived from molecular cloning could be exported. OTHER PROTEINS In addition to the proteins and polypeptides Serum albumin is presently the principal already mentioned, the structural proteins, product of blood plasma fractionation, a change such as the collagens (the most abundant pro- in the way it is manufactured would significant- teins in the body), elastins and keratins (the ly affect that industry. Because a number of compounds of extracellular structures like hair other products (such as clotting factors) are also and connective tissue), albumins, globulins, and derived from fractionation, a growth in the a wide variety of others, may also be susceptible need for plasma-derived albumin could have a to genetic engineering. Structural proteins are significant impact on the availability and the less likely to be suitable for molecular genetic cost of these byproducts. manipulations: On the one hand, their size and

eFor additional information about howurokinase came to play a 7For a detailed discussion of the costs and benefits of using albu- role in therapy, see footnote 1, p. 69. min and the structure of the industry, see footnote 1, p. 69, Ch. 4—The Pharmaceutical industry . 75

Antibiotics industrially important higher fungi, the presence of chromosomal aberrations in micro-orga- Antimicrobial agents for the treatment of in-nisms improved by mutation, and a number of fectious diseases have been the largest sellingother problems. Furthermore, natural recom- prescription pharmaceuticals in the world forbination is most advantageous when strains of the past three decades. Most of these agents areextremely diverse origins are mated; the pro- antibiotics—antimicrobials naturally producedprietary secrets protecting commercial strains by micro-organisms rather than by chemicalusually prevent the sort of divergent “competi- synthesis or by isolation from higher organisms.tor” strains most likely to produce vigorous However, one major antibiotic, chlorampheni-hybrids from being brought together. col—originally produced by a micro-organism, is now synthesized by chemical methods. The The technique of protoplasm or cell fusion field of antibiotics, in fact, provides most of theprovides a convenient method for establishing a precedent for employing microbial fermenta-recombinant system in strains, species, and tion to produce useful medical substances. Thegenera that lack an efficient natural means for United States has been prominent in theirmating. For example, as many as four strains of development, production, and marketing, withthe antibiotic-producing bacterium Streptomy- the result that American companies account forces have been fused together in a single step to about half of the roughly $5 billion worth of an-yield recombinant that inherit genes from four timicrobial agents sold worldwide each year.parents. The technique is applicable to nearly The American market share has been growingall antibiotic producers. It will help combine the as new antibiotics are developed and intro-benefits developed in divergent lines by muta- duced every year. tion and selection. For 30 years,high-yielding, antibiotic-pro- In addition, researchers have compared the ducing micro-organisms have been identified byquality of an antibiotic-producing fungus, Ceph- selection from among mutant strains. Initially,alosporimn acremonium, produced by mating to organisms producing new antibiotics are iso-one produced by protoplasm fusion. (See Tech. lated by soil sampling and other broad screen-Note 15, p. 82.) They concluded that protoplasm ing efforts. They are then cultured in the lab-fusion was far superior for that purpose. What oratory, and efforts are made to improve theiris more, protoplasm fusion can give rise to hun- productivity. dreds of recombinants–including one isolate that consistently produced the antibiotic ceph- Antibiotics are complex, usually nonprotein,alosporin C in 40 percent greater yield than the substances, which are generally the end prod-best producer among its parents–without los- ucts of a series of biological steps. While knowl-ing that parent strain’s rare capacity to use in- edge of molecular details in metabolism hasorganic sulfate, rather than expensive methio- made some difference, not a single antibioticnine, as a source of sulfur. It also acquired the has had its complete biosynthetic pathway eluci-rapid growth and sporulation characteristics of dated. This is partly because there is no singleits less-productive parent. Thus, desirable at- gene that can be isolated to produce an antibi- tributes from different parents were combined otic. However, mutations can be induced within in an important industrial organism that had the original micro-organism so that the level ofproved resistant to conventional crossing. production can be increased. Even more significant are the possibilities for Other methods can also increase production, preparation by protoplasm fusion between dif- and possibly create new antibiotics. Microbialferent species or genera of hybrid strains, mating, for example, which leads to naturalwhich could have unique biosynthetic capaci- recombination, has been widely investigated asties. One group is reported to have isolated a a way of developing vigorous, high-yielding an- novel antibiotic, clearly not produced by either tibiotic producers. However, its use has been parent, in an organism created through fusion limited by the mating incompatibility of manyof actinomycete protoplasts, (See Tech. Note 16, 76 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

p. 82.) The value of protoplasm fusion, therefore, ty of organic chemical entities. These drugs, ex- lies in potentially broadening the gene pool. cept for antibiotics, are either extracted from some natural plant or animal source or are syn- Protoplasm fusion is genetic recombination on thesized chemically. a large scale, Instead of one or a few genes being transferred across genus and species bar- Some of the raw materials for pharmaceuti- riers, entire sets of genes can be moved. Success cals are also obtained from plants; micro-orga- is not assured, however; a weakness today is the nisms are then used to convert the material to inherited instability of the “fused” clones. The useful drugs in one or two enzymatic steps. preservation of traits and long-range stability Such conversions are common for steroid hor- has yet to be resolved. Furthermore, it seems mones. that one of the most daunting problems is screening—determining what to look for and In 1949, when cortisone was found to be a how to recognize it. (See Tech. Note 17, p. 82.) useful agent in the treatment of arthritis, the demand for the drug could not be met since no Recombinant DNA techniques are also being practical method for large-scale production ex- examined for their ability to improve strains, isted. The chemical synthesis was complicated Many potentially useful antibiotics do not reach and very expensive. In the early and mid-1950’s, their commercial potential because the micro- many investigators reported the microbial organisms cannot be induced to produce suffi- transformation of several intermediates to com- cient quantities by traditional methods. The syn- pounds that corresponded to the chemical synthesis of certain antibiotics is controlled by thetic scheme. By saving many chemical steps plasmids, and it is believed that some plasmids and achieving higher yields, manufacturers may nonspecifically enhance antibiotic produc- managed to reduce the price of steroids to a tion and excretion. level where they were a marketable commodi- It may also be possible to transfer as a group, ty. A conversion of progesterone, for example, all the genes needed to produce an antibiotic dropped the price of cortisone from $200 to into a new host. However, increasing the num- $6/g in 1949. Through further improvements, ber of copies of critical genes by phage or plas- the price dropped to less than $1/g. The 1980 mid transfer has yet to be achieved in antibiotic- price is $0.46/g. producing organisms because little is known of the potential vectors. The genetic systems of Developments based on genetic techniques to commercial strains will have to be understood increase the production and secretion of key en- before the newer genetic engineering ap- zymes could substantially improve the econom- proaches can be used. Genetic maps have been ics of some presently inefficient processes. Cur- published for only 3 of the 24 or more indus- rently, assessments are being carried out by trially useful bacteria. various companies to determine which of the many nonprotein pharmaceuticals can be man- Since 2,000 of the 2,400 known antibiotics are ufactured more readily or more economically produced by Streptomyces, that is the genus of by biological means. greatest interest to the pharmaceutical industry. Probably every company conducting re- Approximately 90 percent of the pharmaceu- search on Streptomyces is developing vectors, ticals used in the treatment of hypertension are but little of the industrial work has been re- obtained from plants, as well as are miscel- vealed to date. laneous cardiovascular drugs. Morphine and important vasodilators are obtained from the opium poppy, Papaver somniferum. All these Nonprotein pharmaceuticals chemical substances are produced by a series of enzymes that are coded by corresponding genes In both sales and quantity, over 80 percent of in the whole plant. The long-term possibility the pharmaceuticals produced today are not (over 10 years) of using fermentation methods made of protein. Instead, they consist of a varie- will depend on identifying the important genes. Ch. 4—The Pharmaceutical Industry . 77

The genes that are transferred from plant to found in several fungi. Either the enzymes can bacteria must obviously be determined on a be isolated and used directly in a two-step con- case-by-case basis. The case study on acetamino- version or the genes for both enzymes can be phen (the active ingredient in analgesics such as transferred into an organism that can carry out Tylenol) demonstrates the steps in such a feasi- the entire conversion by itself. bility study. (See app. I-A.) Given the cost assumptions outlined in the The first step in such a study is to determine case study and the assumptions on the efficien- whether and where enzymes exist to carry out cy of converting aniline to acetaminophen, the the necessary transformation for a given prod- cost of producing the drug by fermentation uct. Acetaminophen for instance, can be made could be 20 percent lower than production by from aniline, a relatively inexpensive starting chemical synthesis. material. The two necessary enzymes can be

Impacts

Genetic technologies can help provide a varie- mone, which, as a product of solid state chem- ty of pharmaceutical products, many of which ical synthesis, costs as much as $l2,000. Re- have been identified in this report. But the tech- ducing its cost would allow for more extensive nologies cannot guarantee how a product will research on its physiological and therapeutic be used or even whether it will be used at all. qualities. The pharmaceuticals discussed have illustrated By making a pharmaceutical available, genet- the kinds of major economic, technical, social, ic engineering can have two types of impacts. and legal constraints that will play a role in the First, pharmaceuticals that already have med- application of genetic technologies. ical promise will be available for testing. For ex- Clearly, the major direct impacts of genetic ample, interferon can be tested for its efficacy technologies will be felt primarily through the in cancer and viral therapy, and human growth type of products they bring to market. Never- hormone can be evaluated for its ability to heal theless, each new pharmaceutical will offer its wounds. For these medical conditions, the in- own spectrum and magnitude of impacts. Tech- direct, societal impact of applied genetics could nically, genetic engineering may lead to the pro- be widespread. duction of growth hormone and interferon with Second, other pharmacologically active sub- equal likelihood; but if the patient population is stances that have no present use will be avail- a thousandfold higher for interferon, and if its able in sufficient quantities and at a low enough therapeutic effect is to alleviate pain and lower cost to enable researchers to explore their possi- the cancer mortality rate, its impact will be sig- bilities, thus creating the potential for totally nificantly greater. new therapies. Genetic technologies can make available for example, cell regulatory proteins, a Many hormones and human proteins cannot class of molecules that control gene activity and be extensively studied because they are still that is found in only minute quantities in the either unavailable or too expensive. Until the body. The cytokines and lymphokines typify the physiological properties of a hormone are countless rare molecules involved in regulation, understood, its therapeutic values remain un- communication, and defense of the body to known. Recombinant DNA techniques are being maintain health. Now, for the first time, genetic used to overcome this circular problem. In one technologies make it possible to recognize, iso- laboratory, somatostatin is being used as a relate, characterize, and produce these proteins. search tool to study the regulation of the hormonal milieu of burn patients. A single experi- The potential importance of this class of phar- ment may use as much as 25 mg of the hor- maceuticals—the new cell regulatory mole- 78 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals cules—is underscored by the fact that half of cation of available scientific tools. These new the 22 active INDs for new molecular entities molecules are valuable tools for dissecting the that have been rated by FDA as promising im- structure and function of the cell. The knowl- portant therapeutic gains are in the Metabolic edge gained may lead to the development of and Endocrine Division, which oversees such new therapies or preventive measures for drugs. It is reasonable to anticipate that they diseases. will be employed to treat cancer, to prevent or The increased availability of new vaccines combat infections, to facilitate transplantation might also have serious consequences. But the of organs and skin, and to treat allergies and extent to which molecular cloning will provide other diseases in which the immune system has useful vaccines for intractable diseases is still turned against the organism to which it belongs. unknown. For some widespread diseases, such (See table 7.) as amebic dysentery, not enough is known At the very least, even if immediate medical about the interaction between the micro-orga- uses cannot be found for any of these com- nism and the patient to help researchers design pounds, their indirect impact on medical re- a rational plan of attack. For others, such as search is assured. For the first time, almost any trachoma, malaria, hepatitis, and influenza, biological phenomenon of medical interest can there is only preliminary experimental evidence be explored at the cellular level by the appli- that a useful vaccine could be produced. (See table 8.) To date, the vaccine that is most likely cable 7.–Diseases Amenable to Drugs Produced by to have an immediate impact combats foot-and- Genetic Engineering in the Pharmaceutical Industry mouth disease in veterinary medicine. There is little doubt however, that should any one of the Drug potentially produced by vaccines for human diseases become available, Disease or condition genetically engineered organism the societal, economic, and political conse- Diabetese Insulin Atherosclerosis Platelet-derived growth factor quences of a decrease in morbidity and mortali- (PDGF) ty would be significant. Many of these diseases are particularly prevalent in less-developed Virus diseases Interferon Influenza countries. The effects of developing vaccines Hepatitis Polio Herpes Table 8.-Major Diseases for Which Vaccines Common cold Need To Be Developed Cancer Interferon Parasitic diseases Hodgkin’s disease Hookworm Leukemia Trachoma Breast cancer Malaria Schistosomiasis (river blindness) Anovulation Human chorionic gonadotropin a Sleeping sickness Dwarfism Human growth hormone Pain Enkephalins and endorphins viruses Wounds and burns Human growth hormone Hepatitis Inflammation, Influenza rheumatic diseasesa Adrenocorticotrophic hormone Foot-and-mouth disease (for cloven-hoofed animals) (ACTH) Newcastle disease virus (for poultry) Bone disorders, e.g., Herpes simplex Paget’s diseasea Calcitonin and parathyroid Mumps hormone Measles Nerve damage Nerve growth factor (NGF) Common cold rhinoviruses Anemia, hemorrhage Erythropoietin Varicella-zoster (shingles) Hemophilia Factor Vlll and Factor IX 8ecterJa Blood clotsa Urokinase Dysentery Shocka Serum albumin Typhoid fever Immune disorders Cytokines Cholera Traveller’s diarrhea alndicates dlsaaaes currently treated by the drugs listed.

SOURCE: Office of Technology Assessment. SOURCE: Office of Technology Assessment. Ch. 4—The Pharmaceutical industry ● 79 for them will be felt on an international scale ican aversion to therapies that require frequent and will involve hundreds of millions of people. injection, for instance, is illustrated by the opinion of some that a drug like ACTH offers few, if The new technologies may also lower the any, advantages over steroids. risks of vaccine production. For example, the FMDV vaccine produced by Genentech is con- The use of ACTH is somewhat greater abroad structed out of 17 of the 20 genes in the entire than in the United States. This is due in part virus—enough to confer resistance, but too few because physicians in other cultures make far to develop into a viable organism. less use of systemic steroids than their Amer- ican counterparts, and in part because frequent The new technology may also supply pharma- injections are more acceptable hence more com- ceuticals with effects beyond therapy. At least mon. Sales of ACTH in Great Britain—with two promise impacts with broad consequences: its much smaller population—equal American MSH/ACTH 4-10 can be expected to be used on a sales. wide scale if it is shown to improve memory; and bombesin and cholecystokinin might ex- At present, the need for injection is a far pand the appetite suppression market. But nei- more likely deterrent to the wider use of ACTH ther of these compounds has yet been found to than the cost of the drug itself. Reports that it be useful. While genetic technologies may pro- can be applied by nasal spray suggest that its vide large supplies of the drugs, they do not use may grow. Implantable controlled-release guarantee their value. dosages may also become available within the next 5 years. This dependence on appropriate Antibody-based diagnostic tests, developed drug delivery mechanisms may lead to another through genetic engineering, may eventually in- line of research–increased attempts to develop clude early warning signals for cancer; they technologies for drug-delivery. should be able to recognize any one of the scores of cancers that cause about a half-million As new pharmaceuticals become available, deaths per year in the United States. If anti- disruption can be expected to occur in the sup- bodies prove successful as diagnostic screening ply of some old ones. Pharmaceuticals whose agents to predict disease, large-scale screening production is tied to the production of others of the population can occur, accelerating the might become increasingly expensive to pro- trend toward preventive medicine in the United duce. Clotting factors, for example, are ex- States. tracted with other blood components from plasma. Nevertheless, producing any of the 14 In addition to drugs and diagnostic agents, currently approved blood plasma products by proteins could be produced for laboratory use. rDNA would reduce the incidence of hepatitis Expensive, complex media such as fetal calf caused by contamination from natural blood serum are presently required for growing most sources. mammalian tissue cells. Genetic cloning could make it possible to synthesize vital constituents Whether new pharmaceuticals are produced cheaply, and could markedly reduce the costs of or new production methods for existing phar- cell culture for both research and production. maceuticals are devised, future sources for the Ironically, genetic cloning could make economi- drugs may change. Currently, the sources are cally competitive the very technology that of- diverse, including many different plants, nu- fers an alternative production method for many merous animal organs, various tissue culture drugs: tissue culture. cells, and a wide range of raw materials used for chemical synthesis. A massive shift to fer- Nevertheless, the mere availability of a phar- mentation would narrow the selection. The im- macologically active substance does not ensure pacts on present sources can only be judged on its adoption in medical practice. Even if it is a case-by-case basis. The new sources—micro- shown to have therapeutic usefulness, it may organisms and the materials that feed them— not succeed in the marketplace. Consumer re- offer the guarantee that the raw materials won’t sistance limits the use of some drugs. The Amer- dry up. If one disappears, another can be found. 80 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Clearly, there is no simple formula to identify Given the assumptions described, the immedi- all the impacts of applied genetics on the phar- ate direct economic impact of using genetic ma- maceutical industry. Even projections of eco- nipulation in the industry, measured as sales, nomic impacts must remain crude estimates. can be estimated in the billions of dollars, with Nevertheless, the degree to which genetic engi- the indirect impacts (sales for suppliers, savings neering and fermentation technologies might due to decreased sick days, etc.) reaching potentially account for drug production in spe- several times that value. cific categories is projected in appendix I-B.

Technical notes

1. Many hormones are simply chains of amino acids (poly - At practically the same time, researchers at Genen- peptides); some are polypeptides that have been mod- tech, in conjunction with their associates at City of ified by the attachment of carbohydrates (glycopep- Hope National Medical Center disclosed the production tides). Hormones usually trigger events in cells remote of an hGH analog. This was the first time that a human from the cells that produced them. Some act over polypeptide was directly expressed in E. coli in func- relatively short distances—between segments in the tional form. The work was supported by Kabi Gen AB, brain, or in glands closely linked to the brain, others and Kabi has the marketing rights. act on distant sites in tissues throughout the body. The level of hGH production reported in the scientif- 2. For peptides about 30 AA in length, the cost may ap- ic account of the Genentech work was on the same preach $1 per mg as the volume approaches the kilo- order as that reported for the insulin fragments— gram level–a level of demand rarely existing today but approximately 186,000 hGH molecules per cell—a level likely to be generated by work in progress. Today, the that might be competitive even before efforts are made cost of the 32 AA polypeptide, calcitonin, which is syn- to increase yield. Genentech stresses the point that de- thesized chemically and marketed as a pharmaceutical sign, rather than classical mutation and selection, is the product by Armour, is probably in the range of$20 per logical way to improve the system, since the hormone’s mg, since the wholesale price in vials containing ap- “blueprint” is incorporated in a plasmid that can be proximately 0.15 mg is about $85/mg. (That price is an moved between strains of E. coli, between species, or educated guess, since such costs are closely guarded even from simple bacteria into more complex orga- secrets and since the price of a pharmaceutical in- nisms, such as yeast. cludes so many variables that the cost of the agent 5. Since erythropoietin is a glycoprotein, it may not be itself is a small consideration. ) feasible to synthesize the active hormone with present- 3. In addition to those helped by the National Pituitary ly available rDNA techniques. Agency, another 100 to 400 patients are treated with 6. Antigens are surface components of pathogenic orga- hGH from comercial sources. The commercial price nisms, toxins, or other proteins secreted by pathogenic is approximately $15 per unit (roughly $30/mg). The micro-organisms. They are also the specific counter- production cost at the National Pituitary Agency is parts of antibodies: antibodies are formed by the about $0.75/unit ($1.50/mg). The National Pituitary body’s immune system in response to their presence. Agency produces 650,000 international units (lU) Antibodies are synthesized by white blood cells and are (about 325 g) of hGH, along with the thyroid-stimulat- created in such a way that they are uniquely struc- ing hormone, prolactin, and other hormones, from tured to bind to specific antigens. about 60,000 human pituitaries collected each year. 7. Many of the most devastating infectious diseases in- That is enough hGH both for the current demand and volve complex parasites that refuse to grow under lab- for perhaps another 100 hypopituitary patients. oratory conditions. The first cultivation of the most 4. Workers at the Howard Hughes Medical Institute of malignant of the species of protozoa that causes ma- the University of California, San Francisco, isolated laria, using human red blood cells, was described in messenger RNA from a human pituitary tumor and 1976 by a Rockefeller University parasitologist, William converted it into a DNA-sequence that could be put into Rager. Experimental immunogens were prepared and E. coli. The sequence, however, was a mixture of hGH showed promise in monkeys, but concern about the ex- and non-hGH material. It has been reported that Eli Lil- istence of the red blood cell remnants—which could ly & Co., which has provided some grant money to the give rise to autoimmune reactions–curtailed the pros- Institute, has obtained a license to the patents relating pect for making practical vaccines by that route. Sever- to this work. Grants from the National Institutes of al biotechnology firms are currently trying to synthe- Health and the National Science Foundation were also size malaria antigens by molecular cloning. This effort acknowledged in the publication. may produce technical solutions to such scourges as Ch. 4—The Pharmaceutical industry ● 81

schistosomiasis (bilharzia), filariasis (onchocerciasis tute (for work done under the then Department of and elephantiasis), Ieshmaniasis, hookworm, amebic in- Health, Education, and Welfare funding) on the pro- fections, and trypanosomiasis (sleeping sickness and duction of monoclinal antibodies against tumor cells. Chagas’ disease). In a number of examples, these researchers demon- 8. Another potential use of antigens is suggested by the strated that an animal can be immunized with tumor experimental treatment of stage I lung cancer patients cells, and that hybridomas derived from that animal with vaccines prepared from purified human lung will produce antibodies that demonstrate a specificity cancer antigens, which appears to substantially pro- for the tumor. long survival. And the Salk Institute is expanding clin- The final sentence of the patent text provides the ra- ical trials in which a procine myelin protein prepared tionale for the use of antibodies in both cancer and in- by Eli Lilly & Co. is injected into multiple sclerosis pa- fectious disease therapies: “If the (tumor) antigen is tients to mop up the antimyelin antibodies that those present, the patient can be given an injection of an anti- patients are producing. Fifteen to forty-two g of myelin body as an aid to react with the antigen. ” (U.S. have been injected without adverse effects, suggesting 4,172,124.) a new therapeutic approach to auto-immune diseases. 12. Myeloma cells grow vigorously in culture and have the The protein appears to suppress the symptoms of ex- unique characteristic of producing large quantities of perimental allergic encephalomyelitis, an animal dis- antibodies. Each spleen cell of the immune type, on the ease resembling multiple sclerosis. Should this re- other hand, produces an antibody that recognizes a search succeed, the use of molecular clones to produce single antigen, but these do not grow well in culture. human protein antigens seems inevitable. When normal immune spleen cells are fused with mye- 9. There are at least two distinct kinds of “classical” inter- loma cells, the resulting mixture of genetic capacities ferons—leukocyte interferon and fibroblast interferon, forms a cell, called a “hybridoma,” which displays the so-called for the types of cells from which they are ob- desired characteristics of the parent cells: 1) it secretes tained. A third kind, called lymphoblastoid because it is the antibody specified by the genes of the spleen cell; produced from cells derived from a Burkitt’s lympho- and 2) it displays the vigorous growth, production, and ma, appears to be a mixture of the other two inter- longevity that is typical of the myeloma cell. feron. All produce the antiviral state and are induced 13. The use of high-correlation antibody assays in cancer by viruses. A fourth kind, known as “immune” inter- studies has only just begun. Antibodies that have been feron, is produced by lymphocytes. Some evidence in- treated so they can be seen with X-rays and that are dicates that it may be a more potent antitumor agent specific for a tumor, can be used early to detect the oc- than the classical types. Currently, interferon is ob- currence or spread of tumor cells in the body. Because tained chiefly from white blood cells (leukocytes) from some 785,000 new cancer cases will be detected in the blood bank in Helsinki that serves all of Finland, or 1980 with current diagnostic methods, because cancer from fibroblasts grown in cell culture. will cause 405,000 deaths, and because early detection 10. Recently, G. D. Searle & Co. announced that new tech- is the major key to improving survival, the implications nology developed at its R&D facility in England has in- are indeed enormous. creased the yield of fibroblast interferon by a factor of 14. In the late 1950’s, Lederle Laboratories marketed a 60. On the basis of this process, Searle expects to sup- preparation of 95-percent pure streptokinase (a bac- ply material for the first large-scale clinical trial of terially produced enzyme that dissolves blood clots) for fibroblast interferon. Abbott Laboratories also recently intravenous administration. They withdrew the prod- announced plans to resume production of limited uct from the market around 1960 because it caused quantities of fibroblast interferon for clinical studies it allergic reactions, which dampened clinical enthusiasm plans to sponsor. for its therapeutic potential. Unlike leukocytes and specially treated fibroblasts, The presence in human urine of urokinase, an en- which can be used only once, lymphoblasts derived zyme also capable of removing blood clots, was also dis- from the tumor Burkitt’s lymphoma grow freely in covered in the early 1950’s. Urokinase was purified, suspension and produce the least costly interferon crystallized, and brought into clinical use in the mid- presently obtainable. However, they also produce a dis- 1960’s. From the beginning it was apparent that “an in- advantageous mixture of both leukocyte and fibroblast tense thrombolytic state could be achieved with a interferon. The Burroughs-Wellcome Co. produces much milder coagulation defect than occurred with lymphoblastoid interferon in 1,000-1 fermenters and streptokinase; no pyrogenic or allergic reactions were has begun clinical trials in England, but the U.S. FDA noted, and no antibodies resulted from its administra- has generally resisted efforts to make use of products tion . . . There did not appear to be as great variation in derived from malignant cells. It is used extensively in patient responsiveness.” In 1967-68 and 1970-73, the research, and FDA is considering evidence from Bur- National Heart and Lung Institute organized clinical roughs-Wellcome that may lead to a relaxation of the trials that compared urokinase with streptokinase and prohibition, under pressure from the National Cancer heparin, an anticoagulant, in the treatment of pul- Institute. monary embolism. The trials indicated that strep- 11. What may be a landmark patent has been issued to tokinase and urokinase were equivalent and superior Hilary Koprowski and Carlos Croce of the Wistar Insti- to heparin over the short term, although their long- 82 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

term benefits were not established. Since then, clinical 15. Intergeneric hybrids have extremely interesting pos- investigation of urokinase has been hampered by sibilities. For example, it would be beneficial to cepha- domestic regulatory problems, which have raised the Iosporin-process technology to combine in one orga- cost of production and restricted its availability in the nism the acyltransferase from Penicillium chrysogenum United States. and the enzymes of C. acremonium, which does not in- In January 1978, Abbott Laboratories obtained a corporate side chain precursors onto cephalosporin new drug application for urokinase and introduced the like P. chrysogenum does for penicillins. product Abbokinase; by that time, however, the sales 16. Another example of recombination between species is of urokinase in Japan were already pushing $90 million that reported for two species of fungi, Aspergillus nidu- per year. Recently, Sterling Drug has begun marketing lans and A. rugulosus, subsequent to protoplasm fusion. a urokinase product (Breokinase) manufactured by The only report of a successful cross between Green Cross of Japan: “According to Japanese reports, genera using protoplasm fusion technology has been be- urokinase is the first Japanese-made drug formulation tween the yeasts Candida tropicalis and Saccario- to receive production and sales approval from FDA. mycopsis fibuligera, which took place at low frequency Green Cross estimates that within 3 years of the start and gave rise to types intermediate between the of Sterling’s marketing activities, the value of uro- parents. kinase exports will reach Yen 500 million ($2.12 mil- 17. An example of screening is provided by the new lion) per month, and considers that its profits from ex- B-lactam (penicillin-like) antibiotics. Using older porting a finished product will probably be better than screening methods, no new B-lactams were found those from bulk drug sales or the licensing of technol- from 1956 until 1972 when a new method was devised. ogy. ” The Green Cross product is made from human A new series of these antibiotics was thus found. urine collected throughout Korea and Japan, and takes Within the past year, 6 new B-lactams have been advantage of technology licensed from Sterling. Ab- commercialized and at least 12 more are in clinical bott’s product, on the other hand, is derived from trials around the world. The sales forecasts for these kidney-cell culture. new agents are estimated to be over $1 billion. Chapter 5 The Chemical Industry Chapter 5

Page Background ...... 85 10. Summary of Recent Estimates of Primary Overview of the Industry ...... 85 U.S. Cost Factors in the Production of Fermentation and the Chemical Industry. . . . . 87 Lysine Monohydrochloride by Fermentation New Process Introduction...... , , . . . . 89 and Chemical Synthesis...... Characteristics of Biological Production 11. Some Commercial Enzymes and Their U Technologies...... 90 12. Expansion of Fermentation Into the Renewable Resources...... 90 Chemical Industry, ...... Physically Milder Conditions...... 91 13. The Potential of Some Major Polymeric One-Step Production Methods...... 91 Materials for Production Using Biotechnology 95 Reduced Pollution...... 91 14. Some Private Companies With Industrial Chemicals That May Be Produced by Biotechnology Programs ., ...... , . 98 Biological Technologies...... 92 15. Distribution of Applied Genetics Activity Fertilizers, Polymers, and Pesticides...... 94 in Industry ...... , ...... 100 Constraints on Biological Production Techniques 96 16. Manpower Distribution of a Firm With An Overview of Impacts ...... 97 Applied Genetics Activity...... 100 Impacts on Other Industries...... 97 17. Index to Fermentation Companies...... 100 Impacts on University Research...... 98 18. Fermentation Products and Producers . . . . . 101 The Social Impacts of Local Industrial Activity . 99 19. U.S. Fermentation Companies ...... 103 Impacts on Manpower...... , ...... 99 Figures

Figure No. Tables Page 24. Flow of industrial Organic Chemicals From Table No. Page Raw Materials to Consumption ...... 86 9. Data for Commercially Produced Amino 25. Diagram of Alternative Routes to Organic Acids...... 88 Chemicals , ...... 90 Chapter 5 The Chemical Industry

Background

The organic substances first used by humans chemical industry. Rather, both coal and bio- to make useful materials such as cotton, linen, mass will be examined for their potential roles silk, leather, adhesives, and dyes were obtained on a product-by-product basis. 1 from plants and animals and are natural and re- The chemical industry is familiar with the newable resources. In the late 19th century, coal tar, a nonrenewable substance, was found technology of converting coal to organic chemicals, and a readily available supply exists. Coal- to be an excellent raw material for many organic compounds, When organic chemistry devel- based technologies will be used to produce a wide array of organic chemicals in the near fu- oped as a science, chemical technology im- ture. * Nevertheless, economic, environmental, proved. At about the same time relatively cheap petroleum became widely available. The indus- and technical factors will increase the industry’s interest in biomass as an alternative source for try shifted rapidly to using petroleum as its ma- probably jor raw material. raw materials. Applied genetics will play a major role in enhancing the possibilities The chemical industry’s constant search for by allowing biomass and carbohydrates from cheap and plentiful raw materials is now about natural sources to be converted into various to come full circle. The supply of petroleum, chemicals. Biology will thereby take on the dual which presently serves more than 90 percent of role of providing both raw materials and a proc- the industry’s needs, is severely threatened by ess for production. both dwindling resources and increased costs. It has been estimated that at the current rate of consumption, the world’s petroleum supplies ‘For further details see Ener,qv From Biologica/ Processes, ~wl. 1, will be depleted in the middle of the next cen- OTA.E-124 (Washington, D. C.: office of ‘rechnolo~v Assessment, July 1980). tury. Most chemical industry analysts, there- *Most important organic intermediiites (chemical compounds fore, foresee a shift first back to coal and then, used fol. the illdustl’iitl s.vnthesis of c~n~n]et.ciiil produ(’ts SU(’11 iis once again, to the natural renewable resources pliist i(:s itnd fihers) can be obtained from coal as an iilterniit i\(l Iiiii nliiteriiil. (kirrentl. v, methods iire heing dtn’elopecl to (’onirrt {’oiil referred to as biomass. The shifts will not into “s.vnthetic gas, ” which (;i]n then h{’ used ils ]’ii}~ l]]i~t[>}”ii]l fo]. necessarily occur sequentially for the entire turther conivwions.

Overview of the industry

The chemical industry is one of the largest ing inorganic chemicals such as lime, salt, am- and most important in the world today. The U.S. monia, carbon dioxide, chlorine gas, and hydro- market for synthetic organic chemicals alone, chloric and other acids. excluding primary products made from petroleum, natural gas, and coal tar, exceeded $35 The other third, which is the target for bio- billion in 1978. technology, produces organic chemicals. Its output includes plastics, synthetic fibers, organic The industry’s basic function is to transform solvents, and synthetic rubber. (See figure 24.) low-cost raw materials into end-use products of In general, petroleum and natural gas are first greater value. The most important raw materi- converted into “primary products” or basic or- als are petroleum, coal, minerals (phosphate, ganic chemicals such as the hydrocarbons ethyl- carbonate), and air (oxygen, nitrogen). Roughly ene and benzene. These are then converted into two-thirds of the industry is devoted to produc- a wide range of industrial chemicals. Ethylene

76-565 0 - 81 - 7 86 . Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

Figure 24.-Flow of Industrial Organic Chemicals From Raw Materials to Consumption

Organic resources

80% raw material from petroleum/ natural gas

20°/0 raw material from coal, coke, and renewable resources

SOURCE: U.S. Industrh?l Outlook (W.shlngton, D. C.: Department of Commerce, 1978); Kline Gu/de to Chemioel Industry, Fairfield, N.J., adapted from Tong, 1979. Ch. 5—The Chemical industry ● 87 alone serves as the basic chemical for the manu- has contributed to the economic viability of the facture of half of the largest volume industrial process. The production of citric and lactic chemicals. Each of the steps in a chemical con- acids and various amino acids are among the version process is controlled by a separate reac- processes that have benefited from genetics. tion, which is often performed by a separate Lactic acid is produced both synthetically and company. by fermentation. Over the past 10 to 20 years, manufacture by fermentation has experienced Evaluating the competitiveness both of a competition from chemical processes. process and of the market is critical for the chemical industry, which is intensive for cap- The organisms used for the production of lac- ital, energy, and raw materials. Its plants use tic acid are various species of the bacterium Lac- large amounts of energy and can cost hundreds tobacillus. Starting materials may be glucose, su- of millions of dollars to build, and raw material crose, or lactose (whey). The fermentation per costs are generally 50 to 80 percent of a prod- se is efficient, resulting in 90 percent yields, de- uct’s cost. If a biological process can use the pending on the original carbohydrate. Since same raw materials and reduce the process cost most of the problems in the manufacture of lac- by even 20 percent, or allow the use of inexpen- tic acid lie in the recovery procedure and not in sive raw materials, it could provide the industry fermentation, few attempts have been made to with a major price break. improve the industrial processes through genetics. Fermentation and Citric acid is the most important acidulant, the chemical industry and historically has held over 55 to 65 percent of the acidulant market for foods. * It is also The production of industrial chemicals by used in pharmaceuticals and miscellaneous in- fermentation is not new. Scores of chemicals dustrial applications. It is produced commercial- have been produced by micro-organisms in the ly by the mold Aspergillus niger. Surprisingly lit- past, only to be replaced by chemical produc- tle work has been published on improving citric tion based on petroleum. In 1946, for example, acid-producing strains of this micro-organism. 27 percent of the ethyl alcohol in the United Weight yields of 110 percent have recently been States was produced from grain and grain prod- reported in A. niger mutants obtained by ir- ucts, 27 percent from molasses, a few percent radiating a strain for which a maximum yield of each from such materials such as potatoes, pine- 29 percent had been reported. apple juice, cellulose pulp, and whey, and only 36 percent from petroleum. Ten years later Amino acids are the building blocks of pro- almost 60 percent was derived from petroleum. teins. Twenty of them are incorporated into proteins manufactured in cells, others serve Even more dramatically, fumaric acid was at specialized structural roles, are important meta- one time produced on a commercial scale bolic intermediates, or are hormones and neu- through fermentation, but its biological produc- rotransmitters. All of the amino acids are used tion was stopped when a more economical syn- in research and in nutritional preparations, thesis from benzene was developed. Frequently, with most being used in the preparation of after a fermentation product was discovered, pharmaceuticals. Three are used in large quan- alternative chemical synthetic methods were tities for two purposes: glutamic acid to manu- soon developed that used inexpensive petro- facture monosodium glutamate, which is a fla- leum as the raw material.

Nevertheless, for the few chemical entities ● The other two important acidukmts, or acidifying agents, are still produced by fermentation, applied genetics phosphoric acid (20 to 25 percent) and maltc acid (S percent), 88 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

vor enhancer particularly in oriental cooking;* plished by using two methods: I) manipulating and lysine and methionine as animal feed ad- microbial growth conditions, and 2) isolating ditives. nat urally occurring mutants. Conventional technology for producing glu- Although microbial production of all the tamic acid is based on pioneering work that was amino acids has been studied, glutamic acid and subsequently applied to other amino acids. The L-lysine* * are the ones produced in significant production employed microbial strains to pro- quantities by fermentation processes. (See table duce amino acids that are not within their nor- 9.) The production of L-lysine is an excellent ex- mal biosynthetic capabilities. This was accom- “ *The lack of a single amino acid can retard protein synthesis, and therefore growth, in a mammal. The limiting amino acid is a “Monosodium glutamate is the sodium salt of glutamic acid. In function of the animal and its feed. The major source of animal 1978, about 18,000 tonnes were manufactured in the United feed in the United States is soybean meal. The limiting amino acid States and about 11,000 tonnes imported. The food industry con- for feeding swine is lysine, the limiting amino acid for feeding sumed 97 percent. The fermentation plant of the Stauffer Chem- poultry is methionine. Because of increased poultry demand, ical Co. in San Jose, Calif., is the sole U.S. producer. The microbes world demand for Iysine is climbing, Eurolysine is spending $27 used in glutamic acid fermentation (Corynebacferium glutamicum, million to double its production capacity in Amiens, France, to 10 C. Iileum, and Brevibacterium j?avum) produce it in 60 percent of thousand tonnes. The Asian and Mideast markets are ;stimated to theoretical yield. Thus, there is some but not great potential for increase to 3 thousand tonnes in 1985. Some bar .eria produce the use of applied genetics to improve the yield. Many of the ge- lysine at over 90 percent of theoretical yield, L:[tle genetic im- netic approaches have already been thoroughly investigated by in- provement is likely in this conversion yield, however, significant dustrial scientists. improvement can be made in the rate and final concentration.

Table 9.—Data for Commercially Produced Amino Acidsa

Price March Potential for application of 1980 (per kg Production 1978 biotechnology (de novo synthesis or Amino acid pure L) Present source (tonnes) bioconversion; organisms and enzymes) Alanine...... $ 80 Hydrolysis of protein; 10-50(J) b – chemical synthesis Arginine ...... 28 Gelatin hydrolysis 200-300 (J) Fermentation in Japan Asparagine...... 50 Extraction 10- 50(J) – Aspartic acid ...... 12 Bioconversion of fumaric acid 500-1,000 (J) Bioconversion Citrulline...... 250 10-90 (J) Fermentation in Japan Cysteine...... 50 Extract ion 100- 200(J) – Cystine...... 60 Extraction 100- 200(J) – DOPA (dihydrophenylalanine) . 750 Chemical 100- 200(J) — Glutamic...... 4 Fermentation 10,000-100,000 (J) De novo: Micrococcus gutamicus Glutamine...... 55 Extraction 200-300 (J) Fermentation in Japan Histidine...... 160 — 100-200 Fermentation in Japan Hydroxyproline ...... 280 Extraction from collagen 10-50 — Isoleucine...... 350 Extraction 10-50 (J) Leucine...... 55 — 50-100(J) Fermentation in Japan Lysine...... 350 Fermentation (800A) 10,000 (J) (80% by fermentation) De novo: Chemical (20%) Corynebacterium glutamicum and Brevibacterium flavum Methionine...... 265 Chemical from acrolein 17,000(D,L) c — 20,000 (D,L) (J) Ornithine ...... 60 — 10-50 (J) Fermentation in Japan Phenylalanine ...... 55 Chemical from 50-100 (J) Fermentation in Japan benzaldehyde Proline ...... 125 Hydrolysis of gelatin 10-50 (J) Fermentation in Japan Serine...... 320 — 10-50 (J) Bioconversion in Japan Threonine...... 150 — 50-10 (J) Fermentation in Japan Tryptophan...... 110 Chemical from indole 55 (J) Tyrosine...... 13 Extraction 50- 100(J) - Valine ...... 60 — 50-100 (J) Fermentation in Japan

Laproduction data largely from Japan because of relative Small U.S. production. ‘Japan.

CD and L forms. SOURCE: Massachusetts Institute of Technology. Ch. 5—The Chemical Industry . 89 ample of the competition between chemical and mostly from Japan and South Korea. Recent biotechnological methods. Fermentation has estimates of primary U.S. cost factors in the been gradually replacing its production by competing production methods are summarized chemical synthesis; in 1980, 80 percent of its in table 10. Fermentation costs are lower for all worldwide production is expected to be by mi- three components of direct operating costs: crobes. It is not produced in the United States, labor, material, and utilities. which imported about 7,000 tonnes in 1979,

Table 10.—Summary of Recent Estimates of Primary U.S. Cost Factors in the Production of L= Lysine Monohydrochloride by Fermentation and Chemical Synthesis

Cost factors in Production of 98% L-lysine monohvdrochloride By fermentation By chemical synthesis Requirement Estimated 1976 cost Requirement Estimated 1976 cost (units per unit per unit product (units per unit per unit product (product) Cents/lb Cents/kg product) Cents/lb Cents/lb c Total Iabor ...... — 8 18 — 9 20 Materials Molasses ...... 44 7 16 — — — Soy beanmeal, hydrolyzed. . . 0.462 4 9 — — — Cyclohexanol...... — — 0.595 17 37 Anhydrous ammonia...... — — — 0.645 6 14 Other chemicalsd...... — 7 15 — 4 10 Nutrients and solvents. . . . . — — — — 4 8 Packaging, operating, and maintenance materials. . . 10 22 — 9 21 Total materials...... — 28 62 — 45 90 Total utilities ...... — 6 12 — 7 16 Total direct operating cost — 42 92 — 56 126 Plant overhead, taxes, and insurance ...... — 10 21 — 10 21 Total cash cost ...... — 52 11 — 66 147 Depreciation...... — 16 35 — 13 28 Interest on working capital — 1 3 — 1 3 Total costg ...... — 69 151 — 80 178 aAggumes a 23-percent yield on molasses. bAssumes a 65-percent yield on cyclohexanol. clncludes operating, maintenance, and control laboratory labor. dFor both the proce~~ of fermentation and chemical synthesis, assumed use of hydrochloric acid (~ percent) and ammonia (n percent). For fermentation includes dSO potassium diphosphate, urea, ammonium suifate, calcium carbonate, and magnesium sulfate. For chemical synthesis also includes nitrosyl chloride, sulfuric acid, and a credit for ammonium sulfate byproduct. ~otal utiiities for both processes include cooling water, steam process water, and electricity. For chemical synthesia, natural gas is also included. fTen percent ~r year of fixed capital costs for a new 20 million lb per year U.S. plant built in 1975 at assumed capital cost of $36.6X 10’ for fOrmOntatiOn and $32.5x 10’ for chemical synthesis exclusive of land costs. SOURCE: Stanford Research Institute, Chemical Economics Handbook 563:3401, May 1979.

New process introduction

The development of biotechnology should be second will allow fermentation-derived prod- viewed not so much as the creation of a new in- ucts to enter the chemical conversion chains, dustry as the revitalization of an old one. Both and will compete directly with traditional chem- fermentation and enzyme technologies will ical transformations. (See figure 25. ) Fermenta- have an impact on chemical process develop- tion, by replacing various production steps, ment. The first will affect the transition from could act as a complementary technology in the nonrenewable to renewable raw materials. The overall manufacture of a chemical. 90 ● Impacts of Applied Genetics-Micro-Organisms, Plants,and Animals

Figure 25.—Diagram of Alternative Routes to Organic Chemicals

Petroleum Carbohydrates

% followed by number indicatea iength of carbon chain. SOURCE: (3. E. Tong, “industrial Chemicals From Fermentation Enzymes,” M/croLr. TechrroL, voi. 1,1979, PP. 173-179.

Characteristics of biological atmosphere into carbohydrates, some of which production technologies are used for their own energy needs. The rest are accumulated in starches, cellulose, lignins, The major advantages of using commercial and other materials called the biomass, which is fermentation include the use of renewable re- the foundation of all renewable resources. sources, the need for less extreme conditions during conversion, the use of one-step produc- The technologies of genetic engineering could tion processes, and a reduction in pollution. A help ease the chemical industry’s dependence micro-organism might be constructed, for ex- on petroleum-based products by making the use ample, to transform the cellulose in wood di- of renewable resources attractive. All micro- rectly into ethanol. * (App. 1-D, a case study of organisms can metabolize carbohydrates and the impact of genetics on ethanol production, convert them to various end products. Exten- elaborates these points. ) sive research and development (R&D) has RENEWABLE RESOURCES already been conducted on the possibility of Green plants use the energy captured from using genetically engineered strains to convert sunlight to transform carbon dioxide from the cellulose, the major carbohydrate in plants, to commercial products. The basic building block “If IWILI(WI I“OIS iil)~)]S()\’i~l 01 SU(SI1 illl it(s(:oilll)lislllllel~l I)$v I’DNA Icchniqut?s wits sulmlilted to lht> Ik:otl]l)iiliii)l DNA Advisory of cellulose—glucose—can be readily used as a (kmlnlilltw ill the %?pl. 25, 1980 mt?eling. raw material for fermentation. Ch. 5—The Chemical industry ● 91

other plant carbohydrates include corn- In biological systems, micro-organisms often starch, molasses, and lignin. The last, a polymer complete entire synthetic schemes. The conver- found in wood, could be used as a precursor for sion takes place essentially in a single step, the biosynthesis of aromatic (benzene-like) although several might occur within the orga- chemicals, making their production simpler and nisms, whose enzymes can transform the pre- more economical. Nevertheless, the increase in cursor through the intermediates to the desired the price of petroleum is not a sufficient reason end product. Purification is not necessary. for switching raw materials, since the cost of REDUCED POLLUTION carbohydrates and other biological materials has been increasing at a relative rate. Metal catalysts are often nonspecific in their action; while they may promote certain reac- PHYSICALLY MILDER CONDITIONS tions, their actions are not ordinarily limited to In general, there are two main ways to speed making only the desired products. Consequent- chemical reactions: by increasing the reaction ly, they have several undesirable features: the temperature and by adding a catalyst. A catalyst formation of side-products or byproducts; the (usually a metal or metal complex) causes one incomplete conversion of the starting materi- specific reaction to occur at a faster rate than al(s); and the mechanical and accidental loss of others in a chemical mixture by providing a sur- the product. face on which that reaction can be promoted. The last problem occurs with all types of syn- Even using the most effective catalyst, the con- thesis. The first two represent inefficiencies in ditions needed to accelerate industrial organic the use of the raw materials. These necessitate reactions often require extremely high tem- the separation and recycling of the side-prod- peratures and pressures—several hundred de- ucts formed, which can be difficult and costly grees Celsius and several hundred pounds per because they are often chemically and physi- square inch. cally similar to the desired end products. (Most Biological catalysts, or enzymes, on the other separation techniques are based on differences hand can speed-up reactions without the need in physical properties—e.g., density, volatility, for such extreme conditions. Reactions occur in and size.) dilute, aqueous solutions at the moderate condi- When byproducts and side-products have no tions of temperature, pressure, and pH (a meas- value, or when unconverted raw material can- ure of the acidity or alkalinity of a solution) that not be recycled economically, problems of are compatible with life. waste disposal and pollution arise. Their solu- ONE-STEP PRODUCTION METHODS tion requires ingenuity, vigilance, energy, and In the chemical synthesis of compounds, each dollars. Many present chemical processes create reaction must take place separately. Because useless wastes that require elaborate degrada- most chemical reactions do not yield pure prod- tion procedures to make them environmentally ucts, the product of each individual reaction acceptable. In 1980, the chemical industry is ex- must be purified before it can be used in the pected to spend $883 million on capital outlays next step. This approach is time-consuming and for pollution control, and well over $200 million expensive. If, for example, a synthetic scheme on R&D for new control techniques and re- that starts with ethylene (a petroleum-based placement products. These figures do not in- product) requires 10 steps, with each step yield- clude the millions of dollars that have been ing 90 percent product (very optimistic yields in spent in recent years to clean up toxic chemical chemical syntheses), only about one-third of the dumps and to compensate those harmed by ethylene is converted into the final end product. poorly disposed wastes, nor do they include the Purification may be costly; often, the chemicals cost of energy and labor required to operate involved (such as organic solvents for extrac- pollution-control systems. tions) and the byproducts of the reaction are A genetically engineered organism, on the toxic and require special disposal. other hand, is designed to be precursor- and 92 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals product-specific, with each enzyme having compounds are produced by both methods. essentially 100-percent conversion efficiency. However, in most beverage distilling operations, An enzymatic process that carries out the same pollution has been reduced to almost zero with transformation as a chemical synthesis pro- the complete recovery of still slops as animal duces no side-products (because of an enzyme’s feeds of high nutritional value. Such control high specificity to its substrate) or byproducts procedures are generally applicable to most (because of an enzyme’s strong catalytic power). fermentation processes. (App. I-C describes the Consequently, biological processes eliminate pollutants that may be produced by current many conventional waste and disposal problems chemical processes and those expected from at the front end of the system-in the fer- biologically based processes.) menter. This high conversion efficiency reduces The Environmental Protection Agency has the costs of recycling. In addition, the efficiency estimated that the U.S. Government and indus- of the biological conversion process generally try combined will spend over $360 billion to simplifies product recovery, reducing capital control air and water pollution in the decade and operating costs. Furthermore, by their from 1977 through 1986. The share of the nature, biologically based chemical processes, chemical and allied industries is about $26 bil- tend to create some waste products that are bio- lion. Genetic engineering technology may help degradable and valuable as sources of nutrients. alleviate this burden by offering cleaner proc- Specific comparisons of the environmental esses of synthesis and better biological waste hazards produced by conventional and biologi- treatment systems. The monetary savings could cal systems are difficult. Data detailing the be tremendous. As pure speculation, if just 5 pollution parameters for various current chem- percent of the current chemical industry were ical processes exist, but much less information affected, spending on pollution could be re- is available for fermentation processes, and few duced by about $100 million per year.

Industrial chemicals that may be produced by biological technologies

Despite the benefits of producing industrial In principle, virtually all organic compounds chemicals biologically, thus far major fermenta- can be produced by biological systems. If the tion processes have been developed primarily necessary enzyme or enzymes are not known to for a few complex compounds such as enzymes. exist, a search of the biological world will prob- (See table 11.) Biological methods have also been ably uncover the appropriate ones. Alterna- developed for a few of the simpler commodity tively, at least in theory, an enzyme can be chemicals: ethanol, butanol, acetone, acetic engineered to carry out the required reaction. acid, isopropanol, glycerol, lactic acid, and citric Within this framework, the potential appears to acid. be limited only by the imagination of the bio- technologist—even though certain chemicals Two questions are critical to assessing the that are highly toxic to biological systems are feasibility or desirability of producing various probably not amenable to production. chemicals biologically: Three variables in particular affect the I. Which compounds can be produced bio- answer to the second question: the availability logically (at least theoretically)? of an organism or enzymes for the desired 2. Which compounds may be primarily de- transformation; the cost of the raw material; pendent on genetic technology, given the and the cost of the production process. When costs and availability of raw materials? specific organisms and production technologies Ch. 5—The Chemical Industry ● 93

Table 1 1.—Some Commercial Enzymes and Their Uses

Enzyme Source Industry and application Amylase ...... Animal(pancreas) Pharmaceutical digestive aids Textile: desizing agent Plant(barley malt) Baking: flour supplement Brewing, distilling, and industrial alcohol mashing Food: precooked baby foods Pharmaceutical digestive aids Textile: desizing agent Fungi (Aspergillus niger,A. oryzae) Baking: flour supplement Brewing, distilling, and industrial alcohol mashing Food: precooked baby foods, syrup manufacture Pharmaceutical digestive aids Bacteria (Bacillus subtilis) Paper starch coatings Starch: cold-swelling laundry starch Bromelin...... Plant (pineapple) Food: meat tenderizer Pharmaceutical digestive aids Cellulase and hemicellulase . . Fungi (Aspergillus niger) Food: preparation of liquid coffee concentrates Dextransucrase...... Bacteria(Leuconostoc mesenteroides) Pharmaceutical preparation of blood-plasma extenders, and dextran for other uses Ficin ...... Plant (fig latex) Pharmaceutical: debriding agent Glucose oxidase (plus catalase or peroxidase) ...... Fungi (Aspergillus niger) Pharmaceutical test paper for diabetes Food: glucose removal from egg solids Invertase...... Yeast (Saccharomyces cerevisiae) Candy: prevents granulation of sugars in soft-center candies Food: artificial honey Lactase...... Yeast (Saccharomyces fragilis) Dairy: prevents crystallization of lactose in ice cream and concentrated milk Lipase...... Fungi (Aspergillus niger) Dairy: flavor production in cheese Papain ...... Plant(papaya) Brewing: stabilizes chill-proof beer Food: meat tenderizer Pectinase ...... Fungi (Aspergillus niger) Wine and fruit juice: clarification Penicillinase ...... Bacteria(Bacillus cereus) Medicine: treatment of allergic reaction to penicillin, diagnostic agent Pepsin ...... Animal (hog stomach) Food: animal feed supplement Protease...... Animal (pancreas) Dairy: prevents oxidized flavor Food: protein hydrolysates Leather: bating Pharmaceutical: digestive aids Textile: desizing agent Animal (pepsin) Brewing: beer stabilizer Animal (rennin, rennet) Dairy: cheese Animal (trypsin) Pharmaceutical: wound debridement Fungi (Aspergillus oryzae) Baking: bread Food: meat tenderizer Bacteria (Bacillus subtilis) Baking: modification of cracker dough Brewing: clarifier Streptodornase ...... Bacteria (Streptococcus pyrogenes) Pharmaceutical: wound debridement

SOURCE: David Perlman, “The Fermentation Industries,” American Society for Microbiology News 39:10, 1973, p. 653. have been developed, the cost of raw materials organism. Finally, the production process itself becomes the limiting step in production. If a is a factor. After fermentation, the desired prod- strain of yeast, for example, produces 5 percent uct must be separated from the other com- ethanol using sugar as a raw material, the proc- pounds in the reaction mixture. As an aid to re- ess might become economically competitive if covery, the production conditions might be the cost of sugar drops or the price of petro- altered and improved to generate more of a de- leum rises. Even if prices remain stable, the sired compound. micro-organisms might be genetically improved to increase their yield; genetic manipulation More than one raw material can be used in a might solve the problem of an inefficient fermentation process. If, in the case of ethanol, 94 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

the price of sucrose (from sugarcane or sugar Table 12.—Expansion of Fermentation Into beets) is not expected to change, the production the Chemical Industry technology is being run at optimum efficiency, Examples and the micro-organism is producing as much Aerobic fermentation ethanol as it can, the hurdle to economic com- Enzymes ...... Amylases, proteases petitiveness might be overcome if a less expen- Vitamins ...... Riboflavin B,z Pesticides...... Bacillus thuringiensis sive raw material—cellulose, perhaps—were Growth regulators ...... Gibberellin used. But cellulose cannot be used in its natural Amino acids ...... Glutamic, Iysine state: physical, chemical, or biological methods Nucleic acids must be devised to break it down to its glucose Acids...... Malic acid, citric acid Anaerobic fermentation (also a sugar) components. Solvents ...... Ethanol, acetone, n-butanol The constraints vary from compound to com- Acids...... Acetic, propionic, acrylic pound. But even though the role of genetics SOURCE: Office of Technology Assessment. must be examined on a product-by-product basis, certain generalizations can be made. Over- these products, ethylene glycol, by the all, genetic engineering will probably have an Cetus Corp. in Berkeley, Calif. The process impact on three processes: is claimed to be more energy efficient and ● Aerobic fermentation, which produces en- less polluting. If it proves successful when zymes, vitamins, pesticides, growth regula- run at an industrial scale, the technology tors, amino acids, nucleic acids, and other could become significant to a [J. S. market speciality chemicals, is already well-estab- totaling $21/2 billion per year. lished. Its use should continue to grow. Al- The chemical industry produces a variety of ready, complex biochemical like antibiot- likely targets for biotechnology. Tables I-B-27 ics, growth factors, and enzymes are made through 1-B-32 in appendix I-B present projec- by fermentation. Amino acids and nucleo- tions of the potential economic impacts of ap- tides—somewhat less complicated mole- plied genetics on selected compounds that cules—are sometimes produced by fer- represent large markets, and the time frames mentation. Their production is expected to for potential implementation. Table I-B-7 lists increase. one large group of organic chemicals that were ● Anaerobic fermentation, which produces identified by the Genex Corp. and Massachu- organic acids, methane, and solvents, is the setts Institute of Technology (MIT) as amenable industry’s area of greatest current growth. to biotechnological production methods. They Already, 40 percent of the ethanol man- are in agreement on about 20 percent of the ufactured in the United States is produced products cited, which underscores the uncer- in this way. The main constraint on the tain nature of attempting to predict so far into production of other organic acids and sol- the future. vents is the need for cheaper methods for converting cellulose to fermentable sugars. Fertilizers, polymers, and pesticides ● Chemical modification of the fermentation products of both aerobic and anaerobic Gaseous ammonia is used to produce nitrogen fermentation, which to date has rarely fertilizers. About 15 billion tonnes of ammonia been used on a commercial scale, is of were produced chemically for this purpose, in great interest. (See table 12.) Chemical pro- 1978; the process requires large amounts of duction technologies that employ high tem- natural gas. Nitrogen can also be converted, or peratures and pressures might be replaced “fixed,” to ammonia by enzymes in micro-orga- by biological technologies operating at at- nisms; about 175 billion tonnes are fixed per mospheric pressure and ambient tempera- year. For example, one square yard of land ture. A patent application has already been planted with certain legumes (such as soybeans) filed for the biological production of one of can fix up to 2 ounces of nitrogen, using bac- Ch. 5—The Chemical Industry ● 95 teria associated with their roots. Currently, mi- Table 13.—The Potential of Some Major Polymeric crobial production of ammonia from nitrogen is Materials for Production Using Biotechnology not economically competitive. Aside from the Domestic production 1978 difficulties associated with the enzyme’s sen- Product (thousand tonnes) sitivity to oxygen and the near total lack of Plastics understanding of its mechanism, it takes the Thermosetting resins Epoxy...... 135 equivalent of the energy in 4 kilograms (kg) of Polyester...... 544 sugar to make 1 kg of ammonia. Since ammonia Urea...... 504 costs $0. 13/kg and sugar costs $0.22/kg, it is un- Melamine ...... 90 Phenolic ...... 727 likely that the chemical process will be replaced Thermoplastic resins in the near future. On the other hand, the genes Polyethylene for nitrogen fixation have now been transferred Low density ...... 3,200 High density...... 1,890 into yeast, opening up the possibility that agri- Polypropylene ...... 1,380 culturally useful nitrogen can be made by fer- Polystyrene...... 2,680 mentation. Polyamide, nylon type...... 124 Polyvinyl alcohol ...... 57 A large segment of the chemical industry en- Polyvinyl chloride...... 2,575 Other vinyl resins...... 88 gaged in the manufacture of polymers is shown Fibers in table 13. A total of 4.3 million tonnes of Cellulosic fibers fibers, 12 million tonnes of plastics, and I. I mil- Acetate ...... 139 lion tonnes of synthetic rubber were produced Rayon ...... 269 Noncellulosic fibers in the United States in 1978. All were derived Acrylic...... 327 from petroleum, with the exception of the less Nylon...... 1,148 than 1 percent derived from cellulose fibers. Olefins...... 311 Polyester...... 1,710 The most likely ones are polyamides (chemically Textile glass ...... 418 related to proteins), acrylics, isoprene-type rub- Other ...... 7 ber, and polystyrene, Because most monomers, Rubbers the building blocks of polymers, are chemically Styrene-butadiene...... 628 Polybutadiene ...... 170 simple and are presently available in high yield Butyl ...... 69 from petroleum, their microbial production in Nitrile...... 33 the next decade is unlikely. Polychlorophene ...... 72 Ethylene-propylene ...... 78 While biotechnoloqy is not ready to replace Polyisoprene...... 62 the present technology, its eventual impact on SOURCE: Office of Technology Assessment. polymer production will probably be large. Biopolymers represent a new way of thinking. Conceivably, biotechnology could enable the Most of the important constituents of cells are modification of their function and form. polymers: proteins (polypeptides from amino acid monomers), polysaccharides (from sugar Pesticides include fungicides, herbicides, in- monomers), and polynucleotides (from nucleo- secticides, rodenticides, and related products tide monomers). Since cells normally assemble such as plant growth regulators, seed disinfec- polymers with extreme specificity, the ideal in- tants, soil conditioners, and soil fumigants. The dustrial process would imitate the biological largest market (roughly $500 million annually) production of polymers in all possible respects— involves the chemical and microbial control of using a single biological machine to convert a insects. Although microbial insecticides have raw material, e.g., a sugar, into the monomer to been around for years, they comprise only 5 polymerize it, then to form the final product. A percent of the market. However, recent suc- more likely application is the development of cesses in developing viruses and bacteria that new monomers for specialized applications. produce diseases in insects, and the negative Polymer chemistry has largely consisted of the publicity given to chemical insecticides, have study of how their properties can be modified. encouraged the use of microbial insecticides. 96 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

The other two species can be produced by con- Ventional fermentation techniques. They have been useful because they form spores that can be mass-produced easily and are stable enough to be handled commercially. The actual substances that cause toxicity to the insect are toxins synthesized by the microbes. Genetic engineering should make it possible to construct more potent bacterial insecticides by increasing the dosage of the genes that code for the synthesis of the toxins involved. Mix- tures of genes capable of directing the synthesis of various toxins might also be produced.

Constraints on biological production techniques ____

The chief impediments to using biological A second major impediment is associated production technology are associated with the with the purification stage of production. Most need for biomass. 2 They include: chemical products of fermentation are present in extremely dilute solutions, and concentrating ● competition with food needs for starch and these solutions to recover the desired product is sugar; ● highly energy-intensive. Problems of technology cyclic availability; and cost will continue to make this stage an im- ● biodegradability and associated storage portant one to improve. problems; ● high moisture content for cellulosics, and The developments in genetics show great high collection and storage costs; promise for creating more versatile micro-orga- ● mechanical processing for cellulosics; nisms, but they do not by themselves produce a ● the heterogeneous nature of cellulosics (mix- cheaper fuel or plastic. Associated technologies tures of cellulose, hemicellulose, and lig- still require more efficient fermentation facil- nin); and ities and product separation processes; mi- ● The need for disposal of the nonferment- crobes may produce molecules, but they will able port ions of the biomass. not isolate, purify, concentrate, mix, or package them for human use. For food-related biomass sources, such as sugar, corn, and sorghum, few technological bar- The interaction between genetic engineering riers exist for conversion to fermentable sugars; and other technologies is illustrated by the but subsidies are needed to make the fermenta- problems of producing ethanol by fermentation of sugars as profitable as their use as food. tion. The case study presented in appendix I-D For cellulosic biomass sources such as agricul- identifies those steps in the biomass-to-ethanol tural wastes, municipal wastes, and wood, tech- scheme that need technological improvements nological barriers exist in collection, storage, before the process can become economical. pretreatment, fermentation, and waste disposal. Genetic engineering is expected to reduce In addition, biomass must always be trans- costs in many production steps. For certain formed into sugars by either chemical or en- ones—such as the pretreatment of the biomass before fermentation can zymatic processes to make it fermentable—genetics will probably begin. not play a role: physical and chemical technologies will be responsible for the greatest ad- ‘ljnf~r,qv F’r(ml ~io/(J,gif’if/ PrOct’s.st*,s, 01). (’it. vances. For others, such as distillation, genetic Ch. 5—The Chemical Industry . 97 technologies should make it possible to engineer ogies, such as the immobilization of whole cells organisms that can ferment at high tempera- in reactor columns, could be developed in paral- tures (82° to 85° C) so that the fermentation and lel with genetic technologies to increase the sta- at least part of the distillation can both take bility of cells in fermenters. place in the same reactor. Various technol-

“’1’11(~1.lllfjl]llilit (’tllill}ol 1)1’()(111(’(’1’S llil\’f’ illl’(’il(l~r 1)[’[’11 IIescrikd ‘l”lIt? ild\’illltit#?S Ot” SllCh Ilwrnmphilic I“t’l’lll(’lllilt iollS ill’t’ signif’i- ill IIW g(~tlus [,’l(jslri(fiunl [i. (’., (;. fhf’rr?~()(:f;ll[~n~). Ill i![ldil ion, gciwl i- (’illll: I’t?l’lllt%ltiitioll I inw is CollSi&Will)l}” l’t’(111(’tXl; IIIP risk ot (’oll- (’ill 11’ wlgilwf’1’(~d ol’~illlisllls, (Ies(’ril)e(i ii!+ il (’1’OSS 1)(’ltt’tWll V(!ilSIS Iillllillill ion is llt?ill’1.V f?litllitlille(l: illl(l cooling” t.t’(lltil.(’lllt’tlls ill’(’ ;111( i I I)ol’lllol)ll” i] it’ I)il(’1 f>l’lil, (’iIll I’pl’lllf)tll il I 700 (:. I[)w’t?t’ (111(+ 10 llle lli@el. lt?lll~t?l’iitll l’(’ ot” III(’ t’msnlt’nt in~ 1)1’0111.

An overview of impacts

The cost of raw materials may become cheap- zymes may expand and drive this sector of the er than the petroleum now used—especially if industry. cellulose conversion technologies can be developed. The source of raw materials would also Impacts on other industries be broader since several kinds of biomass could be interchanged, if necessary. For small quan- Although genetic engineering will develop tities of chemicals, the raw material supply new techniques for synthesizing many sub- would be more dependable, particularly be- stances, the direct displacement of any present cause of the domestic supply of available bio- industry appears to be doubtful: Genetic engi- mass. For substances produced in large quanti- neering should be considered simply another in- ties, such as ethanol, the supply of biomass dustrial tool. As such, any industry’s response could limit the usefulness of biotechnology. should be to use this technique to maintain its positions in its respective markets. The point is Raw materials, such as organic wastes, could illustrated by the variety of companies in the be processed both to produce products and pharmaceutical, chemical, and energy indus- reduce pollution. Nevertheless, the impact on tries that have invested in or contracted with total imported petroleum will be low. Estimates genetic engineering firms. Some large com- of the current consumption of petroleum as a panies are already developing inhouse genetic raw material for industrial chemicals is approx- engineering research capabilities. imately 5 to 8 percent of the total imported. The frequent, popular reference to the small, Impacts on the process include relatively innovative “genetic engineering companies” as a cheaper production costs for selected com- major new industry is somewhat misleading. pounds. For these, lower temperatures and The companies (see table 14) arose primarily to pressures can be used, suggesting that the proc- convert micro-organisms with little commercial esses might be safer. Chemical pollution from use into micro-organisms with commercial po- biotechnology may be lower, although methods tential. A company such as the Cetus Corp. ini- of disposal or new uses must be found for the tially used mutation and selection to improve micro-organisms used in fermentation. Finally, the biological processes will demand the devel- strains, whereas other pioneers such as Genen- tech, Inc., Biogen, S. A., and Genex Corp. were opment of new technologies for the separation founded to exploit recombinant DNA (rDNA) and purification of the products. technology. Part of their marketing strategy in- impacts on the products include both cheaper cludes the sale or licensing of genetically engi- existing chemicals as well as entirely new prod- neered organisms to large established commer- ucts. Since biotechnology is the method of cial producers in the chemical, pharmaceutical, choice for producing enzymes, new uses for en- food, energy, and mining industries. Each engi- 98 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Table 14.—Some Private Companies With Biotechnology Programs

Approximate Research capacity Company Founded employees 1979 Ph. D.s 1979 Recombinant DNA Hybridomas Atlantic Antibodies...... 1973 50 2 x Bethesda Research Laboratories ...... 1976 130 x x Biogen ...... 1978 30 (50b) (18b) (3)(5) x x Bens Bio Logicals ...... 1979 15 10 x x Centocor...... 1979 20(1)-10(4) x Cetus ...... 1972 250 50 x x Clonal Research ...... 1979 6 1 x Collaborative ResearchC...... 1961 85 15 x x (Collaborative Genetics) ...... (1979) (4) (3) x Genentech ...... 1976 90 30 x Genex ...... 1977 30 12 x Hybritech ...... 1978 33(1) 6 x Molecular Genetics...... 1979 6(4) x x Monoclinal Antibodies...... 1979 6 x NewEngland Biolabs ...... 1974 22(22)-5 (4) x q=.E~r9t~t~co. estimates. bEXwtedby~cem~rl~. ccollaboratlve Research isamajorowner of Collaborative Genetics. Thedivision batweenthem isnotyetdistinct. SOURCES: (l)Sc/ence206, p.692.693, 1960(52 peopletoexpandto100 by1961). (2) Science 206, p. 692-693, 19S0 (20 senior peraona). (3) Science 206, p. 692-693, 1960(16 scientists, 30 employees). (4) Dun & Bradstreet, Inc. (5) Chemlcalarrd Engineering News, Mar. 19,1960. Office of Technology Assessment. neering firm also intends to manufacture some beginning to examine the possibility of convert- products itself. It is likely that the products re- ing them into useful products. This approach served for inhouse manufacture will be low- (which is somewhat more developed in Europe) volume, high-priced compounds like interferon. assumes that with the proper technology the waste materials can become a resource. Genetic engineering by itself is a relatively small-scale laboratory operation. Consequently, Some industries, including manufacturers of genetic engineering firms will continue to offer agitators (drives), centrifuges, evaporators, fer- services to companies that do not intend to menters, dryers, storage tanks and process develop this capacity in their own inhouse lab- vessels, and control and instrumentation sys- oratories. Specifically, a genetic engineering tems, might profit by producing equipment company may contract with a firm to develop a associated with fermentation. biological production method for its products. At the same time, larger companies might establish inhouse staffs to develop biological methods Impacts on university research for both old and new products. (Several larger From the beginning, genetic engineering companies already have more inhouse genetic firms established strong ties with universities. engineering personnel than some of the inde- These were responsible for providing most of pendent genetic engineering companies.) the scientific knowledge that formed the basis In addition, suppliers of genetic raw materials for applied genetics as well as the initial scien- may decide to expand into the production of tific workforce: genetically engineered organisms. Suppliers of restriction endonuclease enzymes for example, Cetus Corp. established a pattern by re- which are used in constructing rDNA, have cruiting a prestigious Board of Scientific already entered the field. Diagnostic firms could Advisors who remain in academic posi- develop new bioassays for which they them- tions. selves would guarantee a market. Finally, com- Genentech, Inc., cofounded by a professor panies with byproducts or waste products are at the University of California at San Fran- Ch. 5—The Chemical Industry . 99

cisco, initially depended largely on outside useful to industry-such as chemistry and phys- scientists. ics—have managed to achieve a balance be- ● Biogen, S. A., was organized by professors tween secrecy and openness. at Harvard and MIT plus six European scientists, and placed R&D contracts with aca- The social impacts of local demic researchers. industrial activity ● Collaborative Genetics has a Nobel prize winner from MIT as the chairman of its sci- Despite the extensive media coverage of entific advisory board. rDNA and other forms of genetic engineering, ● Hybritech, Inc., has as its scientific nucleus there is little evidence that people who live near a University of California, San Diego, pro- companies using such techniques are still great- fessor complemented by scientists at the ly concerned about possible hazards. This may Salk Institute. be partly owing to a lack of awareness that a particular company is doing genetic research In addition to these companies, others have also been establishing closer ties with the academic and partly because companies thus far have community. adhered to the National institutes of Health (NIH) Guidelines. Some companies have placed Much of the research that will be useful to in- individuals on their institutional biosafety com- dustry will continue to be carried out in univer- mittees who are respected and trusted mem- sity laboratories. At present, it is often difficult bers of the local community. By involving the to decide whether a research project should be local citizens with no vested corporate interest, classified as “basic” (generally more interesting a mechanism for oversight has been provided. to an academic researcher) or “applied” (gener- (For a more detailed discussion, see ch. 11.) ally more interesting to industry). E.g., a change in the genetic code, which increases gene activi- Impacts on manpower ty, would be just as exciting to a basic scientist as to an industrial one. Two types of impacts on workers can be expected: This dialog between the universities and industry—both through formal and informal ar- ● The creation of jobs that replace those held rangements –has fostered innovation. Although by others. E.g., a worker involved in the number of patents applied for is not a direct chemical production might be replaced by reflection of the level of innovation, it is still one one producing the same product biologi- indication. By the end of 1980, several hundred cally. patent applications were filed for genetically ● The creation of new jobs. engineered micro-organisms, their products, Workers in three categories would be af- and their processes. fected: University research has clearly affected in- ● those actually involved in the fermentation- dustrial development, and has in turn been af- production phase of the industry; fected by industry. Although the benefits are ● those involved in the R&D phase of the in- easily recognized, some drawbacks have been dustry, particularly professionals; and suggested. The most serious is the concern that . those in support industries. university scientists will be restrained in their academic pursuits and in their exchange of in- Projections of manpower requirements are formation and research material. To date, anec- only as accurate as the projections of the level of dotal information suggests that some scientists industrial activity. In the past 5 years, about 750 are being more circumspect about sharing in- new jobs have been created within the small ge- formation. Still, secrecy is not new to highly netic engineering firms (including monoclinal competitive areas of biomedical research. In ad- antibody producers). Of these, approximately dition, scientists in other academic disciplines one-third hold Ph. D. degrees. 100 . Impacts of Applied Genetics—Micro-0rganisms, Plants, and Animals

Data obtained through an (OTA survey of 284 Table 17.—lndex to Fermentation Companies firms indicate that the pharmaceutical industry 1. Abbott Laboratories, North Chicago, 111. employs the major share of personnel working 2. American Cyanamid, Wayne, N.J. in applied genetics programs. (See table 15. ) The 3. Anheuser-Busch, Inc., St. Louis, Mo. average number of Ph. D.s in each industry is 4. Bristol-Myers Co., Syracuse, N.Y. 5. Clinton Corn Processing Co., Clinton, Iowa given in table 16. A rough estimate of profes- 6. CPC International, Inc., Argo, Ill. sional scientific manpower at this level includes: 7. Dairyland Laboratories, Inc., Waukesha, Wis. 6 in food, 45 in chemical, 120 in pharmaceutical, 8. Dawe’s Laboratories, Inc., Chicago Heights, Ill. 9. Grain Processing Corp., Muscatine, Iowa and 18 in specialty chemicals-a total of 189. If 10. Hoffman-LaRoche, Inc., Nutley, N.J. the number of research support personnel is 11. IMC Chemical Group, Inc., Terre Haute, Ind. approximately twice the number of Ph. D.s, the 12. Eli Lilly & Co., Indianapolis, Ind. 13. Merck & Co., Inc., Rahway, N.J. total rises to about 570. If $165,000 per year is 14. Miles Laboratories, Inc., Elkhart, Ind. required to support one Ph. D. in industry, the 15. Parke, Davis & Co., Detroit, Mich. total value of such manpower is approximately 16. S. B. Penick & Co., Lyndhurst, N.J. 17. Pfizer, Inc., New York, N.Y. $31 million. 18. Premier Malt Products, Inc., Milwaukee, Wis. 19. Rachelle Laboratories, Inc., Long Beach, Cal if. Estimates of the number of companies en- 20. Rohm & Haas, Philadelphia, Pa. gaged in applied genetics work in 1980 can be 21. Schering Corp., Bloomfield, N.J. compared with the total number of firms with 22. G. D. Searle & Co., Skokie, Ill. 23. E. R. Squibb& Sons, Inc., Princeton, N.J. fermentation activities. A tabulation of firms on 24. Standard Brands, Inc., New York, N.Y. a worldwide basis in 1977 revealed 145 com- 25. Stauffer Chemical Co., Westport, Corm. panies, of which 27 were American. (See table 26. Universal Foods Corp., Milwaukee, Wis. 27. The Upjohn Co., Kalamazoo, Mich. 17. ) These companies produced antibiotics, en- 28. Wallerstein Laboratories, Inc., Morton Grove, Ill. zymes, solvents, vitamins and growth factors, 29. Wyeth Laboratories, Philadelphia, Pa.

SOURCE: Office of Technology Assessment.

Table 15.—Distribution of Applied Genetics nucleosides, amino acids, and miscellaneous Activity in Industry products. (See table 18.) The only chemical firm Distribution of applied listed was the Stauffer Chemical Co. Ten firms genetics activity by Percent are listed as having the ability to produce food a Classification company class of total and feed yeast. (See table 19. ) Correcting for Food ...... (6146) 13 firms listed twice, at least 38 U.S. firms were Chemical ...... (9/52) 17 Pharmaceutical . . . . . (12/25) 48 engaged in significant fermentation activity for Specialty chemicalb . (6/68) 9 commercial products, excluding alcoholic bev- algnoreg small firing specializing in genetic research. erages, in 1977. Not all have research expertise bFood ingredients, reagents, enzymes. in fermentation or biotechnology, much less a SOURCE: Office of Technology Assessment. regular genetics program: 10 to 20 were in the chemical industry; 25 to 40 in fermentation (enzyme, pharmaceutical, food, and specialized Table 16.—Manpower (Iow.(average)-high) Distribution chemicals); and 10 to 15 in biotechnology (genet- of a Firm With Applied Genetics Activity ic engineering)—or about 45 to 75 firms in all. Ph. D. M.S. Bachelors If average manpower numbers are used, the Food...... ql)-2 0-(1 )-2 O-(2)-8 Chemical ...... 3-(5)-7 0-(1 )-2 2-(5)-7 total number of professionals involved in com- Pharmaceutical...... 2-(10)-24 l-(4)-9 1-(8)-20 mercial applied genetics research is: Specialty...... l-(3)-8 l-(3)-4 2-(2)-4 Biotechnology Ph. D.s: 300-450 Genetic engineering. 3-(15)-32 2-(1 1)-20 5-(15)-25 Others: 600-900 Hybridoma...... l-(3)-6 0-(2)-0 0-(20)-0 900-1,350 Other ...... O-(2)-4 2-(4)-6 8-(10)-13 Average ...... 1-(6)-12 l-(4)-6 3-(8)-12 The number of workers that will be involved SOURCE: Office of Technology Assessment. in the production phase of biotechnology repre- Ch.5—The Chemical Industry ● 101

Table 18.-Fermentation Products and Producers

Some producers Product Some producers Product

Amino acids Capreomycin ...... L-alanine...... Cephalosporins...... 4,12 L-arginine...... Chromomycin A3 ...... L-aspartic acid...... Colistin ...... 27 L-citrulline ...... Cycloheximide...... 11 L-glutarnic acid ...... 25 Cycloserine ...... 13 L-glutamine ...... Dactinomycin...... L-glutathione ...... Daunorubicin...... L-histidine ...... Destomycin ...... L-homoserine...... Enduracidin ...... 17,27 L-isoleucine ...... Erythromycin ...... L-leucine...... Fortimicins...... L-lysine ...... Fumagiliin ...... L-methionine ...... Fungimycin ...... L-ornithine ...... Fusidic acid ...... 21 L-phenylalanine...... Gentamicins...... 28 L-proline...... Gramicidin A ...... L-serine...... Gramicidin J(S) ...... L-threonine...... Griseofulvin ...... 12 L-tryptophan...... Hygromycin B ...... L-tyrosine ...... Josamycin ...... 4 L-valine...... Kanamycins...... Kasugamycin...... Miscellaneous products and processes Kitasatamycin...... Acetoin ...... Lasalocid ...... 10 Acyloin ...... 13 Lincomycin...... 27 Anka-pigment(red) ...... Lividomycin ...... Blue cheese flavor...... 7 Macarbomycin...... Desferrioxamine ...... Mepartricin...... Dihydroxyacetone ...... 17,21,28 Midecamycin...... Dextran ...... Mikamycins ...... Diacetyl (from acetoin) ...... Mithramycin...... 17 Ergocornine ...... Mitomycin C...... 4 Ergocristine ...... Mocimycin ...... Ergocryptine ...... Monensin ...... Ergometrine ...... Myxin ...... 10 Ergotamine...... Neornycins...... 16,17,23,27 Bacillus thuringiensis insecticide...... 1 Novobiocin...... 27 Lysergic acid ...... Nystatin ...... 23 Paspalic acid ...... Oleandomycin...... 17 Picibanil ...... Oligomycin...... Ribose...... Paromomycins...... 15 Scteroglucan ...... Penicillin G...... 4,12,13,17,23,29 Sorbose(from sorbitol)...... 10,17 Penicillin V ...... 1,4,12,17,23,29 Starter cultures ...... 7,13,14 Penicillins(semisynthetic)...... 4,13,17,23,29 Sterol oxidations...... 22,27 Pentamycin ...... Steroid oxidations...... 21,23,27,29 Pimaricin ...... Xanthan ...... 13,17 Polymyxins...... 17 Antibiotics Polyoxins ...... Adriamycin...... Pristinamycins...... Amphomycin ...... Quebemycin...... Ribostamycin...... Amphotericin B...... 23 Avoparcin ...... 2 Rifamycins...... Azalomycin F ...... Sagamicin...... Bacitracin...... 11,16,17 Salinomycin, ...... Bambermycins...... Siccanin ...... Siomycin...... Bicyclomycin...... 21 Blasticidin S...... Sisomicin ...... Spectinomycin...... 27 Bleomycin ...... 13,17,29 Cactinomycin Streptomycin...... Candicidin B...... 16 Tetracyclines Candidin ...... Clortetracycline...... 19

76-565 0 - 81 - 8 102 . Impacts of Applied Genetics—Micro-Organkms, Plants, and Animals

Table 18.-Fermentation Products and Producers

Product Some producers Product Some producers

Demeclocycline...... 2 Lactase ...... 28 Oxytetracycline...... 17,19 Lipase ...... 20 Tetracycline...... 2,4,17,19,23,27 Microbial rennet...... 17,28 Tetranactin...... Naringinase ...... 28 Thiopeptin ...... Pectinase ...... 20,28 Thiostrepton ...... 23 Pentosanase ...... 20,28 Tobramycin ...... 12 Proteases ...... 14,17,18,20,28 Trichomycin...... Streptokinase-streptodornase...... 2 Tylosin ...... 12 Uricase ...... Tyrothricin ...... 16,28 Organic acids Tyrocidine ...... Citric acid ...... 14,17 Uromycin ...... Comenic acid...... 17 Vaildamycin ...... Erythorbic acid...... Vancomycin ...... 12 Gluconic acid...... 4,17,18 Variotin...... Itaconic acid...... 17 Viomycin...... 2-keto-D-giuconic acid ...... 17 Virginiamycin...... ~-ketoglutaric acid...... Enzymes Lactic acid ...... 5 Amylases ...... 5,19,20,24,28 Malic acid ...... Amyloglucosidase...... 5,6,14,28 Urocanic acid...... Anticyanase...... Solvents L-asparaginase ...... Ethanol ...... 9 Catalase ...... 8,14 2,3-butanediol ...... Cellulase...... 6,20,28 Dextranase...... Vitamins and growth factors 1,12,13 ‘Diagnostic enzymes’ ...... Gibbereliins ...... 13 Esterase-lipase ...... 28 Riboflavin ...... Vitamin B ...... 13 Glucanase ...... 28 12 11 Glucose dehydrogenase...... Zearalanol...... Glucose isomerase ...... 3,5,14,24 Nucleosides and nucleotides Glucose oxidase ...... 8,14 5-ribonucieotides and nucleosides . . . . . Glutamic decarboxylase...... 18 Orotic acid ...... Hemi-celiulase...... 14,20,28 Ara-A-(9-B-D-arabino-furanosyl) ...... 15 Hespiriginase...... 6-azauridine ...... lnvertase...... 24,26,28 aBlankm=n~ noU.S.pr~ucer in 1977;therefore, l~produeedbyoneormore foreign flrms(fromatleast 120different firms). SOURCE: OtticeotTechnology Assessment. sents a major impact of genetic engineering. To produced by genetically engineered strains in estimate this number these two calculations 10 years and $7.1 billion in 20 years. Table must be made: I-B-l0 in appendix I-B lists the potential markets for pharmaceuticals. Excluding methane pro- ● the value or volume of chemicals that duction, the total potential market for products might be produced by fermentation, and obtained from genetically engineered orga- ● the number of production workers needed nisms is approximately $14.6 billion. per unit volume of chemicals produced. If the production of chemicals having this Any prediction of the potential volume of value is carried out by fermentation, it impossi- chemicals is necessarily filled with uncertain- ble to calculate how many workers will be ties. The approximate market value of organic needed. Data obtained from industrial sources chemicals produced in the United States is given reveal that 2 to 5 workers, including those in in appendix I-B. Total U.S. sales in 1979 were supervision, services, and production, are re- calculated to be over $42 billion. On the basis of quired for $l million worth of product. Hence, the assumptions made, $522 million worth of 30,000 to 75,000 workers would be required for bulk organic chemicals could be commercially the estimated $14.6 billion market. Ch. 5—The Chemical lndustry ● 103

Table 19.—U.S. Fermentation Companies indicates that approximately the same number of workers will be required per unit of output. Producers of Baker’s yeast and food/feed yeast in the United States in 1977 Estimates of the number of workers are di- Baker’s yeast: American Yeast Co., Baltimore, Md. vided into: 1) workers directly involved in the Anheuser-Busch, Inc., St. Louis, Mo. growth of the organisms; and 2) workers in- Federal Yeast Co. (now Diamond Shamrock), volved in the "recovery" phase, where the Baltimore, Md. Fleischmann Yeast Co., New York, N.Y. organisms are harvested and the chemical prod- Universal Foods Corp., Milwaukee, Wis. uct is extracted, purified, and packaged. Based Food/feed yeast: on industry data, the number of workers in the Amber Laboratories, Juneau, Wis. Amoco Foods Co., Chicago, Ill. fermentation phase is approximately 30 percent Boise-Cascade, Inc., Portland, Oreg. of the total, and those in recovery approximate- Diamond Mills, Inc., Cedar Rapids, Iowa ly 50 percent. Hence, about 9,000 to 22,500 Fleischmann Yeast Co., New York, N.Y. Lakes States Yeast Co., Rhinelander, Wis. workers might be expected to hold jobs in the Stauffer Chemical Co., Westport, Corm. immediate fermentation area, and about 15,000 Enzyme producers, 1977 to 37,500 workers would be involved in han- Clinton Corn Processing Co., Clinton, lowa Miles Laboratories, Inc., Elkhart, Ind. dling the production medium (with or without Premier Malt Products, Inc., Milwaukee, Wis. the organisms).

SOURCE: Compiled by Perlman, Arner/can Soc/ety for M/crobio/ogy News 43:2, Estimates of the number of totally new jobs 1977, pp 82-89. that would be created are highly speculative; they should allow for estimates of increases in Since the chemicals considered above are the quantity of chemicals currently being pro- currently being produced, any new jobs in bio- duced and the production of totally new com- technology will displace the old ones in the pounds. According to estimates by Genex, the chemical industry. Whether the change will renew and growth markets may reach $26 billion sult in a net loss or gain in the number of jobs is by the year 2000, which would add 52,000 to difficult to predict. However, a rough estimate 130,000 jobs to the present number. chapter 6 The Food Processing Industry Chapter 6

Page Introduction—The Industry ...... 107 21. Classification of Yeast-Related U.S. Patents , , 108 Single-Cell Protein...... 107 22. Comparison of Selling Price Ranges for Genetic Engineering and SCP Production. ... 109 Selected Microbial, Plant, and Animal Commercial Production...... 109 Protein Products...... 108 Genetics in Backing, Brewing, and Winemaking .110 23, Raw Materials Already Tested on a Microbial Polysaccharides. ., ...... 111 Laboratory or Small Plant Scale. ... , , .. ...109 Enzymes...... , ...... 111 Genetic Engineering and Enzymes in the Figure Food Processing Industry ...... 112 Figure No. Page Sweeteners, Flavors, and Fragrances ...... 112 26. The Use of Hybridization To Obtain a Yeast Overview ...... 113 Strain for the Production of Low- Carbohydrate Beer ...... 110 Tables

Table No. Page 20. Estimated Annual Yeast Production, 1977...108 Chapter 6 The Food Processing Industry

Introduction—the industry

The food processing industry comprises Traditionally, micro-organisms have been those manufacturers that transform or process used to stabilize, flavor, and modify various agricultural products into edible products for properties of food. More recently, efforts have market. It is distinguished from the production, been made to control microbial spoilage and to or farming and breeding portions of the agricul- ensure that foods are free from micro-orga- tural industry. nisms that may be hazardous to public health. These are the two major ways in which micro- Genetics can be used in the food processing biology has been useful. industry in two ways: to design micro-organisms that transform inedible biomass into food Historically, most efforts have been devoted for human consumption or for animal feed; and to improving the ability to control the harmful to design organisms that aid in food processing, effects of micro-organisms. The industry recog- either by acting directly on the food itseIf or by nized the extreme heat resistance of bacterial providing materials that can be added to food. spores in the early 20th century and sponsored or conducted much of the early research on the Eight million to ten million people work in the mechanisms of bacterial spore heat resistance. meat, poultry, dairy, and baking industries; in Efforts to exploit the beneficial characteristics canned, cured, and frozen food plants; and in of micro-organisms, on the other hand, have moving food from the farm to the dinner table. been largely through trial-and-error. Strains In 1979, the payroll was over $3.2 billion for the that improve the quality or character of food meat and poultry industries, $2.6 billion for generally have been found, rather than de- baking, and $1.9 billion for food processing. signed.

Single-cell protein

The interest in augmenting the world’s sup- The idea of using SCP as animal feed or ply of protein has focused attention on micro- human food is not new; yeast has been used as bial sources of protein as food for both animals food protein since the beginning of the century. and humans. * Since a large portion of each However, in the past 15 years, there has been a bacterial or yeast cell consists of proteins (up to dramatic increase in research on SCP and in the 72 percent for some protein-rich cells), large construction of large-scale plants for its pro- numbers have been grown to supply single-cell duction, especially for the production of yeast. protein (SCP) for consumption, The protein can (See table 20.) Interest in this material is re- be consumed directly as part of the cell itself or flected in the numerous national and interna- can be extracted and processed into fibers or tional conferences on SCP, the increasing meat-like items. By now, advanced food proc- number of proceedings and reviews published, essing technologies can combine this protein and the number of patents issued in recent with meat flavoring and other substances to years. (See table 21.) produce nutritious food that looks, feels, and tastes like meat. The issues addressed have covered topics such as the economic and technological factors *AS an example of the potential significance of SCP, the Soviet influencing SCP processes, nutrition and safety, IInion, which is one of the largest producers, expects to produce enough fodder yeast from internally available raw materials to be and SCP applications to human or animal foods. self-sufficient in animal protein foodstuffs by 1990. Thus far, commercial use has been limited by

107 108 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Table 20.-Estimated Annual Yeast Production, 1977 Table 22.—Comparison of Selling Price Ranges (dry tonnes) for Selected Microbial, Plant, and Animal Protein Products Baker’s yeast Dried yeasta Europe...... 74,000 b 160,000 b Crude Price range North America ...... 73,000 53,000 protein 1979 Us. The Orient...... 15,000 25,000 Product, substrate, and quality content dollars/kg United Kingdom...... 15,500 (c) Single cell proteins South America...... 7,500 2,000 Candida utilis, ethanol, food grade 52 1.32-1.35 Africa...... 2,700 2,500 Kluyveromyces tragilis, cheese Totals ...... 187,700 242,500 whey, food grade...... 54 1.32 Saccharomyces cerevisiae: aDrigd yeast includes food and fodder yeasts; data for petroleum-grown Yeasts Brewer’s, debittered, food grade 52 1.00-1.20 are not available. Feed grade...... 52 bproduction figures for U.S.S.R. not reported. 0.39-0.50 cNone reported. Plant proteins SOURCE: H. J. Peppier and D. Perlman (eds.), Microbial Technology, vol. 1 (Lon- Alfalfa (dehydrated) ...... 0.12-0.13 don: Academic Press, 1979), p. 159. Soybean meal, defatted...... 0.20-0.22 Soy protein concentrate...... 70-72 0.90-1.14 Table 21.—Classification of Yeast-Related Soy protein isolate ...... 90“ 92 1.96-2.20 U.S. patents (1970 to July 1977) Animal proteins Fishmeal (Peruvian) ...... 65 0.41-0.45 Meat and bonemeal ...... 50 Category Number issued 0.24-0.25 Dry skim milk...... 37 0.88-1.00 Yeast technology (apparatus, processing) . . . . 22 Growth on hydrocarbons. ., ...... 28 SOURCE: Office of Technology Assessment. Growth on alcohols, acids, wastes...... 22 Production of chemicals...... 14 Agriculture (USDA), total domestic and export Use of baking and pasta products ...... 24 supply for U.S. soybeans will grow 73 percent Condiments and flavor enhancers ...... 18 Reduced RNA...... , ...... 11 by 1985. Yeast modification of food products ...... 13 Isolated protein ...... 5 Soybeans are primarily consumed as animal Texturized yeast protein ...... 7 feed. But while only 4 percent of their annual Lysates and ruptured cells ...... 7 Animal feed supplements ...... 12 production are directly consumed by humans, Total. ., ...... 183 the market is growing significantly. The introduction of improved textured soy protein in SOURCE: H. J. Peppier, “Yeast,” Annual Report on Fermentation Processes, D. Perlman (cd.), vol. 2 (London: Academic Press, 1978), pp. 191-200. cereals, in meat substitutes and extenders, and in dairy substitutes has increased the use of soy several factors. For each bacterial, yeast, or products. Nevertheless, the market does not de- algal strain used, technological problems (from mand soy products in particular but protein the choice of micro-organisms to the use of cor- supplements, vegetable oils, feed grain supple- responding raw material) and logistical prob- ments, and meat extenders in general. Other lems of construction and location of plants have protein and oil sources could replace soybeans arisen. But the primary limitation so far has if the economics were attractive enough. Fish- been the cost of production compared with the meal, dry beans, SCP, and cereals are all poten- costs of competing sources of protein. (The com- tial competitors. As long as a substitute can parative price ranges in 1979 for selected meet the nutritional, flavor, toxicity, and regula- microbial, plant, and animal protein products tory standards, competition will be primarily are shown in table 22.) based on price. The costs of manufacturing SCP for animal The competition between soybeans and SCP feed in the United States are high, particularly illustrates one of the paradoxes of genetic engi- relative to its major competing protein source, neering. While significant research is attempt- soybeans, which can be produced with little fer- ing the genetic improvement of soybeans, ge- tilizer and minimal processing. The easy avail- netic techniques are also being explored to inability of this legume severely limits microbial crease the production of SCP. Consequently, the SCP production for animal feed or human food. same tool—genetic engineering—encourages In fact, according to the U.S. Department of competition between the two commodities. Ch. 6—The Food Processing Industry ● 109

Genetic engineering and nisms that produce the enzyme cellulase, SCP production which degrades cellulose to glucose. Despite the microbial screening studies that Most of the significant genetic studies on the have been conducted and the wealth of basic production of cellulase by micro-organisms are genetic knowledge available about common just beginning to appear in the literature. The yeast (a major source of SCP), genetic engineer- most recent experiments have been successful ing has had little economic impact on SCP proc- in creating fungal mutants that produce excess esses until recently. Today, a variety of sub- amounts. stances are being considered as raw materials for conversion. Commercial production

● Petroleum-based hydrocarbons.—Until re- Of the estimated 2 million tons of SCP pro- cently, the wide availability and low cost of duced annually throughout the world, most petrochemicals have made the n-alkane hy- comes from cane and beet molasses, with about drocarbons (straight chain molecules of 500,000 tons from hydrolyzed wood wastes, carbon and hydrogen), which are petro- corn trash, and papermill wastes. (See table 23.) chemical byproducts, potential raw materi- Integrated systems can be designed to couple als for SCP production. At British Petro- the production of a product or food with SCP leum, mutants of micro-organisms have production from wastes. E.g., the waste saw- been obtained having an increased protein dust from the lumber industry could become a content. Mutants have also been found source of cellulose for micro-organisms. ICI’s with other increased nutritive values, e.g., successful genetic engineering of a micro-orga- vitamin content. nism to increase the usefulness of one raw ● Methane or methanol-Relatively few ge- material (methanol) should encourage similar netic studies have been directed at investi- attempts for other raw materials. gating the genetic control of the microbial use of methane or methanol. However, one But while SCP can be obtained from a wide recent application of genetic engineering variety of micro-organisms and raw materials, has been reported by the Imperia] Chem- the nutritional value and the safety of each ical Industries (ICI) in the United Kingdom, micro-organism vary widely, as do the costs of where the genetic makeup of a bacterium competing protein sources in regional markets. (Methylophilus methylotrophus) has been Consequently, accurate predictions cannot be altered so that the organism can grow made about the likelihood that SCP will displace more readily on methanol. The increase in traditional protein products, overall. Displace- growth provides increased protein and has ments have and will continue to occur on a case- made its production less expensive. The by-case basis. genetic alteration was accomplished by transferring a gene from Escherichia coli to M. methylotrophus. ● Carbohydrates. —Many carbohydrate sub- Table 23.-Raw Materials Already Tested on a strates—from starch and cellulose to beets Laboratory or Small Plant Scale and papermill wastes—have been investi- Agave juices Pulpmill wastes gated. Forests are the most abundant Barley straw Sawdust source of carbohydrate in the form of cel- Cassava Sunflower seed husks Citrus wastes (treated) lulose. But before it can be used by micro- Date carbohydrates Wastes from chemical organisms, it must be transformed into the Meatpacking wastes production of maleic carbohydrate, glucose, by chemical or en- Mesquite wood anhydride zymatic pretreatment. Many of the SCP Peat (treated) Waste polyethylene (treated) processes that use cellulose employ orga- SOURCE: Office of Technology Assessment. 110 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Genetics in baking, brewing, and winemaking

The micro-organism of greatest significance Figure 26.—The Use of Hybridization To Obtain in the baking, brewing, and winemaking indus- a Yeast Strain for the Production of tries is common yeast. Because of its impor- Low-Carbohydrate Beer tance, yeast was one of the first micro-organisms to be used in genetic research. Neverthe- less, the surge in studies in yeast genetics has not been accompanied by an increase in its practical application, for three reasons: ● industries already have the desired efficient strains, mainly as a result of trial-and- error studies; ● new genetic strains are not easily bred; they are incompatible for mating and the genetic characteristics are poorly understood; and ● many of the important characteristics of industrial microbes are complex; several genes being responsible for each. Changing technologies in the brewing industry and increased sophistication in the molec- SOURCE: Office of Technology Assessment. ular genetics of yeast have made it possible for researchers to achieve novel goals in yeast breeding. one strain that has already been con- ried out with wine yeasts. Interestingly, within structed can produce a low-carbohydraate beer the past 10 years, scientists have isolated in- suitable for diabetics. (See figure 26. ) duced mutants of wine yeasts that have: I) an increased alcohol tolerance and the capacity to The baking industry is also undergoing tech- completely ferment grape extracts of unusually nological revolution, and yeasts with new prop- high sugar content; 2) improved sedimentation erties are now needed for the faster fermenta- properties, improving or facilitating separation tion of dough. New strains with improved bio- of yeasts from the wine; and 3) improved per- logical activity, storage stability, and yield formance in the production of certain types of would allow improvements in the baking proc- wines. Hybridization studies of wine yeasts ess. have been actively pursued only recently. In the past, most genetic applications have Progress in developing strains of yeast with come in the formation of hybrid yeasts. The novel properties is limited by the lack of enough newel* genetic approaches, which use cell fu- suitable approved systems for using recombi- sion now open up the possibility of hybrids de- nant DNA (rDNA) technology. Eventual approv- veloped from strains of yeast that carry useful al by the Recombinant DNA Advisory Commit- genes but cannot mate normally, tee is expected to boost applied research for the Classical genetic research has also been car- brewing, baking, and winemaking industries. Ch. 6—The Food Processing Industry ● 177

Microbial polysaccharides

The food processing industry uses polysac- A wide variety of polysaccharides could theo- charides (polymeric sugars) to alter or control retically be produced for use in food processing, the physical properties of foods. Many are in- Applied genetics may increase their production, corporated into foods as thickeners, gelling modify those that are produced, eliminate the agents, and agents to control ice crystal forma- degradative enzymes that break them down, or tion in frozen foods. They are used in instant change the microbes that produce them. How- foods, salad dressings, sauces, whips, toppings, ever, as with other microbial processes, the ap- processed cheeses, and dairy products. New plication of genetics depends on an understand- uses are constantly appearing. The annual mar- ing of both the biochemical pathway for synthe- ket in the United States is reported to be over sis of a given polysaccharide and the systems 36,000 tons, not including starches and deriva- that control microbial production. For many mi- tives of cellulose. crobial polysaccharides, this information does not yet exist; furthermore, little is known about Since many of the polymeric sugars now used the enzymes that may be used to modify poly - in food processing are derived from plant saccharides to more useful forms. Progress will sources, microbial polysaccharides have had only be able to occur when these information limited use. To compete economically, a micro- gaps are filled. bial polysaccharide must offer new properties, meet all safety requirements, and be readily Iization Research and Oeveiopment Dii’ision ol LISL)A’S large nli- crohia! culture co]]ection. Xanthiin gunl produced h+v Xanlhomo- available. Very few have reached the level of nas campestris NRRI, B-1459 was found to ha\re chiiracterist ics t hilt commercial applications; the only one in large- rendered it very promising as a commercial product. in 1960, the scale commercial production is xanthan gum. * Kelco division of Merck &. (:0., inc., carried out pilot Plii[lt feasihili - t,v studies, and substantial commercial production t)e~iin in 1964. Although much Of the work to diit~ hiis I}(+pll (’ii].ri[d OLII wiih ● The history of the development of xanthan gum indicates that polvsaccharides from one Pitrti[;uliir stritin, t here is increilsing e\i- the commercially significant organisms resulted from an extensive dence to suggest thilt ihey could iilso he produced from other screening program for gum producers stored in the Northern Uti- S1 rains.

Enzymes

Enzymes are produced for industrial, med- ucts; it also shows that it is easier to discover a ical, and laboratory use both by fermentation new enzyme than to create a profitable applica- processes that employ bacteria, molds, and tion for it. * yeasts and by extraction from natural tissues. Most industrial enzymes are used in the de- The present world market for industrial en- tergent industry and the food processing in- zymes is estimated to be $l50 million to $174 million; the technical (laboratory) market adds ● The enzyme literature is extensive i~tld comprises well over 10,00( papers per year. Although less thitll 5(1 percent of these another $20 million to $40 million. Fewer than publications are concerned with microhial enzymes iind mos[ are so microbial enzymes are of industrial impor- found to have no industrial interest, iI few Ihousand papers per tance today, but patents have been granted for year are of potential interest for the industriill development of enzymes. Less than 100 papers dealing with industrial processes ap- more than a thousand. This reflects the increas- pear every year, and few desct~be processes of great economic sig- ing interest in developing new enzyme prod- nificance. 112 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals dustry, particularly for starch processing. En- plastic-like wraps may provide its largest indus- zymes began to be used in quantity only 20 trial market, although that market has not yet years ago. In the early 1960’s, glucoamylase en- been developed. zyme treatment began to replace traditional [f applications for the products made by acid treatment in processing starch; around pullulanase can be developed, genetic engineer- 1965, a stable protease (an enzyme) was ining can be used to insert this enzyme into introduced into detergent preparations to help dustrially useful organisms and to increase its break down certain stains; and in the 1970’s, production. However, the food processing in- glucose isomerase was used to convert glucose dustry is permitted to use only enzymes that are to fructose, practically creating the high-fruc- obtained from sources approved for food use. tose corn syrup industry. Since the chief source of pullulanase is a pathogenic bacterium, KlebsiellaII aerogenes, no signifi- Genetic engineering and enzymes in cant efforts have been made to apply genetics to the food processing industry improve its production or quauality. Molecular genetics could ultimately transfer the pullula- Biotechnology applied to fermentation proc- nase trait from K. aerogenes to a micro-organism esses will make available larger quantities of ex- approved for food use, if approved micro-orga- isting enzymes as well as new ones. (See ch. 5.) nisms that manufacture pullulanase cannot be The role of genetic engineering in opening com- found. mercial possibilities in the food processing industry is illustrated by the enzyme, pullulanase. Sweeteners, flavors, and fragrances This enzyme degrades pullulan, a polysaccharide, to the maltose or high-maltose syrups that Biotechnology has already had a marked im- give jams and jellies improved color and bril- pact on the sweetener industry. The availability liance. They reduce off-color development proof the enzymes glucose isomerase, invertase, duced by heat in candies and prevent sandiness and amylase has made the production of high- in ice cream by inhibiting sugar crystallization. fructose corn sweeteners (HFCS) profitable. Pro- Mahose has several unique and favorable char- duction of HFCS in the United States has in- acteristics. It is the least water-absorbent of the creased from virtually nothing in 1970 to 10 maltose sugars and, although it is not as sweet percent of the entire production of caloric as glucose, it has a more acceptable taste. It is sweeteners in 1980 (11 lb per capita). The price also fermentable, nonviscous, and easily solu- advantage of HFCS is expected to cause its con- ble. It does not readily crystallize and gives de- tinued growth, particularly in the beverage in- sirable browning reactions. dustry. In fact, the Coca Cola Co. announced in 1980 Pullulanase can also break down another car- that fructose will soon constitute as much as 50 percent of the sweetener used in its name bohydrate, amylopectin, to produce high amy - lose starches. These starches are used in indus- brand beverage. try as quick-setting, structurally stable gels, as Biotechnology can be used to produce other binders for strong transparent films, and as sweeteners as well. While it is unlikely that su- coatings. Their acetate derivatives are added to crose will ever be made by micro-organisms (al- textile finishes, sizing, adhesives, and binders. though improvements in sugarcane and sugar In food, amylose starches thicken and give tex- beet yields may result from agricultural genetic ture to gumdrop candies and sauces, reduce fat studies, see ch. 8.), the microbial production of and grease in fried foods, and stabilize the pro- low-caloric sweeteners is a distinct possibility. tein, nutrients, colors, and flavors in reconsti- Three new experimental sweeteners—aspar- tuted products like meat analogs. tame, monellin, and thaumatin—are candidates. In view of the current shortages of petro- Aspartame is synthesized chemically from leum-derived plastics and the need for a biode- the amino acids, aspartic acid and phenylala- gradable replacement, amylose’s ability to form nine, which can themselves be made by fermen- Ch. 6— The Food Processing Industry ● 113

tation. The possibility of using microbes to COU- Other flavors and fragrances show less prom- ple the two amino acids is being investigated in ise at present. Although the chemistry of sev- at least one biotechnology research firm. Chem- eral flavors and aromas has been identified, too ical production of aspartame is expensive and little research into their use has been con- benefits from biotechnology are possible. ducted. * Monellin and thaumatin are natural substances—proteins obtained from West African plants. Both are intensely sweet–up to 100,000 times sweeter than table sugar—and the sensation of sweetness can last for hours. Their ● KW’elll ttr(}l’k 011 1116? 101’lllilliOll 1)}1 Illi(’1’()-{)l’gilllisllls ot” tliii’01’ microbial production may be competitive with illld ill’olllil (’h~llli(’ill S hllol$’11 iiS lil(’lollt>S iill(l terpfmoids tlil S I)(WI1 their extraction from plants. Since the physical I’(?p{)l’td. [.il[Ytoll~S 0(:(:111” ilS !]iil’ol’-(’oll~ l’lt)lltlll~” (’Olllpollf?lltS ill and biological properties of thaumatin are IIUII1.V l’~1’il)t?tltiitiotl produ(’ts, \\’h[’l’(> III(’}I ii[’(~ lk)IvN(d I)v microhial reiictions. [)it’t’erent ])i~th~iii.vs exist !(N- t heir n~icrohial i“ornlation. known, it might also be produced through ge- k;.g., galllllla-t)llty l’ola{’toll[” , mfhich is I“ol’llled during J~CilSt ternlen- netic engineering. Such an approach would not tittion, is 1(}uIM1 in sherry, wine, iind hf?er. /IS eiIt.1.v iis Ig3(J, an ot’- only increase the available supply, but would giinism Wiis isolated f]xNll orange leiii,t?s that hii{j a pea(+-lik(+ ~d(]r iimi Wiis thougll[ to he Sporobdonl.vces resew. The kwtones, ZI- offer new molecules for investigating the physi- decmolide ii[ld (:is-6-docle(’ell-4-olici(’” Mere found to he responsi- ology of taste. I)le.

Overview

The application of genetic engineering will af- duce the amount of required testing by fect the food processing industry in piecemeal transferring the desired gene into micro- fashion. Isolated successes can be expected for organisms that already meet FDA stand- certain food additives, such as aspartame (not ards. yet approved by the Food and Drug Administra- Nevertheless, the application of new genetic tion (FDA) for sale in the United States) and fruc- technologies will probably accelerate. Techno- tose, and for improvements in SCP production. logically sophisticated companies are being But an industrywide impact is not expected in drawn into the business. Traditionally capital- the near future because of several conflicting intensive companies such as Union Carbide, forces: ITT, General Electric, Corning Glass, and ● The basic genetic knowledge of character- McDonnell-Douglas can be expected to intro- istics that could improve food has not been duce automation and more sophisticated engi- adequately developed. neering to food processing, modernizing the in- ● The food processing industry is conserva- dustry’s technology. As has been noted by one tive in its research and development ex- industry observer: 1 penditures for improved processes, gener- You don’t work on a better way to preserve ally allocating less than half as much as fish. You try to change the system so that you more technologically sophisticated indus- no longer catch fish; you “manufacture” them tries. and, if possible, do it right on top of your mar- ● Products made by new microbial sources ket so that you don’t have to preserve them at must satisfy FDA safety regulations, which all. include undergoing tests to prove lack of harmful effects. * It may be possible to re-

“ fi;.g., all food additites and nli(’1.()-{)1.gar)isr~ls used in food proc- IM. L. Kastens, “The Coming Food Industry,” Chemtech, April msing must he approtwd its geIWriill&V regarded iIs sate. 1980, pp. 215-217. 114 ● Impacts of Applied Genetics-Micro-Organisms, Plants, and Animals

You don’t worry about processing bacon Genetic engineering can be expected to aid in without nitrites, you engineer a synthetic bacon the creation of novel food preparations through with designed-in shelf life. effects on both the food itself and the additives You don’t try to educate people to eat a “bal- used for texturizing, flavoring, and preserving. anced diet;” you create a “whole” food with the proper balance of nutrients and supplements, and you make it taste like something people already like to eat. Chapter 7 The Use of Genetically Engineered Micro-Organisms in the Environment Chapter 7

Page Page Mineral Leaching and Recovery ...... , . . 117 Pollution Control...... 123 Microbial Leaching ...... 117 Enhancing Existing Microbial Degradation Applied Genetics in Strain Improvement . . , . . 118 Activity...... 1.24 Metal Recovery . . . . , ...... 118 Adding Microbes to Clean Up Pollution ...... 124 Oil Recovery ...... 119 Commercial Applications—Market Size and Enhanced Oil Recovery ...... 119 Prospects . , . . . . . , ...... 125 Microbial Production of Chemicals Used Genetic Research in Pollution Control ...... 126 in EOR . . . . , ...... , . . . . . , . , , 120 Federal Research Support for Engineering In Situ Use of Micro-Organisms. . . , ...... 121 Microbes to Detoxify Hazardous Substances . 127 EOR and Genetic Engineering ...... 122 Summary...... , . . . . , ...... 127 Constraints to Applying Genetic Issue and Options—Biotechnology, ...... 128 Engineering Technologies in EOR...... 122 Genetic Engineering of Micro-Organisms for Use in other Aspects of Oil Recovery Figure and Treatment...... 123 Overview of Genetic Engineering in Mining Figure No. Page and Oil Recovery ...... , ...... 123 27. chemical Flooding Process ...... 120 Chapter 7 The Use of Genetically Engineered Micro-Organisms in the Environment

Although most genetically engineered micro- ● less control over the behavior and fate of organisms are being designed for contained fa- the micro-organisms; cilities like fermenters, some are being exam- ● a possibility of ecological disruption; and ined for their usefulness in the open environ- ● less basic research and development (R&D) ment for such purposes as mineral leaching and —and a higher degree of speculation—than recovery, oil recovery, and pollution control. the industries previously discussed. All three applications are characterized by:

● the use of large volumes of micro-organisms;

Mineral leaching and recovery

All micro-organisms interact with metals, be leached by one of two general mechanisms: Two interactions that are of potential economic either the bacteria act directly on the ore to ex- and industrial interest are leaching metals from tract the metal or they produce substances, their ores, and concentrating metals from such as ferric iron and sulfuric acid, which then wastes or dilute mixtures. The first would allow extract the metal. It appears that simply adding the extraction of metals from large quantities of acid is not as efficient as using live bacteria. low-grade ores; the second would provide meth- Although acid certainly plays a role in metal ex- ods for recycling precious metals and control- traction, it is possible that direct bacterial attack ling pollution caused by toxic metals. on some ores is also involved. In fact, some of the bacteria that are known to be involved in Microbial leaching mineral leaching have been shown to bind tena- ciously to those minerals. In microbial or bacterial leaching, metals in ores are made soluble by bacterial action. Even The application of the leaching process to before bacterial leaching systems became ac- uranium mining is of particular interest be- cepted industrial practice, it was known that cause of the possibility of in situ mining. Instead dissolved metals could be recovered from mine of using conventional techniques to haul urani- and coal wastes. Active mining operations cur- um ore to the surface, microbial suspensions rently based on this process (such as those in can extract the metal from its geological setting. Rio Tinto, Spain) date back to the 18th century. Water is percolated through underground Presently, large-scale operations in the United shafts where the bacteria dissolve the metals. States use bacterial leaching to recover copper The solution is then pumped to the surface “rem waste material. Estimates for the contri- where the metal is recovered. This approach, bution of copper leaching to the total annual also called “underground solution mining, ” is mines, J.S. production range from 11.5 to 15 percent. already used in Canadian uranium where it began almost by chance. In 1960, after Leaching begins with the circulation of water only 2 years of operation, researchers at the through large quantities–often hundreds of Stanrock Uranium Mine found that the natural ins—of ore. Bacteria, which are naturally asso- underground water contained large amounts of iated with the rocks, then cause the metals to leached uranium. In 1962, over 13,000 kilo-

117

76-565 0 - 81 - 9 118 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals grams (kg) of uranium oxide were obtained more information is slow because less than a from the water. Thereafter, water was circu- dozen laboratories in the Nation are actively lated through the mines as part of the mining performing research. operation. It has been suggested that extending But even when the scientific knowledge is this practice to most mines would have signifi- gathered, two obstacles to the use of genetically cant environmental benefits because of the engineered micro-organisms will remain. The minimal disruption of the land surface. first is the need to develop engineered systems Although the process is slower than the technol- on a scale large enough to exploit their biologi- ogy currently employed, the operating costs cal activities. A constant interchange must take might be lower because of the simplicity of the place between microbial geneticists, geologists, system, since no grinding machinery is needed. chemists, and engineers. E.g., the geneticists Furthermore, deeper and lower grade deposits must understand the needs identified by the could be mined more readily. geologists as well as the problems faced by the Bacterial leaching can also extract sulfur-con- engineers, who must scale-up laboratory-scale taining compounds, such as pyrite, from coal, processes. The complex nature of the problem producing coal with a lower sulfur content. can be approached most successfully by an Sulfur-containing coals from such areas as Ohio interdisciplinary group that recognizes the and the Appalachian Mountains are now less de- needs and limitations of each discipline. sirable than other coals because of the sulfur The second obstacle is environmental. In- dioxide they release during burning. They often troducing large numbers of genetically engi- contain up to 6 percent sulfur, of which 70 per- neered micro-organisms into the environment cent can be in the form of pyrite. According to raises questions of possible ecological disrup- recent data, mixed populations of different bac- tion, and liability if damage occurs to the envi- teria, rather than a single species, are respon- ronment or human health. sible for the most effective removal of sulfur—a finding that may lead to the genetic engineering In summary, the present lack of sufficient of a single sulfur-removing bacterium in the scientific knowledge, scientists, and interdis- future. ciplinary teams, and the concerns for ecological safety present the major obstacles to the use of Applied genetics in strain improvement genetic engineering in microbial leaching.

The bacterium most studied for its leaching Metal recovery properties has been Thiobacillus ferrooxidians (which leaches copper), but others have also The use of micro-organisms to concentrate been identified in natural leaching systems. metals from dilute solutions such as individual Although leaching ability is probably under waste streams has two goals: to recover metals genetic control in these organisms, practically as part of a recycling process; and to eliminate nothing is known about the precise mecha- any metal that may be a pollutant. The process nisms. This is largely because little information makes use of the ability of micro-organisms to exists in two critical areas: the chemistry of in- bind metals to their surfaces and then concen- teraction between the bacteria and rock sur- trate them internally. faces; and the genetic structure of the micro- organisms. The finding that mixed populations Studies at the Oak Ridge National Laboratory of bacteria interact to increase leaching efficien- in Tennessee have shown that micro-organisms cy complicates the investigation. can be used to remove heavy metals from industrial effluents. Metals such as cobalt, nickel, Because of the lack of genetic and biochemi- silver, gold, uranium, and plutonium in concen cal information about these bacteria, the appli- trations of less than 1 part per million (ppm) car. cation of genetic technologies to mineral leach- be recovered. The process is particularly useful ing remains speculative. Progress in obtaining for recovering metals from dilute solutions 01 Ch. 7—The Use of Genetically Engineered Micro-Organisms in the Environment ● 119

10 to 100 ppm, where nonbiological methods As with other biological systems, genetic may be uneconomical. Organisms such as the engineering may increase the efficiency of the common yeast Saccharomyces cerevisiae can ac- extraction process. In the Saccharomyces sys- cumulate uranium up to 20 percent of their tern, differences in the ability to recover the total weight.— metals have been demonstrated within populations of cells. Selection for cells with the genetic The economic competitiveness of biological ability to accumulate large amounts of specific, methods has not yet been proven, but genetic desired metals would be an important step in improvements have been attempted only re- designing a practical system. cently. The cost of producing the micro-organisms has been a major consideration. If it can be reduced, however, the approach might be useful.

Oil recovery

Since 1970, oil production in the United physical association of the trapped oil and the States has declined steadily. The supply can be surrounding geological formations varies signif- increased by: accelerating explorations for new icantly from site to site. The unknown charac- oilfields; by mining oil shale and coal and con- teristics of these variations are largely respon- verting them to liquids; and by developing new sible for the economic risk in an attempted EOR. methods for recovering oil from existing reservoirs. Enhanced oil recovery In primary methods of oil recovery, natural Of the original estimated volume of more expulsive forces (such as physical expansion) than 450 billion barrels (bbl) of U.S. oil reserves, drive the oil out of the formation. In secondary about 120 billion bbl have been recovered by methods of recovery, a fluid such as water or primary and secondary techniques, and another natural gas is injected into the reservoir to force 30 billion bbl are still accessible by these the oil to the well. Approximately 50 percent of methods. The remaining 300 billion bbl how- domestic crude in recent years has been ob- ever, are probably recoverable only by EOR tained through secondary recovery. methods. These figures include the oil remain- Recently, new methods of oil recovery have ing in known sandstone and limestone reser- been added to primary and secondary methods, voirs and exclude tar sands and oil shale. which are called tertiary, improved, or en- Four EOR processes are currently used. All hanced oil recovery (EOR) techniques. They em- are designed to dislodge the crude oil from its ploy chemical and physical methods that in- natural geological setting: it crease the mobility of oil, making easier for ● other forces to drive it out of the ground. The In thermal processes, the oil reservoir is major target for EOR is the oil found in sand- heated, which causes the viscosity of the oil stone and limestone formations. It is here that to decrease, and with the aid of the applied genetics may play a major role, pressure of the air introduced, supports engineering micro-organisms to aid in recovery. the combustion that forces the petroleum to the producing well. Thermal processes Oil susceptible to these processes is localized will not be improved by genetic technol- in reservoirs and pools at depths ranging from ogies. 100 ft to more than 17,000 ft. In these areas, the ● Various crude oils differ in their viscosity— oil is adsorbed on grains of rock, almost always ability to flow. Primary and secondary accompanied by water and natural gas. The methods can easily remove those that flow 120 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

as readily as water, but many of the reser- Figure 27.-Chemical Flooding Process voirs contain oil as viscous as road tar. Miscible processes use injected chemicals injection fluids Oil and water that blend with the crude oil to form mixtures that flow more readily. The chemi- 4 cals used include alcohols, carbon dioxide, petroleum hydrocarbons such as propane and butane-propane mixtures, and petroleum gases. A fluid such as water is generally used to push a “slug” of these chemicals through the reservoir to mix with the

crude oil and move it to the surface. Legend II ● Chemicals are also used in alkaline flooding, polymer flooding, and combined surfactant/polymer flooding. zone SOURCE: Office of Technology Assessment, Enhanced Oil Recovery Potential In alkaline flooding, sodium hydroxide, sodi- in the United States (Washington, D. C.: U.S. Government Printing Of- um carbonate, or other alkaline materials are fice, January 1978). used to enhance the flow of oil. Neither natural nor genetically engineered micro-organisms are found in the early 1960’s and have since been considered useful in this process. responsible for the recovery of more than 2 Polymer flooding is a recent apparently suc- million bbl. Polymers such as polyacrylamide cessful method of recovery. It depends on the and xanthan gum can increase the viscosity of ability of certain chains of long molecules, water in concentrations as low as one part in a known as polymers, to increase the viscosity of thousand. Xanthan gum is readily made in large water. Instead of altering the characteristics of quantities by micro-organisms. Different strains the crude oil, the aim is to make the injected of Enterobacler aerogenes produce a wide varie- water more capable of displacing it. ty of other polymers. A useful biopolymer—one formed by a biological process—might be de- In the combined surfactant/polymer flooding signed specifically to improve oil recovery. technique, a detergent-like material (surfactant) is used to loosen the oil from its surrounding Xanthan gum, produced by Xanthomonas rock, while water that contains a polymer to in- campestris and currently marketed by the Kelco crease its viscosity is used to drive the oil from division of Merck & Co., Inc., is useful but far the reservoir. (See figure 27.) from ideal for oil recovery. While it has excellent viscous properties, it is also very expen- ● Other EOR methods include many novel sive. Furthermore, unless it is exceptionally possibilities, such as the injection of live pure, it can plug reservoir pores, since the fluid micro-organisms into a reservoir. These often has to travel through hundreds of meters may produce any of the chemicals used in of fine pores. To avoid such plugging, the fluid miscible and chemical processes, from sur- must be filtered to remove bacterial debris be- factants and polymers to carbon dioxide. fore it is injected. One target for EOR is the half million strip- per wells (producing less than 10 barrels Nevertheless, micro-organisms can be se- per day (bbl/d) in the United States. lected or genetically engineered to overcome many obvious difficulties. * With improved MICROBIAL PRODUCTION OF CHEMICALS properties, polysaccharides (polymeric sugars) USED IN EOR ● A good organism, for example, might have the following EOR methods that use chemicals tend to be desired properties: nonpathogenic to humans, plants, or animals; rapid growth on simple, cheap raw materials; ease of separation expensive because of the cost of the chemicals. from its products; limited detrimental effect on reservoirs, e.g., Nevertheless, potentially useful polymers were plugging; easy disposal of cells, e.g., byproduct credits; ability to Ch. 7—The Use of Genetically Engineered Micro-Organisms in the Environment . 121 obtained by microbial fermentation could com- IN SITU USE OF MICRO-ORGANISMS pete with those obtained from alternative One alternative to growing micro-organisms sources, especially seaweed. Controlled fermen- in large fermenters then extracting their chem- tation is not affected by marine pollution and ical products and injecting them into wells, is to weather, and production could be geared to inject the micro-organisms directly into the market demand. wells. They could then produce their chemicals in situ. Biological processes have disadvantages primarily in the costs of appropriate raw materials Unfortunately, the geophysical and geochem- and in the need for large quantities of solvent. ical conditions in a reservoir seldom favor the Current efforts to find cheaper raw materials, growth of micro-organisms. High temperature, such as sugar beet pulp and starch, show prom- the presence of sulfur and salt, low oxygen and ise. The need for solvents to precipitate and con- water, extremes of pH, and significant engi- centrate the polymers before shipment from neering hurdles make it difficult to overcome plant to field can be circumvented by producing these limitations. The micro-organisms must be them onsite. fed and the microenvironment must be carefully adjusted to their needs at distances of hun- Micro-organisms can also produce substances dreds to thousands of feet. The oil industry has like butyl and propyl alcohols that can be used already had discouraging experiences with as cosurfactants in EOR. It has been calculated micro-organisms in the past. In the late 1940’s, that if n-butanol were used to produce crude oil for instance, the injection of sulfite-reducing at a level of 5 percent of U.S. consumption, 2 micro-organisms, along with an inadvertently billion to 4 billion lb per year—or four to eight high-iron molasses as a carbon source, resulted times the current butanol production—would in the formation of iron sulfide, which clogged be required. Micro-organisms capable of pro- the rock pores. One oil company developed a ducing such surfactants have been identified, yeast to break down petroleum, but the size of and genetically superior strains were isolated the yeast cells (5 to 10 micrometers, um) was several decades ago at the Northern Regional enough to clog the 1-um pores. Research Laboratories in Illinois. Other chemicals, such as alcohols that increase the rate of Nevertheless, information from geomicrobi- formation and stability of chemical/crude oil ology suggests that this approach is worth pur- mixtures and the agents that help prevent pre- suing. Preliminary field tests have also been en- cipitation of the surfactants, have also been pro- couraging. The injection of 1 to 10 gal of Bacillus duced by microbial systems. or Clostridium species, along with a water- The uncertainties of the technical and eco- suspended mixture of fermentable raw materi- nomic parameters are compounded by the lack als such as cattle feed molasses and mineral nutrients, has resulted in copious amounts of of sufficient field experiments. Laboratory tests cannot be equated with conditions in actual oil carbon dioxide, methane, and some nitrogen in reservoirs. The carbon dioxide made the crude wells. Each oil field has its own set of character- less viscous, and the other gases helped to istics—salinity, pH (acidity and alkalinity), repressurize the reservoir. In addition, large temperature, porosity of the rock, and of the crude oil itself—and an injected chemical be- amounts of organic acids formed additional car- haves differently in each setting. In most cases, bon dioxide through reactions with carbonate minerals. The production of microbial sur- not enough is known about a well’s characteris- factants further aided the process. tics to predict the nature of the chemical/crude oil interaction and to forecast the efficiency of Although previous assessments have argued oil recovery. that reservoir pressure is a significant hin- drance to the growth of micro-organisms, more use water available at site; growth under conditions that discour- recent studies indicate the contrary. The micro- age the growth of unwanted micro-organisms; no major problems organisms must, however, be selected for in- in culturing the bacterium; and genetic stability. creased salt and pH tolerance. 122 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

EOR AND GENETIC ENGINEERING this problem is the lack of sufficient physical, The current research approach, funded by chemical, and biological information about the the Department of Energy (DOE) and, independ- reservoirs, without which it is difficult to see ently, by various oil companies, is a two-phase how a rational genetic scheme can be con- process. The first phase is to find a micro- structed for strains. Clearly, the activities of organism that can function in an oil reservoir micro-organisms under specified field condi- environment with as many of the necessary tions cannot be studied unless researchers characteristics as possible. The second is to alter know what the appropriate conditions are. it genetically to enhance its overall capability. Three institutional obstacles exist. First, publi- The genetic alteration of micro-organisms to cation in this field is limited because most reproduce chemicals used in EOR has been more search is carried out in the commercial world successful than the alteration of those that may and remains largely confidential. Second, nei- be used in situ. * However, recombinant DNA ther the private nor the public sector has been (rDNA) technology has not been applied in ei- enthusiastic about the potential role of micro- ther category. All efforts have employed artifi- organisms in EOR, The biological approach has cially induced or naturally occurring mutations. only recently been given consideration as a way CONSTRAINTS TO APPLYING GENETIC to advance the state of the art of the technology, ENGINEERING TECHNOLOGIES IN EOR and most oil companies still have limited staffs The genetic data base for micro-organisms in microbiology. To date, DOE’s Division of that produce useful polysaccharides is weak. Fossil Fuel Extraction has conducted the main Few genetic studies have been done. Hence, the- Federal effort. Third, any effort to use micro- oretically plausible approaches such as transfer- organisms must be multidisciplinary in nature. ring enzyme-coding plasmids (see ch. 2) for Geologists, microbiologists (including microbial polysaccharide synthesis, cannot be seriously physiologists and geneticists), chemists, and contemplated at present. Only the crudest engineers must interact to evolve successful methods of genetic selection for desirable prop- schemes of oil recovery. Thus far, such teams erties have been used thus far. They remain the do not exist. only avenue for improvement until more is Environmental and legal concerns have also in- learned about the micro-organism’s genetic hibited progress. Microbial EOR methods usual- mechanisms. ly require significant quantities of fresh water The biochemical data base for the character- and thus may compete with municipal and agri- istics of both the micro-organisms and their cultural uses. Furthermore, the use of micro- products is also lacking. The wide potential for organisms introduces concerns for safety. All chemical reactions carried out by microbes re- strains of Xanthomonas, which produce xanthan mains to be explored. At the same time, a sys- gum polymer, are plant pathogens. Other tem must be devised to allow easy characteriza- micro-organisms with potential, such as Scleroti- tion, classification, and comparison of products um rolfii and various species of Aureobasidium derived from a variety of micro-organisms. have been associated with lung disease and wound infections, respectively. The physical data base for oil reservoirs is limited. The uniqueness of each reservoir sug- Immediate environmental and legal concerns, gests that no universal micro-organism or meth- therefore, arise from the potential risks associ- od of oil recovery will be found. Compounding ated with the release of micro-organisms into the environment. When they naturally cause ● Some of the goals have been to: improve polymer properties to enhance their commercial applicability; improve polymer produc- disease or environmental disruption, their use is tion (a major mistake has been to reject a micro-organism in the clearly limited. And when they do not, genetic initial screening because its level of production was too low); im- engineering raises the possibility that they prove culture characteristics, e.g., resistance to phage, rapid growth, ability to use cheaper raw materials; and eliminate en- might. Such concerns have reduced the private zymes that naturally degrade the polymers. sector’s enthusiasm for attempting genetic Ch. 7—The Use of Genetically Engineered Micro-Organisms in the Environment . 123

engineering. (See ch. 10 for a more detailed desirable constituents from the crude oil itself. discussion of risk.) As an indication of recent progress, three distinct microbial systems have been developed to GENETIC ENGINEERING OF MICRO-ORGANISMS help remove aromatic sulfur-containing mate- FOR USE IN OTHER ASPECTS OF OIL RECOVERY AND TREATMENT rial, a major impurity. Two other aspects of microbial physiology deserve attention: the microbial production of Overview of genetic engineering in oil muds or drill lubricants, and the treatment mining and oil recovery of oil once it has been recovered. Drilling muds The underlying technical problem with the are suspensions of clays and other materials use of genetically engineered organisms in that serve both to lubricate the drill and to either mining or oil recovery is the magnitude counterbalance the upward pressure of oil. Mi- of the effort. In both cases, large areas of land crobially produced polysaccharides have been and large volumes of materials (chemicals, flu- developed for this use. Exxon holds a patent on ids, micro-organisms) must be used. The results a formulation based on the production of xan- of testing any new micro-organism in a labora- than gum, from Xanthomonas campestris, while tory cannot automatically be extrapolated to the Pillsbury Co. has developed a polysac- large-scale applications. The change in charide (glucan) from various species of Scler- magnitude is further complicated by the lack of otium. At least two of the small genetic rigid controls. Unlike a large fermenter whose engineering firms have begun research pro- temperature, pH, and other characteristics can grams to develop biologically produced polysac- be carefully regulated, the natural environment charides with the desired lubricant qualities. cannot be controlled. Nevertheless, despite the Interest in the postrecovery microbial treat- formidable obstacles, the potential value of the ment of oil after its extraction centers around products in these areas assures continuing ef- the ability of micro-organisms to remove un- forts.

Pollution control

Life is a cycle of synthesis and degradation— beginning to use micro-organisms to deal with synthesis of complex molecules from atoms and the pollution problems presented by industrial simple molecules and degradation by bacteria toxic wastes. Chemicals in their place can be yeast, and fungi, back to simpler molecules and useful and beneficial; out of place, they can be atoms when organisms die. The degradation of polluting. complex molecules is an essential part of life. Pollution problems can be divided into two Without it, “. . . we’d be knee-deep in dino- 1 categories: those that have been present for a saurs. ” A more quantitative statement is equal- long time in the biosphere-e. g., most hydro- ly thought provoking. Livestock in the united carbons encountered in the petroleum industry States produce 1.7 billion tons of manure an- and human and animal wastes—and those that nually. Almost all of it is degraded by soil micro- owe their origin to human inventiveness-e. g., organisms. certain pesticides. Chemicals of both sorts, For a long time people have exploited micro- through mishap, poor planning, or lack of bial life forms to degrade and detoxify human knowledge at the time of their application sewage. Now, on a smaller scale, science is sometimes appear in places where they are potentially or actually hazardous to human ‘R. B. Grubbs, “Bacterial Supplementation, What It Can and Can- health or the environment. not Do. ” oral presentation to the Ninth Engineering Foundation on Environmental Engineering in the Food Processing Industry, 1979 Pollution can be controlled by microbes in (Available from Flow Laboratories, Inc., Rockville, Md.) two ways: by enhancing the growth and activity 124 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals of microbes already present at or near the site adding microbes to the pollution site. Three of the pollution problem, and by adding more firms–Flow Laboratories, Polybac Corp., and (sometimes new) microbes to the pollution site. Sybron/Biochemicals Corp.—sell microbes for The first approach does not provide an oppor- such use. Two companies select bacteria for en- tunity for applying genetics, but an example will hanced degradation activity and two mutate indicate how it functions. bacteria to the same end, but none of the three firms currently uses genetic engineering tech- Enhancing existing microbial niques. degradation activity Some “formulations” (mixtures) of bacteria Sun Oil successfully exploited indigenous are designed to degrade particular pollutants, microbes to clean up a 6,000 gal underground such as one that was used to digest the 800,000 gal of oily water that lay in the bilges of the gasoline spill that threatened the water supply of a town in Pennsylvania. z 3 First, engineers Queen Mary. After a 6-week treatment with the drilled wells to the top of the water table and formulation, the water from the bilges was judged safe for disposal into the Long Beach, used pumps to skim gasoline from the water Calif., harbor. It was discharged without caus- surface. About half the gasoline was removed in this fashion, but company calculations showed ing an oil slick or harming marine life. q Flow that dissipating the remaining gasoline would Laboratories markets its services to companies require about 100 years. To speedup the proc- with industrial pollution problems. It investi- gates the problem, develops a formulation to ess, it was decided to encourage the growth of indigenous bacteria that could degrade the degrade the pollutants, and sells it. gasoline. In addition to industrial pollution problems, Pollution-control microbes, like all organisms, Flow markets its products and services for use require a number of different elements and in sewerage systems, which collect and hold compounds for growth. If the amount of any human wastes to facilitate degradation and de- nutrient is limited, the microbe will not be able toxification. Sludge bacteria in sewerage plants to metabolize the pollutant at the fastest rate. degrade the waste, but they are not present in The cleanup depended on increasing the the lines that carry wastes to the treatment plant. As a result, greases and oils from fat dis- growth rate of the bacteria by supplying them carded through garbage disposals and from cos- with additional nutrients. In the case of the metic oils and creams coat the inside of sewer- gasoline-degrading bacteria, the gasoline al- 5 ready supplied the hydrocarbon, but the water- age lines and reduce their carrying capacity. gasoline environment was deficient in nitrogen, Cities have resisted using added microbes in phosphate, and oxygen. Those three nutrients sewerage systems. Standard textbooks simply were pumped down to the water table, bacterial state that the ideal bacteria will establish them- growth increased, and the gasoline was metabo- selves in a well-planned and well-managed sys- lized into innocuous chemicals by the bacteria. tem. The idea that “better” bacteria can be As a result, it was degraded in a single year. added to improve the plant operation is not readily accepted. Adding microbes to clean up pollution The value of adding bacteria to large sewer- Genetics may have important applications in age sytems has not been adequately tested. approaches to pollution control that depend on Because of the size of municipal systems (which already contain tons of sludge bacteria), some ‘R. L. Raymond, V. W.Jamison, J. O. Hudson, “Beneficial Stimu- have argued that adding a few additional lation of Bacterial Activity in Groundwaters Containing Petroleum Products,” AIChE symposium series 73:390404, 1976. W. W. Jamison, R. L. Raymond, J. O. Hudson, “Biodegradation of 4Anon., Environmental Science and Technology 13:1180, 1979. High-Octane Gasoline, ” Proceedings of the Third International Bio- ‘R. E. Kirkup and L. R. Nelson, “City Fights Grease ml odor degradation Symposium, J. M. Sharpley and A. M. Kaplan (eds.) problems in Sewer Systems,” Public Works Magazine, October (City????: Applied Science Publishers, 1976). 1977. Ch. 7—The Use of Genetically Engineered Micro-Organisms in the Environment . 125 pounds of bacteria is unlikely to have any effect. Corp. has sold its products to all seven Exxon Thus far, the Environmental Protection Agency biological waste treatment plants to treat chem- (EPA) has not recommended adding bacteria to ical wastes. One of its formulations has been municipal systems; however, EPA suggests that used to degrade toxic dioxins from an herbicide they might be useful in smaller installations and spill. One month’s treatment with the bacterial for specific problems in large systems. formulation reduced the orthochlorophenol concentration from 600 to 25 ppm in a 20,000 - Dry formulations are available for use in gal lagoon.9 cleaning drains and pipes in smaller installations, such as restaurants and other food proc- Sybron/Biochemical, a division of Sybron essing facilities. In restaurants, the bacteria are Corp., sells cultures of bacteria that are in- added to the drain at the end of the workday. tended to aid in the biological oxidation of in- Bacteria have been selected for their inability to dustrial wastewater; this company also lists 20 produce hydrogen sulfide, which means that different cultures for application to specific the degrading process does not produce the un- wastes. Patent number 4,199,444 was granted pleasant odors frequently encountered in the on April 22, 1980, for a process involving the digestion of oils and fats.6 use of a mutant bacterial culture to decolor waste water produced in Kraft paper process- As of November 1979, the pollution control l0 ing. Other patents are pending on a mixture of industry had few plans for the genetic manipu- two strains that degrade grease and a strain that lation of bacteria, except for the selection of degrades “nonbiodegradable” detergents. 11 naturally occurring better performers. Con- sumer resistance to “mutants” is a factor that There is disagreement about the value of add- discourages the move to microbial genetics. ing microbes to decontaminate soils or waters. Probably even more important is the high cost One point of view argues that serious spills fre- of establishing and maintaining microbial genet- quently sterilize soils, and that adding microbes ics laboratories. It has been estimated that the is necessary for any biodegradation. The other cost of carrying a single Ph. D. microbial geneti- contends that encouraging indigenous microbes cist is over $100,000 annually.7 This expense is is more likely to succeed because they are ac- quite high relative to the $2 million to $4 million climated to the spill environment. Added bac- sales of all biological pollution control com- teria have a difficult time competing with the panies in 1978.8 already-present microbial flora. In the case of marine spills, bacteria, yeast, and fungi already Resistance to the use of genetically manip- present in the water participate in degradation, ulated bacteria is not universal. Many industrial no one has been able to demonstrate the useful- wastes are oxidized to nontoxic chemicals by ness of added microbes. biological treatment in aerated lagoons. The process depends on the presence of microbes in the lagoons; over time, those that grow best on Commercial applications—market size the wastes come to dominate the microbial pop- and prospects ulations. Three companies now sell bacteria The estimated market size of pollution-con- that they claim outperform the indigenous trol biological products in 1978 was $2 million to strains found in the lagoons. E.g., the Polybac $4 million, divided among some 20 companies,

‘Anon., Wean That Sewage System With Bugs!” Environmental 9See footnote 6. Science and 7’echno/ogy 13:1 198-1199, 1979. IOL. Davis, J. E. Blair, and C, W. Randall, “Communicant ion: 7Anon., “Biotechnology DNA Research Expenditures in U.S. May Development of Color Removal Potential in Organisms Treating Reach $500 Million in 1980, With About $150-200 Million for Com- Pulp and Paper Wastewater,” J. Water Pollution Control Fed., Feb- mercial Products, ” Hill told. Drug Research Reports, “The Blue ruarv 1978, pp. 382-385. Sheet, ” May 28, 1980, p. 22. i IF. Spraher and N. Tekeocgak, “Foam Control and Degradation aAnon., Business Week, July 5, 1976, p. 280; Chemical Week of Nonionic Detergent,” Industrial Wastes, January/February 1980; 121:47, 1977; and Food Engineering 49: 138, 1977, cited in T. Gass- L. David, J. E. Blair, and C. Randall, ‘(Mixed Bacterial Cultures Leak ner, “Microorganisms for Waste Treatment, ” Microbial Technol- ‘Non-Biodegradable’ Detergent,” industrial Wastes, May/June ~gv, 2 cd., vol. II, (London: Academic Press, 1979), pp. 211-222. 1979. 126 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

and the potential market was estimated to be as taken. Two of the toxic chemicals, pentachoro- much as $200 million.12 These estimates can be phenol and hexachlorocyclopentadiene, are compared to Polybac’s own sales records. In relatively long-lived compounds and present 1976, its first year, its sales totaled $0.5 million long-term problems. A fungus and a bacterium and in 1977, $1.0 million. It expects to reach $5 that can degrade the first compound have been million in 1981. isolated,13 and Sybron/Biochemical already sells a culture specifically for pentachlorophenol To date genetically engineered strains have degradation. The third toxic compound is meth- not been applied to pollution problems. At least yl parathion. Its inclusion is more difficult to one prominent genetic engineering company understand, since it is degraded within a few has decided not to enter the pollution control days after its application as a pesticide. field, concluding that it was improbable that added microbes could compete with indigenous Efforts have been made to isolate bacteria organisms. More specifically, the possibility of that can degrade (2,4-dichlorophenoxy) acetic liability problems make the approach even less acid (2,4-D) and (2,4,5-trichlorophenoxy) acetic attractive. Pollution control requires that “new” acid (2,4,5-T), the components of Agent life forms be released into the environment, Orange.14 Strains of the bacterium Alcaligenes which is already seen as precariously balanced. paradoxus rapidly degrade 2,4-D, and the Such new forms might cause health, economic, genetic information for the degradation activity or environmental problems. The problems of has been located on a plasmid. The investigator liability that might arise from such applications who found that strain, while optimistic about are enough to deter entrepreneurs from con- the opportunities for isolating and transferring templating work in the field at this time. other resistance genes, has been unable to find An additional reason for the reluctance of a bacterium that degrades 2,4,5-T or its very some companies to engage in this activity is that toxic contaminant, 2,3,7,8-tetrachlorodibenzo- para-dioxin (TCDD or dioxin). the opportunities for making money are limited. Selling microbes, rather than their products, By far the best known research in this area is may well be a one-shot opportunity. The mi- that of Dr. Ananda M. Chakrabarty who engi- crobes, once purchased, might be propagated neered two strains of Pseudomonas, each of by the buyer. Nevertheless, at least two small which has the ability to degrade the four classes companies have announced that they are pursu- of chemicals found in oil spills. Chakrabarty ing efforts to use genetic engineering. began with four different strains of Pseudo- monas. The low-key efforts in this field might accel- None of them presented a threat to erate quickly if a significant breakthrough oc- human health, and each could degrade one of curred, To date, no “new” organism has ap- the four classes of chemicals. His research peared that will degrade previously intractable showed that the genes controlling the degrading chemicals. The effect of such a development activities were located on plasmids. Taking ad- might be enormous. vantage of the relative ease of moving such genes among bacteria, he produced two recombinant bacteria. Genetic research in pollution control Chakrabarty has presented evidence that his bacterium degrades complex petroleum mix- The Oil and Hazardous Materials Spills tures such as crude oil or “Bunker C“ oil, and he Branch of EPA currently supports research aimed at isolating organisms to degrade three ‘SN. K. Thuma, P. E. O’Neill, S. G. Brownlee, and R. S. Valentine, “Laboratory Feasibility and Pilot Plant Studies: Novel Biodegrada- specific chemical compounds. The work is being tion Processes for the Ultimate Disposal of Spilled Hazardous carried out on contract; as of November 1979, Materials, ” National Environment Research Center, U.S. Environ- no field trials of the organisms had been under- mental Protection Agency, Cincinnati, Ohio, 1978. MJ. M< pemberton, “pesticide Degrading plasmids: A Biological Answer to Environmental Pollution by Phenoxyherbicides,” Arnbio ‘zSee footnote 8. 8:202-205, 1979. Ch. 7—The Use of Genetically Engineered Micro-Organisms in the Environment ● 127 has proposed a method for using it to clean up that has already attracted some commercial oil spills. The bacteria are to be grown in the research support. Commercial firms are looking laboratory, mixed with straw, and dried. The for large-scale markets, such as sewerage sys- bacteria-coated straw can be stored until tems, or commonly occurring smaller markets, needed, then dropped from a ship or aircraft such as gasoline spills and common industrial onto oil spills. The straw absorbs the oil and the wastes. bacteria degrades it. 15 To completely cleanup a spill will probably require mechanical efforts in Whatever potential exists in identifying, addition to the biological attack. It was the pro- growing, and using naturally occurring mi- duction of one of Chakrabarty’s strains that led crobes for pollution control pales beside the op- to the Supreme Court decision on “the patenting portunities offered by engineering new ones, of life. ” (See ch. 12 for further details.) Unfortunately, the potential risks increase as well. EPA has taken a preliminary step toward The essential difference between the well- assessing the risks by soliciting studies to deter- publicized Chakrabarty approach and a less mine what environmental risks may exist from well-known one is that all the desired activities accidentally or deliberately released engineered in Chakrabarty’s approach are combined in a microbes. single organism; while in the other method, bacteria bearing single activities are mixed together to yield a desired “formulation.” In yet another approach, Sybron/Biochemical uses Summary mutation and selection to produce specialized degradation activities. It also sells mixed While some unreported efforts may be cultures for some applications. underway, genetics has apparently been little applied to pollution abatement. Nevertheless, The single-organism, multiple-enzyme system the production of “new” life forms that offer a has the advantage that every bacterium can at- significant improvement in pollution control is a tack a number of compounds. The mixed for- possibility. The constraints are questions of mulations allow the preferential proliferation of liability in the event of health, economic, or en- bacteria that feed on the most abundant chem- vironmental damage; the contention that added ical; then, as that chemical is exhausted, other organisms are not likely to be a significant im- bacteria, which flourish on the next most abun- provement; and the assumption that selling dant chemical, become dominant. The pref- microbes rather than products or processes is erential survival of only one or a few strains in a not likely to be profitable. mixed formulation might result in no bacteria being available to degrade some compounds. The factors that have discouraged develop- The multienzyme bacteria, on the other hand, ments in this area would probably become less can degrade one chemical after another, or deterring if convincing evidence were found alternatively, more than one at the same time. that microbes could remove or degrade an intractable pollutant. In the meantime, the re- Federal research support for search necessary to produce marked improve- engineering microbes to detoxify ments has been inhibited. Overcoming this in- hazardous substances hibition may require a governmental commitment to support the research, to buy the EPA currently limits its support to research microbes, and to provide for protection against aimed at selecting indigenous microbes, an area liability suits. Such a governmental role would be in keeping with its commitment to protecting lspa~ent specification I 436 573, Mav 19, 1976, Patent office, health and the environment from the toxic ef- London, England. fects of pollutants. 128 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Issue and Options—Biotechnology

ISSUE: How can the Federal Govern- Department of Commerce (DOC), and the De- ment promote advances in bio- partment of Defense (DOD). technology and genetic engineering? Congress has a long history of recognizing areas of R&D that need priority treatment in The United States is a leader in applying ge- the allocation of funds. Biotechnology has not netic engineering and biotechnology to indus- been one of these. Even though agencies like try. One reason is the long-standing commit- NSF receive congressional funding, its Alter- ment by the Federal Government to the funding native Biological Sources of Materials program of basic biological research; several decades of is one of the few applied programs that is not support for some of the most esoteric basic re- congressionally mandated. As a result, the fiscal search has unexpectedly provided the founda- year 1980 budget saw a reduction in the allocation for a highly useful technology. A second is tion of funds, from $4.1 million in 1979 to $2.9 the availability of venture capital, which has al- million. A congressionally mandated program, lowed the formation of small innovative compa- analogous to the successful NSF Earthquake nies that can build on the basic research. Hazard Mitigation program, could be written The argument for Government promotion of into law. other programs, such as the competitive grants program at USDA (or the Office biotechnology and genetic engineering is that of Basic Biological Research at DOE), are also Federal help is needed in those high priority modestly funded. areas not being developed by industry. The argument against such assistance is that Increasing the amount of money in an agen- industry will develop everything of commercial cy’s biotechnology program could bring criti- value without Federal help. cism from other programs within each agency if their levels of funding are not increased com- A look at what industry is now attempting in- mensurately. The Competitive Grants Program dicates that sufficient investment capital is at USDA has similar problems; those who are available to pursue specific manufacturing ob- most critical of it argue that it should not take jectives, such as for interferon and ethanol, but funds from traditional programs. Nevertheless, that some high-risk areas that might be of in- Congress could promote two types of programs: terest to society, such as pollution control, may those with long-range payoffs (basic research), need promotion by the Government. Other and those which industry is not willing to un- areas, such as continued basic biological re- dertake but that might be in the national in- search, might not be profitable soon enough to terest. attract industry’s investment, Specialized education and training are areas in which the Govern- B. Congress could establish a separate Institute ment has already played a major role, although of Biotechnology as a funding agency. industry has both supported university training The merits of a separate institution lie in the and conducted its own inhouse training. possibility of coordinating a wide range of efforts, all related to biotechnology. Among pres- OPTIONS: ent organizations, biotechnology and applied ge- A. Congress could allocate funds specifically for netics cut across several institutes and divisions genetic engineering and biotechnology R&D in within them. Medically oriented research falls the budget of appropriate agencies, such as primarily under the domain of the National In- the National Science Foundation (NSF), the stitutes of Health (NIH). EPA is concerned with U.S. Department of Agriculture (USDA), the the prevention of pollution; while NSF’s effort in Department of Health and Human Services biotechnology has been restricted to modest (DHHS), the Department of Energy (DOE), the support scattered through several divisions. Ch. 7—The Use of Genetically Engineered Micro-Organisms in the Environment ● 129

The creation of an organization such as the Na- October 21, 1980, President Carter signed into tional Technology Foundation (H.R. 6910) would law a bill (S. 1250) that would establish Centers represent the kind of commitment to engineer- for Industrial Technology to foster research ing, in general, that currently does not exist. links between industry and universities. They would be affiliated with a university or non- Competition for funds within other agencies profit institution. would be avoided, since funding would now occur at the level of congressional appropriations. One or more of these centers could be specifi- A separate institute, carrying the stamp of cally designated to specialize in biotechnology. Government recognition, would make it clear to In addition, training grants could be used to the public that this is a major new area with support the education of biotechnologists at the great potential. This might foster greater aca- centers or elsewhere. Currently, there is no na- demic and commercial interest in biotechnology tionwide training program to train students in and genetic engineering. this discipline. Education programs, especially for the postgraduate and graduate training of On the other hand, biotechnology and genetic engineers, could further the idea of using bioengineering cover such a broad range of disci- logical techniques to solve engineering prob- plines that a single agency would overlap the lems. mandates of existing agencies. Furthermore, the creation of yet another agency carries with it all D. Congress could use tax incentives to stimulate the disadvantages of increased bureaucracy and biotechnology. competition for funds at the agency level. The tax laws could be used to stimulate bio- c. Congress could establish research centers in technology in several ways. First, they could ex- universities to foster interdisciplinary ap- pand the supply of capital for small high-risk proaches to biotechnology. In addition, a pro- firms, which are generally considered more in- gram of training grants could be offered to novative than established firms, because of train scientists in biological engineering. their willingness to undertake the risks of innovation. Much of the pioneering work in the The successful use of biological techniques in industrial application of genetic techniques has industry depends on a multidisciplinary ap- been done by such firms. By nature, they are proach involving biochemists, geneticists, mi- speculative, high-risk investments. Second, the crobiologists, process engineers, and chemists. tax law could provide special subsidies to new Little is now being done, publicly or privately, high-technology firms, which cannot use the to develop expertise in this interdisciplinary standard investment incentives, such as the in- area. vestment tax credit, because they usually have In 1979, President Carter proposed the crea- no taxable profits for the first several years tion of generic technology centers (useful to a against which to apply the tax credit. Third, tax broad range of industries) as one way to stim- incentives could be provided for both estab- ulate innovation. The centers would conduct lished and new firms to make the investment of the kind of research that an individual company money for R&D more attractive. might not consider cost effective, but that might ultimately benefit several companies. Each cen- There are a number of ways to expand the supply of venture capital. One is to decrease the ter would be jointly funded by Government and tax rate on capital gains or the period an asset industry, with Government providing the seed must be held for it to be considered a capital money and industry carrying most of the costs gain rather than ordinary income. This change within 5 years. If the centers were established could be limited to stocks in high-technology at universities, startup costs could be mini- firms in order to focus its impact and minimize mized. revenue loss. Other options involving the stock Several congressional bills contain provisions of high-technology companies are: a tax credit for centers similar to these. For example, on to the investor who purchases the stock; defer- 130 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals ment of capital gains taxes on the sales of these lem is that the credit maybe a windfall for firms stocks if the proceeds are reinvested into simi- that would be investing in R&D anyway. Finally, larly qualifying stock; and more liberal capital the credit would subsidize R&D devoted to loss provisions. minor product changes or incremental improvements in addition to R&D directed to more fun- In addition to focusing on the supply of cap- damental breakthroughs. ital, tax policy could attempt to directly increase the profitability of potential growth companies. One of the provisions of a pending congres- Since most are not profitable for several years, sional bill (H.R. 5829) provides for a credit of 25 they cannot take full advantage of the invest- percent for incremental research expenditures ment tax credit—or even the provision for car- above those for a base period. By limiting the rying net operating losses back 3 years and for- credit to incremental expenditures, the bill ward 7 years to offset otherwise taxable profits. would create a more cost-effective credit, if Two proposals may remedy this situation. First, passed. the investment tax credit could be refundable to The final type of tax credit would be for cor- the extent it exceeded any tax liability of the porate contributions to university research. The firm. A preliminary estimate of the revenue loss Treasury Department estimated that a 25 per- for this proposal was $1 billion for 1979. Sec- cent credit for research in all fields would cost ond, new companies could be permitted to $40 million in 1980. This credit would be tar- carry net operating losses forward for 10 years. geted to more fundamental research and not to This change would give new firms the same the subsidy of short-term, incremental projects number of years over which to deduct losses as that are usually a significant part of corporate established firms. R&D budgets. The final type of tax incentive is directed at E. Congress could improve the conditions under increasing R&D expenditures. Two major pro- which U.S. companies can collaborate with posals would permit companies to take tax cred- academic scientists and make use of the tech- its on a certain percentage of their R&D ex- nology developed in universities in whole or in penses, and on contributions to universities for part at the taxpayer expense. research. Developments in genetic engineering have The R&D credit has been advocated for sev- kindled interest in this option. Nevertheless, the eral reasons. First, it would increase the after- Government’s role in fostering university-aca- tax return on R&D investments, making them demic interaction is far from accepted. Such a more attractive. Second, it would reduce the role may limit the flexibility of a cooperative ef- degree of risk on such investments; with a 10- fort. At the very least, disincentives such as pat- percent credit, the real after-tax expense of a $1 ent restrictions could be removed. million investment is $900,000. Finally, it would give firms maximum flexibility in selecting proj- The controversy has been summed up as fol- ects for investment. lows. Questions have been raised about the cost ef- At the next level of involvement, the Govern- fectiveness of the credit. For calendar year ment could identify potential partners, and fa- 1980, the Treasury Department estimated the cilitate negotiations. A more active role would cost of a lo-percent R&D credit to be $1.9 mil- involve the Government’s providing startup lion. Since R&D costs average only 10 to 20 per- funds. Finally, the Government could be a third partner, sharing costs with industry and the cent of the total cost of bringing a new product university. In this case, too large a Government or process to the market, the net reduction in role could lead to Federal intervention in activ- the cost of commercializing an invention would ities that should be the responsibility of busi- be 1 to 2 percent. Moreover, the commercial ness and industry. stage of innovation is thought to be riskier and costlier than the technical stage. Another prob- ‘Dennis Prager, G. S. omenn, Science 207: 379-384, 1980, Ch. 7—The Use of Genetically Engineered Micro-Organisms in the Environment . 131

Certainly the Government can facilitate com- revenue-producing research; and 3) distortion munication; in the health field, NIH, for in- in the direction of research and in the training stance, is an effective stimulus for contacts of graduate students. among scientists. F. Congress could mandate support for specific The possible advantages and disadvantages of research tasks, such as pollution control using university-industry interaction is illustrated by microbes. a recent case involving a plan by Harvard Uni- Investment in creating microbes to degrade versity to collaborate with a genetic engineering pollutants is slow because the potential market company. The plan had called for the establish- is thought to be small and because of the severe ment of a corporation to commercialize the liability problems that might arise from inten- results of research being done in the laboratory tional release of commercially supplied mi- of a Harvard molecular biologist, who would crobes. have been a principal in the firm. The Univer- sity would not have been involved in financing But microbes may be useful in degrading in- or managing the firm, which would also have tractable waste and pollutants. Genetic deter- been housed separately from the campus. How- minants for desired degradation activities may ever, Harvard would have derived substantial be present in naturally occurring organisms, or income if the company proved successful scientists may have to combine genes from dif- through a gift of 10 to 15 percent of the equity ferent sources into a single organism. Current and a royalty on sales. After much debate research, however, is limited to isolating orga- among the Harvard faculty and educators na- nisms from natural sources or from mutated tionwide, the administration decided not to im- cultures. More elaborate efforts, involving re- plement the plan because of concerns about combinant DNA (rDNA) techniques or other possible adverse impacts on academic values. forms of microbial genetic exchange, will require additional effort. proponents of such arrangements argue that the universities should reap some return from A decision by the Federal Government to sup- the commercialization of research conducted port research and to reduce liability concerns is by their staff. In addition, many universities are probably needed before the potential of micro- pressed for money, and joint ventures or re- bial control of pollution can be realized. Federal search funding arrangements with industry activity might depend on the results of an eval- provide an attractive source of funds for re- uation of the technical feasibility of microbial search programs, especially when Federal sup- pollution control, which could be made by port may decline. In return, industry would either an interagency task force or a special gain access to the kind of fundamental research commission. If the evaluation is negative, Con- that is the foundation for innovation and ap- gress might elect to do nothing to encourage the pears to be especially crucial in the field of ge- technology. If the evaluation is positive, Con- netic engineering, where the gap between basic gress might select from the following sub- research and product development is smaller options: than for other fields. 1. Initiate no research support nor any Fed- Opponents of these arrangements, especially eral relief from or limit on potential liabili- ones involving significant interaction as in the ty claims. This option would not foreclose Harvard plan, fear that the profit-seeking goals private commercial efforts, but it would of industry may be incompatible with academic limit them because of restricted research values. The following possible adverse impacts, funds and large liability questions. If suffi- among others, have been articulated: 1) in- ciently large markets were anticipated or crease in secrecy, to the detriment of the free found, the limitations would be overcome. exchange of ideas so important in academia; 2) 2< Initiate research support programs. Re- discrimination by the university in its hiring and search might be directed at problems promotion policies in favor of those doing the posed by particular pollutants (contract re- 132 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

search). Federal support of biological re- This option, currently the status quo, seems search is managed by several agencies, and to be favored by some industry officials. If it is this course would create few, if any, major worth doing, they argue, industry will do it. To administrative problems. a large extent, the availability of venture capital 3. Guarantee markets for particular prod- in the United States has allowed many com- ucts. In addition to patent protection, panies to pursue projects that are deemed prac- which would be of little value in the case of tical and economically important. The produc- an organism purposefully disseminated tion of interferon, insulin, ethanol, ethylene into the environment, the Government glycol, and fructose are cited as examples of could offer to buy desirable microbes. This successful applications that were motivated by public sector market might provide enough industry. incentive to research to make Federal fund- Generic research, or research that is fun- ing unnecessary, or the market incentive damentally useful to a broad range of com- and research support might be used jointly. panies, will probably not be undertaken by any 4. Fix a limit on liability and set up liability in- one company. When the payoff does not come surance, funded partly or wholly by the soon enough, the Government has traditionally Government. This option would reduce the taken the responsibility for funding the work. financial risk for entrepreneurs who ven- E.g., NIH supported 717 basic research projects ture to clean up pollutants with microbes, involving rDNA in fiscal year 1980 at a cost of Such an insurance scheme would require $91.5 million. Similarly, high-risk research with that a Federal agency (EPA, for instance) be high capital costs would be likely targets for satisfied that little risk was attendent in the Government support. use of the microbe. 5. Arrange a scheme to test micro-organisms Leaving all R&D in industry’s hands would for known and anticipated risks before still produce major commercial successes, but they are released. The Federal Government would not ensure the development of generic might have to bear these costs as part of a knowledge or the undertaking of high-risk proj- research program. ects. 6. Leave most efforts to industry and allow each Government agency to develop programs in the fields of genetic engineering and biotechnology as it sees fit. Part II Agriculture

Chapter 8—The Application of Genetics to Plants ...... , ...... 137 Chapter 9—Advances in Reproductive Biology and Their Effects on Animal Improvement . . . 167 Chapters The Application of Genetics to Plants Chapters Page Page Perspective on Plant Breeding...... 137 Summary ...... 160 The Plant Breeder’s Approach to Issues and Options—Plants ...... 161 Commercialization of New Varieties ...... 138 Technical Notes ...... 162 Major Constraints on Crop Improvement. . . . . 139 Genetic Technologies as Breeding Tools ...... 140 Tables New Genetic Technologies for Plant Breeding .141 Table No. Page Phase I: Tissue Culture to Clone Plants ... , . 141 24. Average Yield per Acre of Major Crops in Phase H: Engineering Changes to Alter 1930 and 1975. , ...... 137 Genetic Makeup; Selecting Desired Traits. . 144 2.5. Some Plants Propagated Through Tissue Phase 111: Regenerating Whole Plants From Culture for Production or Breeding...... 143 Cells in Tissue Culture ...... 146 26. Representative List of Tissue Culture Constraints on the New Genetic Technologies . 149 Programs of Commercial Significance in Technical Constraints...... 149 the United States...... , ...... 143 Institutional Constraints . . . . , ...... , . . . . 150 27. Gene Resource Responsibilities of Federal Impacts on Generating New Varieties ...... 151 Agencies . . , ...... 155 Examples of New Genetic Approaches ...... 152 28. Estimated Economic Rates of Return From Selection of Plants for Metabolic Efficiency .. .152 Germplasm Accessions ...... 156 Nitrogen Fixation...... 152 29. Acreage and Farm Value of Major U. S. Crops Genetic Variability, Crop Vulnerability, and and Extent to Which Small Numbers of Storage of Germplasm...... 154 Varieties Dominate Crop Average ...... 157 The Amount of Genetic Erosion That Has Taken Place ...... 154 Figures The Amount of Germplasm Needed ...... 154 Figure No. Page The National Germplasm System ...... 155 28. The Process of Plant Regeneration From The Basis for Genetic Uniformity ...... 157 Single Cells in Culture ...... 147 Six Factors Affecting Adequate Management 29. A Model for Genetic Engineering of Forest of Genetic Resources...... 158 Trees...... 149 Chapter 8 The Application of Genetics to Plants

Perspective on plant breeding

As primitive people moved from hunting and 1930 and 1975 in table 24 gives a measure of the gathering to farming, they learned to identify contribution of genetics.1 broad genetic traits, selecting and sowing seeds It is impossible to determine exactly to what from plants that grew faster, produced larger degree applied genetics has directly contributed fruit, or were more resistant to pests and dis- to increases in yield, because there have been eases. Often, a single trait that appeared in one simultaneous improvements in farm manage- plant as a result of a mutation (see Tech. Note 1, ment, pest control, and cropping techniques p. 162.) was selected and bred to increase the using herbicides, irrigation, and fertilizers. Var- trait’s frequency in the total crop population. ious estimates, however, indicate that applied Mendel’s laws of trait segregation enabled genetics has accounted for as much as 50 per- breeders to predict the outcomes of hybridiza- cent of harvest increases in this century. The tion and refinements in breeding methods. (See yield superiority of new varieties has been a ma- app. II-A.) Consequently, they achieved breed- jor impetus to their adoption by farmers. Histor- ing objectives faster and with more precision, ically, the primary breeding objective has been significantly increasing production. During the to maintain and improve crop yields. Other past 80 years classical applied genetics has been responsible for: ‘C,. F. Sprague, D. E. Alexander, and J. W. Dudley, “Plant Breed- ing and Genetic Engineering: A Perspective, ” BioscienceSO (I): 17, increased yields; 1980. overcoming natural breeding barriers; increased genetic diversity for specific Table 24.—Average Yield per Acre of Major Crops uses; in 1930 and 1975 expanded geographical limits where crops Average yield per acre Percent can be grown; and 1930 1975 Unit increase improved plant quality. Wheat...... 14.2 30.6 Bushels 115 Rye ...... 12.4 22.0 Bushels 77 Since the beginning of the 20th century, plant Rice...... 46.5 101.0 Bushels 117 breeders have helped increase the productivity Corn ...... 20.5 86.2 Bushels 320 (see Tech. Note 2, p. 162.) of many important Oats ...... 32.0 48.1 Bushels Barley...... 23.8 44.0 Bushels crops for food, feed, fiber, and pharmaceuticals Grain sorghum...... 10.7 49.0 Bushels by successfully developing cultivars (cultivated Cotton ...... 157.1 453.0 Pounds 188 varieties) to fit specific environments and pro- Sugar beets ...... 11.9 19.3 Tons 62 Sugarcane ...... 15.5 37.4 Tons 141 duction practices. Some breeding objectives Tobacco...... 775.9 2,011.0 Pounds 159 have met the needs of the local farmer, while Peanuts ...... 649.9 2,565.0 Pounds 295 other genetic improvements have been applied Soybeans...... 13.4 28.4 Bushels 112 Snap beans ...... 27.9 37.0 Cwt 33 worldwide. The commercial development of Potatoes...... 61.0 251.0 Cwt 129 hybrid corn in the 1920’s and 1930’s and of Onions ...... 159.0 306.0 Cwt 92 “green revolution” wheats in the 1950’s and Tomatoes: Fresh market. . . . . 61.0 166.0 Cwt 172 1960’s are but two examples of how plant Processing...... 4.3 22.1 Tons 413 breeding has affected the supply of food avail- Hops...... 1,202.0 1,742.0 Pounds 45 able to the world market. (See Tech. Note 3, p. SOURCE: U.S. Department of Agriculture, P/ant Genet~c Resources.. Cozwewa- 162.) A comparison of average yields per acre in flon and Use (Washington, D. C.: USDA, 1979).

137 138 ● Impacts Applied Genetics—Micro-Organisms, Plants, and Animals breeding objectives are specific responses to the 6. Producing the seed for distribution to the needs of local growers, to consumer demands, farmer. and to the requirements of the food processing The responsibilities for the different breeding firms and marketing systems. phases are distributed but interactive. In the Developing new varieties does the farmer lit- United States, responsibility for crop improve- tle good unless they can be integrated profitably ment through plant breeding is shared by the into the farming system either by increasing Federal and State governments, commercial yields and the quality of crops or by keeping firms, and foundations,2 Although some specific costs down. The three major goals of crop genes have been identified for breeding pro- breeding are often interrelated. They are: grams, most improvements are due to gradual selection for favorable combinations of genes in ● to maintain or increase yields by selecting superior lines. The ability to select promising varieties for: lines is often more of an art (involving years of —pest (disease) resistance; experience and intuition) than a science. —drought resistance; —increased response to fertilizers; and The plant breeder’s approach is determined —tolerance to adverse soil conditions. for the most part by the particular biological ● to increase the value of the yield by select- characteristics of the crop being bred—e.g., the ing varieties with such traits as: breeder may choose to use a system of inbreed- —increased oil content; ing or outbreeding, or the two in combination, —improved storage qualities; as an approach to controlling and manipulating —improved milling and baking qualities; genetic variability. The choice is influenced by and whether a particular plant in question naturally —increased nutritional value, such as high- fertilizes itself or is fertilized by a neighboring er levels of proteins. plant. To a lesser degree, the breeding objec- ● to reduce production costs by selecting tives influence the choice of methods and the se- varieties that: quence of breeding procedures. —can be mechanically harvested, reducing labor requirements; Repeated cycles of self-fertilization reduce —require fewer chemical protestants or the heterozygosity in a plant, so that after nu- fertilizers; and merous generations, the breeder has homozy- -can be used with minimum tillage sys- gous, pure lines that breed true. (See Tech. Note tems, conserving fuel or labor by reduc- 4, p. 162.) Cross-fertilization, on the other hand, ing the number of cultivation operations. results in a new mixture of genes or increased genetic variability. Using these two approaches in combination produces a hybrid—several lines The plant breeder’s approach to are inbred for homozygosity and then crossed commercialization of new varieties to produce a parental line of enhanced genetic The commercialization of new varieties potential. More vigorous hybrids can be selected for further testing. The effects of hybrid strongly depends on the genetic variability that vigor vary and include earlier germination, in- can be selected and evaluated. A typical plant creased growth rate or size, and greater crop breeding system consists of six basic steps: uniformity. 1. Selecting the crop to be bred. 2. Identifying the breeding goal. A second method for exchanging or adding 3. choosing the methodological approach genes is achieved through altering the number needed to reach that goal. of chromosomes, or ploidy (see Tech. Note 5, p. 4. Exchanging genetic material by breeding. 162.), of the plant. Since chromosomes are 5. Evaluating the resulting strain under field conditions, and correcting any deficiencies ‘National Academy of Sciences, Conservation of Germplasm Re- in meeting the breeding goal. sources: An Imperative, Washington, D. C., 1978. Ch. 8—The Application of Genetics to Plants ● 139 generally inherited in sets, plants whose ploidy plant (such as resistance to pests and diseases), is increased usually gain full sets of new although some of their contributions are small chromosomes. over one-third of domesticated and difficult to assess. Favorable combinations species are polyploids.3 Generally, crop im- of genes result in plants well-adapted to par- provement due to increased ploidy corresponds ticular growing conditions and agronomic prac- to an overall enlargement in plant size; leaves tices. With thousands of genes in a single plant can be broader and thicker with larger flowers, contributing to overall fitness, the possible com- fruits, or seeds. A well-known example is the binations are almost infinite. cultivated strawberry, which has four times more chromosomes than the wild type, and is Most poor combinations of genes are elim- much fleshier. inated by selection of the best progeny; initially favorable combinations are preserved and im- Another technique, called backcrossing can proved. Literally millions of plants may be ex- improve a commercially superior variety by lift- amined each year to find particularly favorable ing one or more desirable traits from an inferior genotypes for development into new breeding one. Generally, this is accomplished by making a stocks. Increasingly sophisticated field testing series of crosses from the inferior to the superi- procedures, as well as advanced statistical anal- or plant while selecting for the desired traits in yses, are now used to evaluate the success of each successive generation. Self-fertilizing the breeding efforts. Overall yield is still the most last backcrossed generation results in some important criterion for success, although con- progeny that are homozygous for the genes be- siderable care is taken to test stress tolerance, ing transferred and that are identical with the pest and disease resistances, mechanical har- superior variety in all other respects. Single vestability, and consumer acceptability. Breed- gene resistance to plant pests and disease-causing programs with specialized goals often use ing agents has been successfully transferred rapid and accurate chemical procedures to through backcrossing. screen lines and progeny for improvements. Because the vigor of the plant depends on the Major constraints on crop interaction of many genes, it has been difficult improvement to identify individual genes of physiological significance in whole plants. As a result, many Two of the many constraints on crop breed- important genes have not been mapped in ing are related to genetics. major crop species. There is little doubt that Many important traits are determined by several breeders would select traits like photosynthetic genes. efficiency (the ability to convert light to such organic compounds as carbohydrates) or miner- The genetic bases for improvements in yield al uptake if the genes could be identified and and other characteristics are not completely manipulated in the same ways that resistance is defined, mainly because most biological traits, selected for pathogens. such as plant height, are caused by the interaction of numerous genes. Although many—per- It is uncertain how much genetic variation for im- haps thousands—of genes contribute to quan- provement exists. titative traits, much variation can be explained Although the world’s germplasm resources by a few genes that have major impact on 4 have not been completely exploited, it has the observable appearance (phenotype) —e.g., become more difficult for breeders to improve the height of some genetic dwarves in wheat many of the highly developed varieties now in can be doubled by a single gene. Many other use-e. g., height reduction in wheat has made genes contribute to the general health of the enormous contributions to its productivity, but

3 further improvement on this basis seems to be W. J. C. Lawrence, Plant Breeding [London: Edward Arnold Ltd., 5 1968). limited. A parallel condition in the potato crop 4J. N. Thompson, Jr., “Analysis of Gene Number and Develop- ‘N. ~. Jensen, “Limits to Growth in World Food Product ion,” Sci- ment in Polygenic Systems, ” Stadler Genetics Symposium 9:63. ence 201:317, 1978. 140 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

base was already somewhat narrow by the time modern potato breeding got under way. The five-fold increase in yield resulting from selection during the last 100 years of potato improvement has produced a group of varieties that are genetically similar and unlikely to respond to further selection for yield. In the long run response to selection for other characteristics is also likely to be limited. As these examples indicate, the level of genetic homogeneity of some crops may make selection for higher yields in general more difficult. Nevertheless, while the genetic basis for overall crop improvement is poorly understood, refinements in plant breeding techniques may increase the potential for greater efficiency in the transfer of genetic information for more precise selection methods, and as a new source of genetic variation. Besides these two constraints, other pressures and limitations may also affect crop pro- Photo credit: U.S. Department of Agriculture ductivity; some are biological (see Tech. Note 6, Bundles of wheat showing variance in height p. 162.), requiring technological breakthroughs, while others are related to environmental, social, and political factors. (See Tech. Note 7, p. was recognized by the National Research Coun- 162.)—e.g., it has been argued that the agri- cil’s Committee on Genetic Vulnerability of Ma- cultural rate of growth is declining: In 1976, the 6 jor Crops: U.S. Department of Agriculture (USDA) esti- If we bear in mind the fairly recent origin of mated that the total-factor productivity of U.S. modern potato varieties and that they are, for agriculture increased by 2 percent per year the most part, derived from the survivors of the from 1939 to 1960, but by only 0.9 percent from 7 late blight epidemics of the 1840’s in Europe and the period of 1960 to 1970.

North America, it seems likely that the genetic 7 11. S. Department of Agriculture, Economics, Statistics, and Co- ‘Niitbnal Academy of Sciences, Genefic Vulnerability of Major operation Services, Agricultural Productivi?,v: EKpanding the Limits, Crops, Washington, D.[I., 1972. Agriculture Information Bulletin 431, Washington, D. C., 1979.

Genetic technologies as breeding tools

The new technologies may provide potential- tion—some are capable of overcoming natural ly useful tools, but they must be used in com- barriers such as incompatibility, Yet even bination with classical plant breeding tech- though one new technology-protoplasm fusion niques to be effective. The technologies devel- —allows breeders to overcome incompatibility, oped for classical plant breeding and those of the new plant must still be selected, regener- the new genetics are not mutually exclusive, ated from single-cell culture, and evaluated they are both tools for effectively manipulating under field conditions to ensure that the genetic genetic information through methods that have change is stable and the attributes of the new been adapted from genetic recombination ob- variety meet commercial requirements. Evacua- served in nature. Plant breeders have many tion is still the most expensive and time-consum- techniques for artificially controlling pollina- ing step. Ch. 8—The Application of Genetics to Plants . 141

New genetic technologies for not be widespread. If the barriers can be over- plant breeding come, the new technologies will offer a new way to control and direct the genetic character- The recent breakthroughs in genetic engi- istics of plants. neering permit the plant breeder to bypass the various natural breeding barriers that have PHASE 1: TISSUE CULTURE TO CLONE PLANTS limited control of the transfer of genetic in- Tissue culture involves growing cells from a formation. While the new technologies do not plant in a culture or medium that will support necessarily offer the plant breeder the radical them and keep them viable. It can be started at changes that recombinant DNA (rDNA) technol- three different levels of biological organization: ogy provides the microbiologist, they will, in with plant organs (functional units such as theory, speedup and perfect the process of ge- leaves or roots);* with tissues (functioning ag- netic refinement. gregates of one type of cell, such as epidermal cells (outermost layer) in a leaf; and with single The new technologies fall into two catego- cells. Tissue cultures by themselves offer spe- ries: those involving genetic transformations cific benefits to plant breeders; just as fermenta- through cell fusion, and those involving the in- tion is crucial to microbial genetic technologies, sertion or modification of genetic information tissue culture is basic to the application of the through the cloning (exactly copying) of DNA other new genetic technologies for plants. and DNA vectors (transfer DNA). Most genetic transformations require that enzymes digest The idea of growing cells from higher plants the plant’s impermeable cell wall, a process that or animals and then regenerating entire plants leaves behind a cell without a wall, or a proto- from these laboratory-grown cells is not new. plasm. Protoplasts can fuse with each other, as However, a better scientific understanding now well as with other components of cells. In exists of what is needed to keep the plant parts theory, their ability to do this permits a wider alive. exchange of genetic information. In tissue culture, isolated single plant cells are The approach exploiting the new technol- typically induced to undergo repeated cell divi- ogies is usually. a three-phase program. sions in a broth or gel, the resulting amorphous cell clump is known as a callus. If culture condi- Phase 1. Isolated cells from a plant are estab- tions are readjusted when the callus appears, its lished in tissue culture and kept alive. cells can undergo further proliferation. As the Phase 11. Genetic changes are engineered in resulting cells differentiate (become special- those cells to alter the genetic makeup ized), they can grow into the well-organized of the plant; and desired traits are tissues and organs of a complete normal plant. selected at this stage, if possible. The callus can be further subcultured, allowing Phase 111. The regeneration of the altered single mass propagation of a desired plant. cells is initiated so that they grow into entire plants. At this time, it is not uncommon to produce as many as a thousand plants from each gram of This approach contains similarities to the genet- starting cells; 1 g of starting carrot callus rou- ic manipulation of micro-organisms. However, tinely produces 500 plants. The ultimate goal of there is one major conceptual difference. In tissue culturing is to have these plantlets placed micro-organisms, the changes made on the cel- in regular soil so that they can grow and devel- lular level are the goals of the manipulation. op into fully functional mature plants. The com- With crops, changes made on the cellular level plete cycle (from plant to cell to plant) permits are meaningless unless they can be reproduced production of plants on a far more massive in the entire plant. Therefore, unless single cells scale, and in a far shorter period, than is possi- in culture can be grown into mature plants that ble by conventional means. (See table 25 for a have the new, desired characteristics—a procedure which, at this time, has had limited success—the benefits of genetic engineering will *Also referred to as organ culture. 142 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

First stage in plant tissue culturing: inoculation of plant tissue

cies basis. However, several commercial uses of tissue culture already exist. (See table 26.) Storage of Germplasm.—Tissue culture can be used in the long-term storage of specialized germplasm, which involves freezing cells and types of shoots. The culture provides stable genetic material, reduces storage space, and decreases maintenance costs. Carrot tissues have been frozen in liquid nitrogen, thawed 2 years later, and regenerated Shows the gradual development of the plant t issue into normal plants. This technique has also on an agar medium proved successful with morning glories, syca- mores, potatoes, and carnations. Generally, the technique is most useful for plant material that is vegetatively propagated, although if it can be list of some p ants propagated through tissue generally applied it could become important for culture. ) other agriculturally important crops. Each of the four stages of the complete Production of pharmaceuticals and cycle—establishment in culture, organogenesis, Other Chemicals From Plant Cells.—Be- plantlet amplification, and reestablishment in cause plant cells in culture are similar to micro- soil—requires precise biological environments organisms in fermentation systems, they can be that have to be determined on a species-by-spe- engineered to work as “factories” to produce Ch. 8—The Application of Genetics to Plants . 143

Table 25.—Some Plants Propagated Through Tissue Culture for Production or Breeding

Agriculture and Poinsettia horticulture Weeping fig Vegetable crops Rubber plant Asparagus Flowers Beet African violet Brussels sprouts Anthruium Cauliflower Chrysanthemum Eggplant Gerbera daisy Onion Gloxinia Spinach Petunia Sweet potato Rose Tomato Orchid Fruit and nut trees Ferns Almond Australian tree fern Apple Boston fern Banana Maidenhair fern Coffee Rabbitsfoot fern Date Staghorn fern Grapefruit Sword fern Lemon Bulbs Olive Lily Orange Daylily Peach Easter lily Fruit and berries Hyacinth Blackberry Pharmaceutical Grape Atropa Pineapple Ginseng Strawberry Pyrethium Foliage Silver vase Silviculture (forestry) Begonia Douglas fir Cryptanthus Pine Dieffenbachia Quaking aspen Dracaena Redwood Fiddleleaf Rubber tree

SOURCE: Office of Technology Assessment.

Table 26.—Representative List of Tissue Culture Programs of Commercial Significance in the United States

Industry Application Economic benefits Asparagus industry...... Rapid multiplication of seed stock Improved productivity, earliness, and spear quality Chemical and pharmaceutical. Biosynthesis of chemicals Reduced production costs Propagation of medicinal plants High volumes of plants for planting Citrus industry...... Virus elimination Improved quality, high productivity Coffee industry ...... Disease resistance breeding Disease resistance Land reclamation...... Mass propagation Availability of select clones of wild species for revegetation Ornamental horticulture...... Mass propagation Reduced costs of certain species Virus elimination of certain species Introduction of new selections Increased volumes of difficult selections Pineapple industry ...... Mass propagation Improved quality in higher volumes Strawberry industry...... Mass propagation Rapid introduction of new strains

SOURCE: Office of Technology Assessment. 144 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

plant products or byproducts. In recent years, anthraquinones, which are used as dye bases, economic benefits have been achieved from the accumulate in copious amounts over several production of plant constituents through cell weeks in cultures of the mulberry, Morinda citri- culture. Among those currently produced com- folia. mercially are camptothecin (an alkaloid with PHASE II: ENGINEERING CHANGES TO ALTER antitumor and antileukemic activity), proteinase GENETIC MAKEUP; SELECTING DESIRED TRAITS inhibitors (such as heparin), and antiviral sub- The second phase of the cycle involves the stances. Flavorings, oils, other medicinal, and genetic manipulation of cells in tissue culture, insecticides will also probably be extracted from followed by the selection of desired traits, the cells. Tissue culturing, in combination with the new The vinca alkaloids—vincristine and vin- genetic tools, could allow the insertion of new blastine, for instance—are major chemothera- genetic information directly into plant cells. peutic agents in the treatment of leukemias and Several approaches to exchanging genetic infor- lymphomas. They are derived from the leaves mation through new engineering technologies of the Madagascar periwinkle (Catharanthus exist: roseus). Over 2,000 kilograms (kg) of leaves are ● culturing plant sex cells and embryos; required for the production of every gram of ● protoplasm fusion; and vinca alkaloid at a cost of about $250/g. Plant ● transfer by DNA clones and foreign vec- cells have recently been isolated from the peri- tors. winkle, immobilized, and placed in culture. This culture of cells not only continues to synthesize These are then followed by: alkaloids at high rates, but even secretes the ma- . screening for desired traits. terial directly into the culture medium instead of accumulating it within the cell, thus remov- Culturing Plant Cells and Embryos.– ing the need for extensive extraction pro- Culturing the plant’s sex cells—the egg from the cedures. ovary and the pollen from the anther (pollen- secreting organ)—can increase the efficiency of Similarly, cells from the Cowage velvetbean creating pure plant lines for breeding. Since sex are currently being cultured in Japan as a cells contain only a single set of unpaired source of L-Dopa, an important drug in the chromosomes per cell, plantlets derived from treatment of Parkinson’s disease. Cells from the them also contain only a single set. Thus, any opium poppy synthesize both the plant’s normal genetic change will become apparent in the re- alkaloids in culture and, apparently, some alka- generated plant, because a second paired gene loids that have not as yet been found in extracts cannot mask its effect. Large numbers of hap- from the whole plant. loid plants (cells contain half the normal num- Another pharmaceutical, diosgenein, is the ber of chromosomes) have been produced for major raw material for the production of corti- more than 20 species. Simple treatment with costeroids and sex steroids like the estrogens the chemical, colchicine, can usually induce and progestins used in birth control pill. The them to duplicate their genomes (haploid set of large tuberous roots of its plant source, chromosomes) —resulting in fully normal, dip- Dioscorea, are still collected for this purpose in loid plants. The only major crop that has been the jungles of Central America, but its cells have bred by this technique is the asparagus.8 been cultured in the laboratory. If the remaining technical barriers can be Other plant products, from flavorings and overcome, the technique can be used to en- oils to insecticides, industrial organic chemicals, hance the selection of elite trees and to create and sweeteners, are also beginning to be de- hybrids of important crops. Although still rived from plants in cell-cultures. Glycyrrhiza, the nonnutritive sweetener of licorice, has been ‘J. G. Torreey, “Cytodifferentiation in Cultured Cells and Tissues, ” produced in cultures of Glycyrrhiza glabra, and l-/ortScience 12(2):138, 1977. Ch. 8—The Application of Genetics to Plants . 145 primarily experimental, successful plant sex-cell tabacum was fused with a variety of a sexually cultures have been achieved for a variety of incompatible Nicotiana species. The resultant important cultivars, including rice, tobacco, hybrids were easily recognized by their inter- wheat, barley, oats, sorghum, and tomato. How- mediate light green color. They have now been ever, because the technique can lead to bizarre regenerated into adult plants, and are currently unstable chromosomal arrangements, it has had being used as a promising source of hornworm few applications. resistance in tobacco plants.

Embryo cultures have been used to germi- Transfer by DNA Clones and Foreign nate, in vitro, those embryos that might not Vectors.—Recombinant DNA technology otherwise survive because of basic incompatibil- makes possible the selection and production of ities, especially when plants from different more copies (amplification) of specific DNA genera are crossed. Embryos may function as segments. Several basic approaches exist. In the starting material in tissue culture systems re- “shotgun” approach, the whole plant genome is quiring juvenile material. They are being used cut by one or more of the commercially avail- to speed up germination in such species as oil able restriction enzymes. The DNA to be trans- palms, which take up to 2 years to germinate ferred is then attached to a plasmid or phage, under natural conditions. which carries genetic information into the plant Protoplasm Fusion.—In protoplasm fusion, cell.—E.g., a gene coding for a protein (zein) that either two entire protoplasts are brought to- is a major component of corn seeds has been gether, or a single protoplasm is joined to cell spliced into plasmids and cloned in micro-orga- components —or organelles—from a second pro- nisms. It is hoped that the zein-gene sequence toplasm. When the components are mixed under can be modified through this approach to in- the right conditions, they fuse to form a single crease the nutritional quality of corn protein hybrid cell. The hybrids can be induced to pro- before it is reintroduced into the corn plant. liferate and to regenerate cell walls. The functional plant cell that results may often be Foreign vectors are nonplant materials (vi- cultured further and regenerated into an entire ruses and bacterial plasmids) that can be used to plant—one that contains a combination of genet- transfer DNA into higher plant cells. Trans- ic material from both starting plant cell progeni- formation through foreign vectors might im- tors. When protoplasts are induced to fuse, they prove plant varieties or, by amplifying the de- can, in theory, exchange genetic information sired DNA sequence, make it easier to recover a without the restriction of natural breeding bar- cell product from culture. In addition, methods riers. At present, protoplasm fusion still has have been discovered that eliminate the foreign many limitations, mainly due to the instability of DNA from the transformed mixture, leaving chromosome pairing. only the desired gene in the transformed plant. The most promising vector so far seems to be Organelles are small, specialized components the tumor-inducing (Ti) plasmid carried by within the cell, such as chloroplasts and mito- Agrobacterium tumefaciens. This bacterium chondria. Some organelles, called plastids, carry causes tumorous growths around the root their own autonomously replicating genes, as a result, they may hold promise for gene transfer crowns of plants. It infects one major group of plants–the dicots (such as peas and beans), so- and for carrying new genetic information into called because their germinating seeds initially protoplasts in cultures, or possibly for influenc- sprout double leaves. Its virulence is due to the ing the functions of genes in the cell nucleus. Ti plasmid, which, when it is transferred to (See Tech. Note 8, p. 163.) plant cells, induces tumors. Once inside the cell, The feasibility of protoplasm fusion has been a smaller segment of the Ti plasmid, called T- borne out in recent work with tobacco–a plant DNA, is actually incorporated into the recipient that seems particularly amenable to manipula- plant cell’s chromosomes. It is carried in this tion in culture. An albino mutant of Nicotiana form, replicating right along with the rest of the 146 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals chromosomal DNA as plant cell proliferation Screening for Desired Traits.—The bene- proceeds. Researchers have been wondering fits of any genetic alteration will be realized whether new genetic material for plant im- only if they are combined with an adequate sys- provement can be inserted into the T-DNA tem of selection to recover the desired traits. In region and carried into plant cell chromosomes some cases, selection pressures can be useful in in functional form. recovery. g The toxin from plant pathogens, for example, can help to identify disease resistance Adding foreign genetic material to the T-DNA in plants by killing those that are not resistant. region has proved successful in several ex- So far, this method has been limited to identify- periments. Furthermore, it has been found that ing toxins excreted by bacteria or fungi and one type of plant tumor cell that contains their analog; after sugarcane calluses were ex- mutagenized T-DNA can be regenerated into a posed to toxins of leaf blight, the resistant lines complete plant. This new discovery supports that survived were then used to develop new the use of the Agrobacterium system as a model commercial varieties. In theory, however, it is for the introduction of foreign genes into the possible to select for many important traits. single cells of higher plants. Tissue culture breeding for resistance to salts, Many unanswered questions remain before herbicides, high or low temperatures, drought, Agrobacterium becomes a useful vector for and new varieties that are more responsive to plant breeding. Considerable controversy exists fertilizers is currently under study. about exactly where the Ti plasmid integrates Five basic problems must be overcome before into the host plant chromosomes; some inser- any selected trait can be considered beneficial tions might disrupt plant genes required for (see figure 28): growth. In addition, these transformations may not be genetically stable in recipient plants; the trait itself must be identified; there is evidence that the progeny of Ti-plasmid- a selection scheme must be found to iden- containing plants do not retain copies of the Ti tify cells with altered properties; sequence. Finally, Agrobacterium does not read- the properties must prove to be due to ge- ily infect monocots (a second group of plants), netic changes; which limits its use for major grain crops. cells with altered properties must confer similar properties on the whole plant; and Another promising vector is the cauliflower the alteration must not adversely affect mosaic virus (CaMV). Since none of the known such commercially important characteris- plant DNA viruses has ever been found in plant tics as yield. nuclear DNA, CaMV may be used as a vector for introducing genetic information into plant While initial screens involving are easier to cytoplasm. Although studies of the structural carry out than screening tests involving entire organization, transcription, and translation of plants, tolerance at the cellular level must be the CaMV are being undertaken, information confirmed by inoculations of the mature plants available today suggests that the system needs with the actual pathogen under field conditions. further evaluation before it can be considered PHASE III: REGENERATING WHOLE PLANTS an alternative to the Agrobacterium system. FROM CELLS IN TISSUE CULTURE Although work remains to be done on Ti- New methods are being developed to: plasmid and CaMV genetic mechanisms, these ● increase the speed with which crops are systems have enormous potential. Most immedi- multiplied through mass propagation, and ately, they offer ways of examining basic mech- . create and maintain disease-free plants. anisms of differentiation and genetic regulation and of delineating the organization of the Mass Propagation.—The greatest single genome within the higher plant cell. If this can use of tissue culture systems to date has been be accomplished, the systems may provide a for mass propagation, to establish selected way of incorporating complex genetic traits into ‘J. F. Shepard, D. Bidney, and E. Shahin, “potato Protop]asts in whole plants in stable and lasting form. Crop Improvement,” Science 208:17, 1980. Ch. 8—The Application of Genetics to Plants ● 147

Figure 28.—The Process of Plant Regeneration From Single Cells in Culture

Cell multiplication Cell wall removal

Tissue

Virus-free

Field performance tests

+ Root-promoting hormones

The process of plant propagation from single ceils in culture can produce plants with selected characteristics. These selections must be tested in the field to evaluate their performance.

SOURCE: Office of Technology Assessment.

which it multiplies and the reduced costs of stock plant maintenance. A tissue culture stock of only 2 square feet (ft2) can produce 20,000 plants per month. 10 Currently, mass production of such cultivars as strawberries (see Tech. Note 9, p. 163.), asparagus, oil palms, and pineapples is being carried out through plant tissue cultures. 11 Very recently, alfalfa was propagated in the same way, giving rise to over 200,000 plants, several thousand of which are currently being tested in

Photo credit: Plant Resources Institute field trials. Also, 1,300 oil palms, selected for high yield and disease resistance, are being Multiplying shoots of jojoba plant in tissue culture on a tested in Malaysia. 12 Other crops not produced petri dish. These plants may potentiality be selected for higher oil content by this method but for which cell culture is an important source of breeding variation include culture because of the increased speed with sources of improved seed or cutting material. ‘OD. P. Holdgate, ‘(Propagation of Ornamental by Tissue Cul- (See table 26.) In some cases, producing plants ture, ” in Plant Cell, Tissue, and Organ C’ulture, .1. Feinert and Y. P. S. by other means is simply not economically com- Bajaj (eds.) (New’ York: Sprin@r-\rerla~, 1977). petitive. A classic example is the Boston fern, “T. Murashige, “Current Status of Plant Cell and organ Cul- tures, ” FfortScience )2(2):127, 1977. which, while it is easy to propagate from runner ‘z’’ The Second Green Revolution, ” special report, Business Week, tips, is commercially propagated through tissue /W~. 25, 1980. 148 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals beets, brussels sprout, cauliflower, tomatoes, uated. Thus, tissue culture production of trees citrus fruits, and bananas. Various horticultural has become an area of considerable interest. plants—such as chrysanthemums, carnations, Already, 2,500 tissue-cultured redwoods have African violets, foliage plants, and ferns—are been grown under field conditions for compari- also being produced by in vitro techniques. son with regular, sexually produced seedlings. (See app. II-B.) Loblolly pine and Douglas fir are Accelerating propagation and selection in also being cultured; the number of trees that culture is especially compelling for economical- can be grown from cells in 100 liters (]) of media ly important forest species for which traditional in 3 months are enough to reforest roughly breeding approaches take a century or more. 13 120,000 acres of land at a 12 x 12 ft spacing. To Trees that reach maturity within 5 years re- date, 3,000 tissue-cultured Douglas firs have acquire approximately 50 years to achieve a useful tually been planted in natural soil conditions. homozygous strain for further breeding. Spe- (See figure 29.) cies such as the sequoia, which do not flower until they are 15 to 20 years old, require be- 13D. J. Durzan, “Progress and Promise in Forest Genetics,” in tween 1 and 2 centuries before traits are sta- Proceedings, 50th Anniversary Symposium Paper, Science and bilized and preliminary field trials are eval- Technology. . . The Cutting Edge (Appleton, Wis: Institute of Paper Chemistry, 1980).

A plantlet of Ioblolly pine grown in Weyerhaeuser Co.’s tissue culture laboratory. The next step in this procedure is to transfer the plant let from its sterile and humid environment to the soil Ch. 8—The Application of Genetics to Plants . 149

Figure 29.—A Model for Genetic Engineering virus-free stock often appear as larger flowers, of Forest Trees more vigorous growth, and improved foliage quality. a. Today, virus-free fruit plants are maintained and distributed from both private and public repositories. Work of commercial importance has been done with such plants as strawberries, sweet potatoes, citrus, freesias, irises, rhubarbs, gooseberries, lilies, hops, gladiolus, geraniums, and chrysanthemums. 14 Over 134 virus-free potato cultures have also been developed by tissue culture.15 f. Constraints on the new genetic technologies Although genetic information has been transferred by vectors and protoplasm fusion, no DNA transformations of commercial value have yet been performed. The constraints on the successful application of molecular genetic technologies are both technical and institutional. TECHNICAL CONSTRAINTS a. Selection of genetic material from germplasm bank Molecular engineering has been impeded by a b. Insertion of selected genes into protoplasts lack of understanding about which genes would c. Regeneration of cells from protoplasts and be useful for plant breeding purposes, as well as multiplication of cell clones by insufficient knowledge about cytogenetics. d. Mass production of embryos from cells In addition, the available tools—vectors and e. Encapsulation to form ‘seeds’ mutants—and methods for transforming plant f. Field germination of ‘seeds’ cells using purified DNA are still limited. g. Forests of new trees Cells carrying traits important to crop productivity must be identified after they have SOURCE: Office of Technology Assessment. been genetically altered. Even if selection for an identified trait is successful, it must be demonstrated that cells with altered properties con- Creation and Maintenance of Disease- fer similar properties on tissues, organs, and, Free Plants.—Cultivars maintained through ultimately on the whole plant, and that the standard asexual propagation over long periods genetic change does not adversely affect yield often pick up viruses or other harmful path- or other desired characteristics. Finally, only ogens, which while they might not necessarily limited success has been achieved in regenerat- kill the plants, may cause less healthy growth. A ing whole plants from individual cells. While the plant’s true economic potential may be reached list of plant species that can be regenerated only if these pathogens are removed—a task from tissue culture has increased over the last 5 which culturing of a plant’s meristem (growing years, it includes mostly vegetables, fruit and point) and subsequent heat therapy can perform. Not all plants produced through these lqh~. NI isa~~,a, K. Sak~IO, hl. ‘1’iinaka, N1. Havashi, and H. SaIII~- methods are virus-free, so screening cells for jima, “Production of Physiologically Acti\w Substances by Plant (kll Suspension (cultures, ” H. E. SIIYXX (cd.), Tissue Culture and viruses must be done to ensure a pathogen-free Plant Science (New York: Academic Press, 1974). plant. In horticultural species, the advantages of jshlul,ashige, op. Cit.

76-565 0 - 81 - 11 150 “ Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals nut trees, flowers, and foliage crops. Some of Competitive Grants Program is shifting support the most important crops—like wheat, oats, and from ongoing USDA programs to new genetics barley–have yet to be regenerated. In addition, research programs that are not aimed at the cells that form calluses in culture cannot always important problems facing agriculture today. be coaxed into forming embryos, which must There is no opposition to supporting the molec- precede the formation of leaves, shoots, and ular approaches as long as they do not come at roots. Technical breakthroughs have come on a the expense of traditional breeding programs, species-by-species basis; key technical discov- and as long as both molecular biologists and eries are not often applicable to all plants. And classical geneticists working with major crop even when the new technologies succeed in plants are assured of enough support to foster transferring genetic information, the changes research groups of sufficient size. can be unstable. At present, funds from nine programs at the The hope that protoplasm fusion would open NSF—primarily in the Directorate for Biologic], extensive avenues for gene transfer between Behavioral, and Social Sciences—support plant distantly related plant species has diminished research. The total support for the plant sci- with the observation of this instability. How- ences may be as high as $25 million, of which ever, if whole chromosomes or chromosome only about $1 million is designated specifically fragments could be transferred in plants where for molecular genetics. sexual hybridization is presently impossible, the The regulation of the release of genetically possibilities would be enormous. altered plants into the environment has not had INSTITUTIONAL CONSTRAINTS much effect to date. As of November 1980, only Institutional constraints on molecular genet- one application [which requested exception ics include those in funding, in regulation, in from the NIH Guidelines (see ch. 11) to release manpower, and in industry. rDNA-treated corn into the environment] has been filed with the Office of Recombinant DNA Federal funding for plant molecular genetics Activities (ORDA). Whether regulation will pro- in agriculture has come from the National duce major obstacles is difficult to predict at Science Foundation (NSF) and from USDA. Re- present. It is also unclear whether restrictions search support in USDA is channeled primarily will be placed on other genetic activities, such through the flexible Competitive Grants Pro- as protoplasm fusion. Currently, at least one gram (fiscal year 1980 budget of $15 million) for other nation (New Zealand) includes such re- the support of new research directions in plant strictions in its guidelines. It is not clear how biology. The panel on genetic mechanisms (an- much the uncertainty of possible ecological dis- nual budget less than $4 million) is of particular ruption and the attendent liability concerns significance for developing new genetic technol- from intentional release of genetically engi- ogies. The panel’s charter specifically seeks pro- neered plants has prevented the industrial sec- posals on novel genetic technologies. The re- tor from moving toward commercial application maining three panels concerned with plants— of the new technology. nitrogen, photosynthesis, and stress—also support projects to define the molecular basis of Only a few universities have expertise in both fundamental plant properties. The success of plant and molecular biology. In addition, only a the USDA Competitive Grants Program is hard few scientists work with modern molecular to assess after just 2 years of operation; how- techniques related to whole plant problems. As ever, its budget over the past 2 years has severe- a result, a business firm could easily develop a ly limited expansion of the program into new capability exceeding that at any individual U.S areas of research. university. However, building industrial labora

16 tories and hiring from the universities could Some private institutions argue that the easily deplete the expertise at the university W. walbot, past, present and Future Trends in Cmp f3medin& level. With the recent investment activity in Vol. H, Working Papers, Impact o~appiied Genetics, NTIS, 1981. bioengineering firms, this trend has already Ch. 8—The Application of Genetics to Plants ● 151 begun; in the long-run it could have serious con- Finally, as a general rule, tradeoffs arise in sequences for the quality of university research. the use of the new technologies that may interfere with their application. It is impossible to get Despite these constraints, progress in over- something for nothing from nature—e.g., in ni- coming the difficulties is continuing. At the trogen fixation the symbiotic relationship bet- prestigious 1980 Gordon Conference, where sci- ween plant and micro-organism requires ener- entists meet to exchange ideas and recent find- gy from the plant; screening for plants that can ings, plant molecular biology was added to the produce and transfer the end products of pho- list of meetings for the first time. In addition, tosynthesis to the nodules in the root more effi- four other recent meetings have concentrated 17 ciently may reduce inorganic nitrogen require- on plant molecular biology. Up to 50 percent ments but may also reduce the overall yield. of the participants at these meetings came from This was the case for the high lysine varieties of nonplant-oriented disciplines searching for fu- corn. (See Tech. Note 10, p. 163.) Farmers in the ture research topics. This influx of investigators United States tended to avoid them because im- from other fields can be expected to enrich the proving the protein quality reduced the yield, variety of approaches used to solve the prob- an unacceptable tradeoff at the market price. lems of the plant breeder. Thus, unless the genetic innovation fits the re- I TGenome Organization and Expression in Plants, NATO s.vm- quirements of the total agricultural industry, posium held in Edinburgh, Scotland, July 1979; Genetic Engineering potentials for crop improvement may not be of Symbiotic Nitrogen Fi&ation and Conservation of Fi~ed Nitrogent June 29-Juiy 2, 1980, Tahoe City, Ca]if; ‘“Molecular Biologists Look realized. al Green Plants,” Si,xfh Annual Symposium, Sepl: 29-oct. 2, lg80, }k?idelberg, West [;ermany; and Fourth /nferna[ional Symposium on Nitro,qm F~xation, Dec. 1-.5, 1980, Canberra, Aust ral iii.

Impacts on generating new varieties

Progress in the manipulation of gene expres- have been few demonstrations in which the in- sion in eukaryotic (nucleus-containing) cells, heritance of a new trait was maintained over which include the cells of higher plants, has several sexual generations in the whole plant. been enormous. Most of the new methodologies have been derived from fruit flies and mam- Because new varieties have to be tested malian tissue culture lines; but many should be under different environmental conditions once directly applicable to studies with plant genes. the problems of plant regeneration are over- There has been great progress in isolating spe- come, it is difficult to assess the specific impacts cific RNA from plants, in cloning plant DNA, and of the new technologies.—E. g., it is impossible to in understanding more about the organization determine at this time whether technical and of plant genomes. Techniques are available for biological barriers will ever be overcome for manipulating organs, tissues, cells, or pro- regenerating wheat from protoplasts. Never- toplasts in culture; for selecting markers; for theless, the impact of genetics on the structure regenerating plants; and for testing the genetic of American agriculture can be discussed with basis of novel traits. So far however, these some degree of confidence. techniques are routine only in a few species. Perfecting procedures for regenerating single Genetic engineering can affect not only what cells into whole plants is a prerequisite for the crops can be grown, but where and how those success of many of the novel genetic technol- crops are cultivated. Although it is a variable in ogies. In addition, work is progressing on production, it usually acts in conjunction with viruses, the Ti plasmid of Agrobacterium, and other biological and mechanical innovations, engineered cloning vehicles for introducing whose deployment is governed by social, eco- DNA into plants in a directed fashion. There nomic, and political factors. 152 ● Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

Examples of new genetic approaches wheat, corn, rice, and forage grasses do not have the capacity to fix atmospheric nitrogen, thus are largely The ways in which the new genetic ap- dependent on chemically produced nitrogen fertilizers. proaches could aid modern agriculture are Because of these crops, it has been estimated that the world demand for nitrogen fertilizers will grow from 51.4 described in the following two examples: million metric tonnes (1979 estimate) to 144 million to 180 8 SELECTION OF PLANTS FOR million tonnes by the year 2000.1 Therefore, geneticists METABOLIC EFFICIENCY are looking into the possibility that the genes for nitrogen fixation present in certain bacteria (called “nif genes”) can Because terrestrial plants are immobile, they live and die be transferred to the major crops. according to the dictates of the soil and weather conditions in which they are planted; any environmental stress can Laboratory investigation has focused on the molecular greatly reduce their yield. The major soil stresses faced by biology of nitrogen fixation in the free living bacterium, plants include insufficient soil nutrients and water or toxic Klebsiella pneumoniae. A cluster of 15 nif genes has been excesses of minerals and salts. The total land area with successfully cloned onto bacterial plasmids using rDNA these conditions approaches 4 billion hectares (ha), or technology. These clones are being used to study the about 30 percent of the land area of the Earth. molecular regulation of nif gene expression and the physical organization of the nif genes on the Klebsiella Traditionally, through the use of fertilizers, lime, chromosome. In addition they have aided the search for drainage, or freshwater irrigation, environments have nitrogen fixation genes in other bacteria. been manipulated to suit the plant. Modern genetic technologies might make it easier to modify the plant to suit the It is thought that a self-sufficient package of nitrogen- environment. fixing genes evolved during the course of plant adaptation, and that this unit has been transferred in a functional Many micro-organisms and some higher plants can tol- form to a variety of different bacterial species, including erate salt levels equal to or greater than those of sea water. Klebsiella and Rhizobium. If the right DNA vector can be While salt tolerance has been achieved in some varieties of found, the nif genes might be transferred from bacteria to plants, the classical breeding process is arduous and lim- plants. The chloroplasts, the cauliflower mosaic virus, and ited. If the genes can be identified, the possibility of actual- the Agrobacterium Ti-plasmid are being investigated as ly transferring those for salt tolerance into plants makes possible vectors. the adaptation of plants to high salt, semiarid regions with high mineral toxicities or deficiencies a more feasible pros- The way that Agrobacteria, in particular, infect cells is pect. In the future, selecting among tissue cultures for similar to the way Rhizobia infect plants and form metabolic efficiency could become important. Tissue nitrogen-fixing nodules. In both cases, the physical attach- culture systems could be used to select cell lines for ment between bacterium and plant tissue is necessary for resistance to salts and for responsiveness to low-nutrient successful infection. In the case of Agrobacteria, tumors levels or less fertilizer. However, too little is known about form when a segment of the Ti-plasmid is inserted into the the biochemistry and physiology of plants to allow a more nuclear genome of the plant cell. Scientists do not yet directed approach at this time. Chances for success would know exactly how a segment of the rhizobial genome is be increased with a better understanding of plant cell transferred into the root tissue to induce the formation of biology. nodules; nevertheless, it is hoped that Agrobacteria will act as vectors for the introduction and expression of foreign Such techniques could be applied to agricultural pro- genes into plant cells, just as Rhizobia do naturally. grams in less developed countries, where, commonly, supplies of fertilizers and lime are scarce, the potential for ir- Other researchers have been investigating the re- rigation is small, and adequate support for technological quirements for getting nif genes to express themselves in innovation is limited. [n addition, the United States itself plants. Nif genes from Klebsiella have already been contains marginal land that could be exploited for forest transferred into common yeast, an organism that can be products and biomass. The semiarid lands of the South- grown in environments without oxygen. Unfortunately, west, impoverished land in the Lake States, and reclaimed the presence of oxygen destroys a major enzyme for mining lands could become cost-effective areas for produc- nitrogen fixation and severely limits the potential application. tions in higher plants. Nevertheless, it is hoped that nif gene expression in yeast will be applicable to higher plants. NITROGEN FIXATION An approach that does not involve genetic engineering, It has been known since the early 1800’s that biological uses improved Rhizobia strains that are symbiotic with fixation of nitrogen is important to soil fertility. In fixation, soybeans. Through selection, Rhizobia mutants are being Rhizobium, micro-organisms, such as the bacterium found that out-perform the original wild strains. Further transform atmospheric nitrogen into a form that plants can use. In some cases—e.g., with legumes this process occurs through a symbiotic relationship between the micro- ‘“F. Ausubel, “Biological Nitrogen E’ixation, ” Supporting Papers: organism and the plant in specialized nodules on the plant World Food and Nurrition Study (Washington, D. C.: National Acad- roots. Unfortunately, the major cereal crops such as emy of Sciences, 1977). Ch. 8—The Application of Genetics to Plants . 153 testing is needed to determine whether the improvement Although it is difficult to make economic pro- can be maintained in field trials, where the improved jections, there are several areas where genetic strains must compete against wild-type Rhizobia already present in the soil. technologies will clearly have an impact if the predicted breakthroughs occur: Another way to improve nitrogen fixation is to select plants that have more efficient symbiotic relationships ● Batch culture of plant cells in automated with nitrogen-fixing organisms. Since the biological proc- systems will be enhanced by the ability to ess requires a large amount of energy from the plant, it engineer and select strains that produce may be possible to select for plants that are more efficient in producing, and then to transfer the end products of larger quantities of plant substances, such photosynthesis to the nodules in the roots. Also existing as pharmaceutical drugs. nitrogen-fixing bacterial strains that can interact with crop ● The technologies will allow development of plants which do not ordinarily fix nitrogen could be elite tree lines that will greatly increase searched for or developed. yield, both through breeding programs Reducing the amount of chemically fixed nitrogen similar to those used for agricultural crops fertilizer–and the cost of the natural gas previously used and by overcoming breeding barriers and in the chemical process—would be the largest benefit of lengthy breeding cycles. Refined methods successfully fixing nitrogen in crops. Environmental benefits, from the smaller amount of fertilizer runoff into of selection and hybridization will increase water systems, would accrue as well. But is it difficult to the potential of short-rotation forestry, predict when these will become reality. Experts in the field which can provide cellulosic substrates for disagree: some feel the breakthrough is imminent; others such products as ethanol or methanol. feel that it might take several decades to achieve. ● The biological efficiency of many economi- The refinements in breeding methods pro- cally important crops will increase. Ad- vided by the new technologies may allow major vances will depend on the ability of the crops to be bred more and more for specialized techniques to select for whole plant charac- uses—as feed for specific animals, perhaps, or teristics, such as photosynthetic soil and to conform to special processing requirements. nutrient efficiency.19 In addition, since the populations in less devel- ● Besides narrowing breeding goals, the oped countries suffer more often from major techniques will increase the potential for nutritional deficiencies than those in industrial- faster improvement of underexploited ized countries, a specific export market of cere- plants with promising economic value. al grains for human consumption, like wheat For such advances to occur, genetic factors with higher protein levels, may be developed. must be selected from superior germplasm, the But genetic methods are only the tools and genetic contributions must be integrated into catalysts for the changes in how society pro- improved cultural practices, and the improved duces its food; financial pressures and Federal varieties must be efficiently propagated for regulation will continue to direct their course. distribution. E.g., the automation of tissue culture systems will decrease the labor needed to direct plant propagation and drastically reduce the cost per plantlet to a level competitive with seed prices for many crops. While such breakthroughs may For the soybean and tomato crops, the research area for im- increase the commercial applications of many proved biological efficiency received the highest allotment of technologies, the effects of a displaced labor funds in fiscal year 1978. Total funding was $12.9 million for soybeans and $2.1 million for tomatoes. The second largest category force and cheaper and more efficient plants are to be funded was control of diseases and nematodes of soybeans at hard to predict. $5.1 million and for tomato at $1.6 million. 154 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Genetic variability, crop vulnerability, and storage of germplasm

Successful plant breeding is based on the ficult to predict future needs. Because pests and amount of genetic diversity available for the in- pathogens are constantly mutating, there is sertion of new genes into plants. Hence, it is always the possibility that some resistance will essential to have an adequate scientific under- be broken down. Even though genetic diversity standing of how much genetic erosion has taken can reduce the severity of economic loss, an epi- place and how much germplasm is needed. Nei- demic might require the introduction of a new ther of these questions can be satisfactorily an- resistant variety. In addition, other pressures swered today. will determine which crops will be grown for food, fiber, fuel, and pharmaceuticals, and how The amount of genetic erosion that they will be cultivated; genetic diversity will be has taken place fundamental to these innovations. Most genetic diversity is being lost because of Even if genetic needs can be adequately iden- the displacement of vegetation in areas outside tified, there is disagreement about how much the United States. The demand for increased germplasm to collect. In the past, its collection agricultural production is a principal pressure has been guided by differences in morphology causing deforestation of tropical latitudes (see (form and structure), which have not often been Tech. Note 11, p. 163), zones that contain exten- directly correlated to breeding objectives. Fur- sive genetic diversity for both plants and thermore, the extent to which the new genetic animals. technologies will affect genetic variability, vul- It has been estimated that several hundred nerability, or the storage of germplasm, has not plant species become extinct every year and been determined. (See app. II-A.) that thousands of indigenous crop varieties (wild types) have already been lost. However, it In addition to its uses in plant improvement, is difficult to measure this loss, not only because germplasm can provide both old and new prod- resources are on foreign soil but because ero- ucts. Recent interest in growing guayule as a sion must be examined on a species-by-species source of hydrocarbons (for rubber, energy basis. In theory, an adequate evaluation would materials, etc.) has focused attention on plants require knowledge of both the quantity of di- that may possibly be underutilized. It has been versity within a species and the breadth of that found that past collections of guayule germ- diversity; this process has in practice, just be- plasm have not been adequately maintained, gun. What is known is that the lost material can- making current genetic improvements more dif- not be replaced. ficult. In addition, half of the world’s medicinal compounds are obtained from plants; maintain- The amount of germplasm needed ing as many varieties as possible would ensure the availability of compounds known to be use- Germplasm is needed as a resource for im- ful, as well as new, and as yet undiscovered proving characteristics of plants and as a means compounds—e.g., the quinine drugs used in the for guaranteeing supplies of known plant treatment of malaria were originally obtained derivatives and potential new ones. Even if from the Cinchona plant. A USDA collection of plant breeders adequately understand the superior germplasm established in 1940 in amount of germplasm presently needed, it is dif- Guatemala was not maintained. As a conse- Ch. 8—The Application of Genetics to Plants ● 155 quence, difficulties arose during the Vietnam The National Germplasm System War when the new antimalarial drugs became less effective on resistant strains of the parasite USDA has been responsible for collecting and and natural quinines were once again used. cataloging seed (mostly from agriculturally important plants) since 1898. Yet it is important to realize that other Federal agencies also have An important distinction exists between pre- responsibilities for gene resource management. serving genetic resources in situ and preserving (See table 27.) Over the past century, over germplasm stored in repositories. Although 440,000 plant introductions from more than 150 genetic loss can occur at each location, evolu- expeditions to centers of crop diversity have tion will continue only in natural ecosystems. been cataloged. With better storage techniques, seed loss and genetic “drift” can be kept to a minimum. Never- The expeditions were needed because the theless, species extinction in situ will continue. United States is gene poor. The economically im-

Table 27.—Gene Resource Responsibilities of Federal Agencies

Type of ecosystems under Federal Agency ownership/control Responsibilities U.S. Department of Agriculture Animal & Plant Health Inspection Service ...... — Controls insect and disease problems of commercially valuable animals and plants. Forest Service ...... Forestlands and Manages forest land and rangeland living resources for rangelands (U.S. production. National Forest) Science & Education Administration . — Develops animal breeds, crop varieties, and microbial strains. Manages a system for conserving crop gene resources. Soil Conservation Service...... — Develops plant varieties suitable for reducing soil erosion and other problems. Department of Commerce National Oceanic & Atmospheric Administration...... Oceans—between 3 Manages marine fisheries. and 200 miles off the U.S. coasts Department of Energy...... — Develops new energy sources from biomass. Department of Health& Human Services National Institutes of Health ...... — Utilizes animals, plants, and micro-organisms in medical research. Department of the Interior Bureau of Land Management...... Forest lands, Manages forest, range, and desert living resources for rangelands, and production. deserts Fish & Wildlife Service ...... Broad range of Manages game animals, including fish, birds, and mammals. habitats, including oceans up to 3 miles off U.S. coasts National Park Service ...... Forest lands, Conserves forestland, rangeland, and desert-living resources. rangelands, and deserts (U.S. National Parks) Department of State ...... — Concerned with international relations regarding gene resources. Agency for International Development. . — Assists in the development of industries in other countries including their agriculture, forestry, and fisheries. Environmental Protection Agency...... — Regulates and monitors pollution. National Science Foundation...... — Provides funding for genetic stock collections and for research related to gene resource conservation.

SOURCE: David Kapton, National Assoclatlon for Gene Resource Conservation. 156 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals portant food plants indigenous to the continen- Table 28.-Estimated Economic Rates of Return tal United States are limited to the sunflower, From Germplasm Accessions cranberry, blueberry, strawberry, and pecan. 1. A plant introduction of wheat from Turkey was found to The centers of genetic diversity, found mostly in have resistance to all known races of common and tropical latitudes around the world, are be- dwarf bunts, resistance to stripe rust and flag smut, plus field resistance to powdery and snow mold. It has lieved to be the areas where progenitors of ma- contributed to many commercial varieties, with jor crop plants originated. Today, they contain estimated annual benefits of $50 million. genetic diversity that can be used for plant im- 2. The highly successful variety of short-strawed wheat, ‘Gaines’ has in its lineage three plant introductions that provement. contributed to the genes for the short stature and for resistance to several diseases. During the 3 years, It is difficult to estimate the financial return 1964-66, about 60 percent of the wheat grown in the from the germplasm that has been collected, State of Washington was with the variety ‘Gaines’. in- but its impact on the breeding system has been creased production with this variety averaged slightly over 13 million bu or $17.5 million per year in the 3-year substantial. A wild melon collected in India, for period. instance, was the source of resistance to pow- 3. Two soybean introductions from Nanking and China dery mildew and prevented the destruction of were used for large-scale production, because they are well-adapted to a wide range of soil conditions. All ma- California melons. A seemingly useless wheat jor soybean varieties now grown in t e Southern United strain from Turkey—thin-stalked, highly sus- States contain genes from one or both of these in- ceptible to red rust, and with poor milling prop- troductions. Farm gate value of soybean crop in the South exceeded $2 billion in 1974. erties—was the source of genetic resistance to 4. Two varieties of white, seedless grapes resulted from stripe rust when it became a problem in the crosses of two plant introductions. These varieties Pacific Northwest. Similarly, a Peruvian species ripen 2 weeks ahead of ‘Thompson Seedless’. Benefits to the California grape industry estimated to be more contributed “ripe rot” resistance to American than $5 million annually. pepper plants, while a Korean cucumber strain — SOURCE: U.S. Department of Aorlculture. Agricultural Research Service. /n- provided high-yield production of hybrid cu- troduct;on, Classitica~ion, Maintenance, Eveiuation, and Docu- mentation of Piant Gerrrrpiasrrr, (ARS) National Research Program No. cumber seed for U.S. farmers. And a gene for 20160 (Washington, D. C., U.S. Government Printing Of fice,1976). resistance to Northern corn blight transferred to Corn Belt hybrids has resulted in an estimated savings of 30 to 50 bushels (bu) per acre, age, 12 new repositories for fruit and nut crops with a seasonal value in excess of $200 million.20 as well as for other important crops, from hops (See table 28.) to mint, were proposed by the National Germ- plasm Committee as additions to the National The effort to store and evaluate this collected Germplasm System (see Tech. Note 12, p. 163). germplasm was promoted by the Agricultural (The development of tissue culture storage Marketing Act of 1946, which authorized re- methods may reduce storage costs for these gional and interregional plant introduction sta- proposed repositories.) tions (National Seed Storage Centers) run coop- eratively by both Federal and State Govern- The National Germplasm System is a vital link ments. The federally controlled National Seed in ensuring that germplasm now existing will Storage Laboratory in Fort Collins, Colo., was still be available in the future. However, the established in 1958 to provide permanent stor- present system was challenged after the South- age for seed. In the 1970’s, it was recognized ern corn blight epidemic of 1970. Many scien- that the system should include clonal material tists questioned whether it was large enough for vegetatively propagated crops, which can- and broad enough in its present form to provide not be stored as seed. Although their storage re- the genetic resources that might be needed. quires more space than comparable seed stor- The devastating effects of the corn blight of 1970 actually led to the coining of the term crop ZOu. s, Department of Agriculture, Agricultural Research SerV- vulnerability. During the epidemic, as much as ice, Introduction, Class[~ication, Maintenance, Evaluation, and Docu- 15 percent of the entire yield was lost. Some mentation of Plant Germplasm, (ARS) National Research Program No. 20160 (Washington, D. C., U.S. Government Printing Office, fields lost their whole crop, and entire sections 1976). of some Southern States lost 50 percent of their Ch. 8—The Application of Genetics to Plants . 157 corn. Epidemics like this one are, of course, not The basis for genetic uniformity new. In the 19th century, the phylloxera disease of grapes almost destroyed the wine industry of Crop uniformity results most often from soci- France, coffee rust disrupted the economy of etal decisions on how to produce food. The Ceylon, and the potato famine triggered exten- structure of agriculture is extremely sensitive to sive local starvation in Ireland and mass emigra- changes in the market. Some of the basic factors tion to North America. In 1916, the red rust de- influencing uniformity are: stroyed 2 million bu of wheat in the United the consumer’s demand for high-quality States and an additional million in Canada. Fur- produce; ther epidemics of wheat rust occurred in 1935 the food processing industry’s demand for and 1953. The corn blight epidemic in the harvest uniformity; United States stimulated a study that led to the the farmer’s demand for the “best” variety publication of a report on the “Genetic Vulner- 21 that offers high yields and meets the needs ability of Major Crops’’. It contained two cen- of a mechanized farm system; and tral findings: that vulnerability stems from ge- the increased world demand for food, netic uniformity, and that some American crops which is related to both economic and pop- are, on this basis, highly vulnerable. (See table ulation growth. 29.) However, genetic variability, is only a hedge New varieties of crops are bred all the time, against vulnerability. It does not guarantee that but several can dominate agricultural produc- an epidemic will be avoided. In addition, path- tion—e.g., Norman Borlaug and his colleagues in ogens from abroad can become serious prob- Mexico pioneered the “green revolution” by lems when they are introduced into new envi- developing high-yielding varieties (HYV) of ronments. As clearly stated in the study, a tri- wheat that required less daylight to mature and angular relationship exists between host, path- possessed stiffer straw and shorter stems. Since ogen, and environment, and the coincidence of the new varieties (see Tech. Note 13, p. 163) their interaction dictates the severity of disease. gave excellent yields in response to applications of fertilizer, pesticides, and irrigation, the in- z INati~nal Academy of Sciences, Genetic Vulnerability of M@r novation was subsequently introduced into Crops, Washington, D. C., 1972. countries like India and Pakistan. When a single

Table 29.—Acreage and Farm Value of Major U.S. Crops and Extent to Which Small Numbers of Varieties Dominate Crop Average (1969 figures)

Value Acreage (millions of Total Major Acreage Crop (millions) dollars) varieties varieties (percent) Bean, dry ...... 1.4 143 25 2 60 Bean, snap...... 0.3 99 70 3 76 Cotton...... 11.2 1,200 50 3 53 C Coma ...... 66.3 5,200 197b 6 71 Millet...... 2.0 ? 3 100 Peanut ...... 1.4 312 15 9 95 Peas ...... 0.4 80 50 2 96 Potato...... 1.4 616 82 4 72 Rice...... 1.8 449 14 4 65 Sorghum...... 16.8 795 ? ? ? Soybean ...... 42.4 2,500 62 6 58 Sugar beet ...... 367 16 2 42 Sweet potato ...... 0.13 63 48 69 Wheat...... 44.3 1,800 269 9 50 acorn k-iclldeg seeds, forage, and sila9e. bRelea9ed public inbreds only. cThere were six major public lines used in breeding the major varieties of corn, so the aCtIJal number of ‘farietieS is higher. SOURCE: National Academy of Sciences, Genetic Vu/nerabi/ity of Major Crops, Washington, D. C., 1972. I&l . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals variety dominates the planting of a crop, there flected in a 1978 recommendation by the Na- is some loss of genetic variability, the resulting tional Plant Genetic Resources Board. It’s recom- uniformity causes crop vulnerability—and the mendation was that four major areas of genetic displacement of indigenous varieties—a real storage—collection, maintenance, evaluation, problem. and distribution—be viewed as a “continuum that sets up a gene flow from source to end The rate of adoption of HYVs levels off below use".24 100 percent in most countries, mainly because of the numerous factors affecting supply and Z. The management of genetic resources is com- demand: 22 plex and costly. ● supply factors: The question of how much germplasm to col- —the present HYVs are not suitable for all lect is difficult and strongly influenced by cost. soil and climatic conditions; Thus far, only a fraction of the available diversi- —they require seeds and inputs (such as ty has been collected. A better scientific under- fertilizers, water, and pesticides) that are standing of the genetic makeup and previous either unavailable or not fully utilized by breeding history of major crops will help deter- every farmer; and mine just how much germplasm should be col- —in some regions, a strong demand still ex- lected. Efforts to give priorities for co11ection25 ists for the longer straw of traditional have been hindered by the scientific gaps in varieties. knowledge about what is presently stored ● demand factors: worldwide. And while attempts have been —consumers may not prefer the HYVs over made to estimate the economic return from in- traditional food varieties; troduction of specific plants (see table 28), the —Government price policies may not en- degree to which agricultural production and courage the production of HYVs. stability are dependent on genetic variability has not been adequately analyzed. For these and other reasons, countries already using a great deal of HYVs will continue to adopt Evaluation of genetic characteristics must be them more slowly. conducted at different ecological sites by multidisciplinary teams. The data obtained will only Six factors affecting adequate be useful if adequately assessed and made avail- management of genetic resources able to the breeding community (see Tech. Note 14, p. 163). 1. Estimating the potential value of genetic resources is dfficult. Germplasm must be adequately maintained to assure viability, “working stocks” must be made Of the world’s estimated 300,000 species of available to the breeding community. The pri- higher plants, only about 1 percent have been mary objective of storing germplasm is to make screened for their use in meeting the diverse the genetic information available to breeders demands for food, animal feed, fiber, and phar- and researchers. maceuticals. 23 Genetic resources not yet col- 3. How much plant diversity can be lost without lected or evaluated are valuable until proven disrupting the ecological balances of natural otherwise, and the efforts to conserve, collect, and agricultural systems is not known. and evaluate plant resources should reflect this assumption. This point of view was strongly re-

=D. G. Dalrymp]e, Oeve/opment and Spread o~Wgh-k’ielding VWi- z4RepOr[ to the Secretarv of Agriculture, bY the Assistant see- eties of Wheat and t?ice in the Less Developed Nations, 6th ed. retary for Conservation, R~search, and Education based on the de- (Washington D. C.: U.S. Department of Agriculture, Office of Inter- liberations and recommendations, Nationai Plant Genetic Re- national Cooperation and Development in cooperation withU.S. sources Board, July 1978. Agency for International Development, 1978). Zssecretaria[, International Board for Plant Genetic Resources, 2SN. Myers, “[conserving Our Global stock, ” Environment Annual Report 1978, Rome; Consultative Group on International 21(9):25, 1979. Agricultural Research, 1979. Ch. 8—The Application of Genetics to Plants ● 159

The arguments parallel those previously dis- Historically, success and failure in breeding cussed in Congress for protection of endan- programs are linked to pests and pathogens gered species (see Tech. Note 15, p. 163). The overcoming resistance. Hence, plant breeders last decade has shown that modes of production try to keep one step ahead of mutations or and development can severely affect the ecolog- changes in pest and pathogen populations; a ical balance of complex ecosystems. What is not plant variety usually lasts only 5 to 15 years on known is how much species disruption can take the market. There is some evidence that patho- place before the quality of life is also affected. gens are becoming more virulent and aggres- sive—which could increase the rate of infection, 4. The extent to which the new genetic technol- enhancing the potential for an epidemic (see ogies will affect genetic variability, germplasm Tech. Note 17, p. 164). storage methodologies, and crop vulnerability has not been determined. 6. Other economic and social pressures affect the use of genetic resources. The new genetic technologies could either increase or decrease crop vulnerability. In theory, The Plant Variety Protection Act has been they could be useful in developing early warn- criticized for being a primary cause of planting ing systems for vulnerability by screening for uniform varieties, loss of germplasm, and con- inherent weaknesses in major crop resistance. glomerate acquisition of seed companies. In its However, the relationship between the genetic opponents’ view, such ownership rights provide characteristics of plant varieties and their pests a strong incentive for seed companies to en- and pathogens is not understood (see Tech. Note courage farmers to buy “superior” varieties that 16, p. 164). can be protected, instead of indigenous varieties that cannot. They also make plant breeding so The new technologies may also enhance the lucrative that the ownership of seed companies, prospects of using variability, creating new is being concentrated in multinational corpora- sources of genetic diversity and storing genetic tions—e.g., opponents claim that 79 percent of material by: the U.S. patents on beans have been issued to ● increasing variability during cell regenera- four companies and that almost 50 once-inde- tion, pendent seed companies have been acquired by ● incorporating new combinations of genetic The Upjohn Co., ITT, and others.26 One concern information during cell fusion, raised about such ownership is that some of ● changing the ploidy level of plants, and these companies also make fertilizer and ● introducing foreign (nonplant) material pesticides and have no incentive to breed for and distantly related plant material by pest resistance or nitrogen-fixation. For the means of rDNA. above reasons, one public interest group has concluded :27 With the potential benefits, however, come risks. Because genetic changes during the devel- [t]hanks to the patent laws, the bulk of the opment of new varieties are often cumulative, world’s food supply is now owned and devel- and because superior varieties are often used oped by a handful of corporations which alone, extensively, the new technologies could in- without any public input, determine which crease both the degree of genetic uniformity strains are used and how. and the rate at which improved varieties dis- Numerous arguments have been advanced place indigenous crop types. Furthermore, it against the above position. Planting of a single has not been determined how overcoming natu- variety, for instance, is claimed to be a function ral breeding barriers by cell fusion or rDNA will of the normal desires of farmers to purchase affect a crop’s susceptibility to pests and dis- the best available seed, especially in the com- eases. 5. Because pests and pathogens are constantly 2’P. R. Mooney, Seed of the Earfh (London: International Coalition tor De\7elopment Action, 1979). mutating plant resistance can be broken down, 2T~1.1ef for PeoP]eS’ ~usilless (hmm ission as Amicus Curiae, requiring the introduction of new varieties. Diamond v. Chalmabarr-v, 100 S. Ct. 2204 (1980), p. 9. 160 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals petitive environment in which they operate. nection between this trend and the plant variety Moreover, hybrid varieties (such as corn), are protection laws is disputed. one explanation is not covered by the plant protection laws; yet that the takeovers are part of the general take- they comprise about 90 percent of the seed over movement that has involved all parts of the trade. economy during the past decade. Since the passage of the 1970 Act, the number of seed com- As for the loss of varieties by vegetation panies, especially soybean, wheat, and cereal displacement, statutory protection has been too grains, has increased.29 While there were six recent to counter a phenomenon that has oc- companies working with soybean breeding curred over a 30- to 40-year period, and avail- prior to 1970, there are 25 at this time.30 able evidence indicates that some crops are actually becoming more diverse. Since most major Thus, to date, although no conclusive connec- food crops are sexually produced, they have tion has been demonstrated between the two only been subject to protection since 1970 when plant protection laws and the loss of genetic the Plant Variety Protection Act was passed; the diversity, the use of uniform varieties, or the first certificates under that Act were not even claims of increasing concentration in the plant issued until 1972. Moreover, at least in the case breeding industry; the question is stilI con- of wheat, as many new varieties were devel- troversial and these complex problems are still oped in the 7 years after the passage of the Plant unresolved. Variety Protection Act as in the previous 17.28 It is clear that large corporations have been ZgHearin@ on H.R. 2844, supra note 35 (Statement of’ Harold Loden, Executive Director of the American Seed Trade Associa- acquiring seed companies. However, the con- tion). JoBrief for pharmace~tica] Manufacturers’ Association as Amicus Curiae, DiarnorId v. Cha)crabarty, 100 S. Ct. 2204 (1980), p. 20H. Rept. No. 96.1115, 96th Cong., 2d S6?SS., p. 5 (June 20J 1980). 26.

Summary

The science and structure of agriculture are ● This lost genetic diversity is irreplaceable. not static. The technical and industrial revolu- ● The world’s major food crops are becoming tions and the population explosion have all con- more vulnerable as a result of genetic uni- tributed to agricultural trends that influence formity. the impacts of the new technologies. Several The solutions—examining the risks and eval- factors affect U.S. agriculture in particular: uating the tradeoffs—are not limited to securing To some degree, the United States depends and storing varieties of seed in manmade on germplasm from sources abroad, which repositories; genetic evolution—one of the keys are, for the most part, located in less devel- to genetic diversity and a continuous supply of oped countries; furthermore, the amount new germplasm—cannot take place on storage of germplasm from these areas that should shelves. Until specific gaps in man’s understand- be collected has not been determined. ing of plant genetics are filled, and until the Genetic diversity in areas abroad is being breeding community is able to identify, collect, lost. The pressures of urbanization, in- and evaluate sources of genetic diversity, it is dustrial development, and the demands for essential that natural resources providing germ- more efficient, more intensive agricultural plasm be preserved. production are forcing the disappearance of biological natural resources in which the supply of germplasm is maintained. Ch. 8—The Application of Genetics to Plants ● 161

Issues and Options—Plants ISSUE: Should an assessment be con- ● Instruct the Agency for International De- ducted to determine how much velopment (AID) to place high priority on, plant germplasm needs to be and accelerate its activities in, assisting less maintained? developed countries to establish biosphere An understanding of how much germplasm reserves and other protected natural areas, should be protected and maintained would providing for their protection, and support associate research and training. make the management of genetic resources ● simpler. But no complete answers exist; nobody Instruct the International Bank for Recon- knows how much diversity is being lost by struction and Development (World Bank) to vegetation displacement in areas mostly outside give high priority to providing loans to the United States. those less developed countries that wish to establish biosphere reserves and other protected natural areas as well as to promote OPTIONS: activities related to biosphere reserve pres- A. Congress could commission a study on how ervation, and the research and manage- much genetic variability is needed or desirable ment of these areas and resources. to meet present and future needs. ● Make a one-time special contribution to UNESCO to accelerate the establishment of A comprehensive evaluation of the National biosphere reserves. Germplasm System’s needs in collecting, evaluating, maintaining, and distributing genetic Such measures for in situ preservation and resources for plant breeding and research could management are necessary for long-term main- serve as a baseline for further assessment. This tenance of genetic diversity. Future needs are evaluation would require extensive cooperation difficult to predict; and the resources, once lost among the Federal, State, and private compo- are irreplaceable. nents linked to the National Germplasm System. C. Congress could commission a study on how to B. Congress could commission a study on the develop an early warning system to recognize need for international cooperation to manage potential vulnerability of crops. and preserve genetic resources both in natural A followup study to the 1972 National Acad- ecosystems and in repositories. emy of Science’s report on major crop vulnerability could be commissioned. Where high This investigation could include an evaluation genetic uniformity still exists, proposals could of the rate at which genetic diversity is being be suggested to overcome it. In addition, the lost from natural and agricultural systems, and avenues by which private seed companies could an estimate of the effects this loss will have. Un- be encouraged to increase the levels of genetic til such information is at hand, Congress could: diversity could be investigated. The study could ● Instruct the Department of State to have its also consider to what extent the crossing of delegations to the United Nations Educa- natural breeding barriers as a consequence of tional, Scientific, and Cultural Organization the new genetic technologies will increase the (UNESCO) and United Nations Environmen- risks of crop vulnerability. tal Program (UNEP) encourage efforts to establish biosphere reserves and other pro- ISSUE: What are the most appropriate tected natural areas in less developed coun- approaches for overcoming the tries, especially those within the tropical various technical constraints latitudes. These reserves would serve as a that limit the success of molec- source for continued natural mutation and ular genetics for plant improve- variation. ment? 162 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Although genetic information has been trans- sis on potential pharmaceuticals derived from ferred by vectors and protoplasm fusion, DNA plants. transformations of commercial value have not c. Establish an institute for plant molecular ge- yet been performed. Molecular engineering has netics under the Science and Education Ad- been impeded by the lack of vectors that can ministration at USDA that would include mul- transfer novel genetic material into plants, by tidisciplinary teams to consider both basic re- insufficient knowledge about which genes search questions and direct applications of the would be useful for breeding purposes, and by technology to commercial needs and practices. a lack of understanding of the incompatibility of chromosomes from diverse sources. Another The discoveries of molecular plant genetics impediment has been the lack of researchers will be used in conjunction with traditional from a variety of disciplines. breeding programs. Therefore, each of the three options would require additional appro- OPTIONS: priations for agricultural research. Existing A. Increase the level of funding for plant molec- funding structures could be used for all three, ular genetics through: but institutional reorganization would be re- 1. the National Science Foundation (NSF)) and quired for options B and C. The main argument for increasing USDA funding is that it is the lead Z. the Competitive Grants Program of the U.S. Department of Agriculture (USDA). agency for agricultural research, for increasing NSF and NIH funding, that they currently have B. Establish research units devoted to plant mo- the greatest expertise in molecular techniques. lecular genetics under the auspices of the Na- Option C emphasizes the importance of the in- tional Institutes of Health (NIH), with empha- terdisciplinary needs of this research.

Technical notes

1. A recent example of such a mutation was the opaque-2 5 Normally, chromosomes are inherited in sets. The gene in corn, which was responsible for increasing the more frequent diploid state consists of two sets in each corn’s content of the amino acid lysine. plant. Because chromosome pairs are homologous Z. There is disagreement about what is meant by produc- (have the same linear gene sequence), cells must main- tivity and how it is measured. Statistical field data can tain a degree of genetic integrity between chromosome be expressed in various ways—e.g., output per man- pairs during cell division. Therefore, increases in hour, crop yield per unit area, or output per unit of ploidy involve entire sets of chromosomes—diploid (2- total inputs used in production. A productivity meas- set) is manipulated to triploid (3-set) or even to urement is a relationship among physical units of pro- tetraploid (4-set). duction. It differs from measurements of efficiency, 6 The estimated theoretical limit to efficiency of photo- which relate to economic and social values. synthesis during the growth cycle is 8.7 percent. How- 3. Nevertheless, some parts of the world continue to lack ever, the record U.S. State average (116 bu/acre, Il- adequate supplied of food. A recent study by the Pres- linois, 1975) for corn, having a high photosynthetic rate idential Commission on World Hunger31 estimates that in comparison to other major crops, approaches only 1 “at least one out of every eight men, women, and chil- percent efficiency. 32 Since a major limiting step in plant dren on earth suffers malnutrition severe enough to productivity lies in this efficiency for the photosyn- shorten life, stunt physical growth, and dull mental thetic process, there is potential for plant breeding ability. ” strategies to improve the efficiency of photosynthesis 4. In theory, pure lines produce only identical gametes, of many other important crops. This would have a tre- which makes them true breeders. Successive cross- mendous impact on agricultural productivity. breeding will result in a mixture of gametes with vary- 7 It is difficult to separate social values from the econom- ing combinations of genes at a given locus on homolog- ic structures affecting the productivity of American ous chromosomes. agriculture. Social pressures and decisions are complex

31 RePO1.t of the pr~sidelltia] (;ornnlission on World Hungfwt ~~v~r- iZoffi~.~ of ‘[”~~:hll[]]t)gy /l SSPSSlllPllt, 11.S. Congress, Energv for comin$ World Hunger: The L’hallenge Ahead, Washington, D. C., Bioiogicai Proresses, Volume 11: Technical Analysis (W’ashinglon, March 1980. 11.(1,: ~1.S. (;mwrnment Printing office, Ju]y 1980). Ch. 8—The Application Of Genetics to Plants ● 163

and integrated—e.g., conflicts between developing If the highest predicted rate of loss continues, half of maximum productivity and environmental concerns the remaining closed forest area will be lost by the year are typified by the removal of effective pesticides from 2000. 33 The significance of this loss is expressed by the market. Applications of existing or new technol- Norman Myers in his report, Conversion of Tropical ogies may be screened by the public for acceptable Moist Forests, prepared for the Committee on Research environmental impact. Conflict also exists between Priorities in Tropical Biology of the National Academy higher productivity and higher nutritive content in of Science’s National Research Council: “Extrapolation food, since selection for one often hurts the other. of figures from well-known groups of organisms sug- 8. A critical photosynthetic enzyme (ribulose biphosphate gest that there are usually twice as many species in the carboylase) is formed from information supplied by tropics as temperate regions. If two-thirds of the different genes located independently in the chloro- tropical species occur in TMF (tropical moist forests), a plast (a plastid) and the nucleus of the cell. It is com- reasonable extrapolation from known relationships, posed of two separate protein chains that must link then the species of the TMF should amount to some 40 together within the chloroplast. The larger of these to so percent of the planet’s stock of species—or some- chains is coded for by a gene in the chloroplast—and it where between 2 million and 5 million species altogeth- is this gene that has been recently isolated and cloned. er. In other words, nearly half of all species on Earth The smaller subunit, however, derives from the plant are apparently contained in a biome that comprises nucleus itself. This cooperation between the nucleus only 6 percent of the globe’s land surface. Probably no and the chloroplast to produce the functional expres- more than 300,000 of these species—no more than 15 sion of a gene is an interesting phenomenon. Because it percent and possibly much less—have ever been given exists, the genetics of the cell could be manipulated so a Latin name, and most are totally unknown. "34 that cytoplasmically introduced genes can influence 12 In 1975, the Committee estimated that $4 million would nuclear gene functions. Perhaps most importantly at be necessary for capital costs of each repository, with this stage, plastid genes are prime candidates to clarify recurring annual expenses of $1.4 million for salaries the basic molecular genetic mechanisms in higher and operations. USDA has allocated $1.16 million for its plants. share of the construction costs for the first facility to 9. The advantages to using mass propagation techniques be constructed at the Oregon State University in Cor- for strawberry plants are that those produced from vallis. tissue culture are virus-free, and a plantlet produced in 13 High yielding varieties (HYVs) can be defined as poten- tissue culture can produce more shoots or runners for tially high-yielding, usually semidwarf (shorter than transplanting. conventional), types that have been developed in na- The disadvantages are that during the first year the tional research programs worldwide. Wheat varieties fruit tends to be smaller and, therefore, less commer- were developed by the International Maize and Wheat cially acceptable; the plants from tissue culture may Improvement Center and rice varieties by interna- have trouble adapting to soil conditions, which can af- tional Rice Research Institute. Many improved varieties fect their vigor, especially during the first growing sea- of major crops of conventional height are not currently son; and the price per plantlet ready for planting from considered HYV types, but they have often been incor- tissue culture systems may be more expensive than porated into HYV breeding. HYVs, because of biological commercial prices for rooted shoots or runners bought and management factors, rarely reach their full in bulk. harvest potential. 10. Wheat protein is deficient in several amino acids, in- 14 Although the National Germplasm System successfully cluding lysine. Considerable attention has been de- handles some 500,000 units to meet annual germplasm voted in the past 5 to 10 years to improving the nutri- requests, many accessions–like the 35,000 to 40,000 tional properties of wheat. Thousands of lines have wheat accessions stored at the Plant Genetics and been screened for high protein, with good success, and Germplasm Institute at Beltsville, Md.–have yet to be high lysine genes with poor success. Some high protein examined. Furthermore, the varieties released for sale varieties have been developed, but adoption by the by the seed companies are not presently evaluated for farmer has been mediocre at best, partly because of their comparative genetic differences. reduced yield levels. There are some “exceptions; —e.g., 15. For comparison, the National Germplasm System func- the Variety “Plainsman V“ has maintained both high tions on less than $10 million annually, whereas the En- protein and yield levels, which indicates that there is no dangered Species Program had a fiscal year 1980 budg- consistent relationship between low protein and high et of over $23 million. The funds allocated to the En- yields in some varieties. jsRep~rt t. the Prmident bv a 11 .S. Interagency Task Force 011 Some 42 percent of the total land area in the tropics, 11 Tropical Forests, 7’he Worldk Tropical Forests: A Policy, S[rategv, consisting of 1.9 billion hectares, contains significant and Program for the C hited Slates, State Departnlent publication forest cover. It is difficult to measure precisely the No. 9117, }Vashin#on, [1.(; ., hla~~, 1980. amount of permanent forest cover that is being lost; W,V, ~lver.s, [;onktersjon Of ‘rro~jcal Moist Forests, report for the however, it has been estimated that 40 percent of Conmitiee on Research Priorities in Tropical Biology of the Na- “closed” forest (having a continuous closed canopy) has tional Research Council, National Academy of Sciences, Washing- already been lost, with 1 to 2 percent cleared annually. ton, D.(:., 1980. 164 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

dangered Species Program are used for such activities 17. Expressed in genetic terms, cases exist ‘(where the in- as listing endangered species, purchasing habitats for troduction of novel sources of major gene resistance protection, and law enforcement. into commercial cultivars of crop plants has resulted in 16. The uses of pest-resistant wheat and corn cultivars on an increase in their frequency of corresponding viru- a large scale for both diseases and insects are classic lence genes in the pathogen’ ’.” This has been reported success stories of host- plant resistance. However, re- in Australia with wheat stem rust, barley powdery mil- cent trends in the Great Plains Wheat Belt are disturb- dew, tomato leaf mold, and lettuce downy mildew. Evi- ing. The acreage of Hessian fly-resistant wheats in Kan- dence suggests that there is considerable gene flow in sas and Nebraska has decreased from about 66 percent the various pathogen populations-e.g., asexual trans- in 1973 to about 42 percent in 1977. Hessian fly infesta- fer can quickly alter the frequency of virulence genes. tions have increased where susceptible cultivars have Furthermore, pressures brought about in the evolu- been planted. In South Dakota in 1978, in an area not tionary process have developed such a high degree of normally heavily infested, an estimated 1.25 million complexity in both resistance and virulence mech- acres of spring wheat were infested resulting in losses anisms, that breeding approaches, especially those only of $25 million to $50 million. An even greater decrease using single gene resistance, can be easily overcome. in resistant wheat acreage is expected in the next 2 to 5 years as a result of releases of cultivars that have improved agronomic traits and disease resistance but that are susceptible to the Hessian fly. Insect resistance has not been a significant component of commercial breeding programs.35 36R, ~;. Shattock, B. ~. Janss~n, R, Whitbread, and D. S. Shaw, ~sotfi{:[l ~,f ‘l’(~chno]o~v Assessment, 11. S. (Mgl’ess, Pest Manage- “h Interpretation of the Frequencies of Host Specific Phenotypes rnenf Sfra~egies in Crop Protection (vol. 1, Washington, D. C.: IJ. S. of Phytophthora infestans in North Wales, ” Ann. Appli. Biol. 86:249, (kn’ernnwnt Printing office, octoher 1979), p. 73. 1977. chapter 9 Advances in Reproductive Biology and Their Effects on Animal Improvement Chapter 9

Page Page Background ...... 167 Other Species ...... 187 The Scientific Era in Livestock Production . . . . . 167 Other Technologies ...... 188 Controlled Breeding...... 168 Aquiculture ...... 189 Scientific Production ...... 170 Poultry Breeding...... 189 Resistance to Change . , , ...... 171 Issue and Options for Agriculture-Animals . . . . 190 Some Future Trends ...... 172 Technologies...... 173 Technologies That Are Presently Useful...... 174 Tables Sperm Storage ...... 174 Artificial Insemination . . , ...... 174 Table No. Page

Estrus Synchronization...... 176 30. Heritabilit y Estimates of Some Economically Superovulation , ...... , 176 Important Traits ...... 169 Embryo Recovery . . , ...... 176 31. Results of Superovulation in Farm Animals . . 174 Embryo Transfer ...... 177 32. Experimental Production of Identical Embryo Storage ...... 177 offspring ...... 178 Sex Selection...... 177 33. National Co\v-Year anci A\7erages for All Twinning ...... 177 Official Herd Records, by Breed May 1, 1978- More Speculative Technologies ...... 178 Apr. 30, 1979...... , ...... , 181 In Vitro Fertilization , . . , , . , , , , ...... 178 34 P1.e{{i(;te(j Diffc?l’enc[? of klilk Yield of Act i~w Parthenogenesis ...... , . . 178 /\l Blllls . . . . . , ...... , ...... 181 Cloning...... 178 Cell Fusion ...... , ...... 179 Figures Chimeras ...... , ...... , . . . . . 179 Recombinant DNA and Gene Transfer...... 179 Figure No. Page Genetics and Animal Breeding...... 179 30. Eras in LJ. S. Beef Production ...... 168 The National Cooperative Dairy Herd 31. Milk Yield/Cow and Cow Population, [Jnit&i Improvement Program ...... 180 States, 1875 -1975 ...... , ...... 170 Other Species. . , ...... , ...... 181 32. Milk Production per Cow in 1958-78 . . . . 17[) Conclusion ...... 183 33. The Way the Reproductive Technologies Impacts on Breeding ...... , . . . 183 Interrelate. , ...... , ...... 175 Dairy Cattle...... 183 34. Change in the Potential Number of ProgcIlj~ Beef Cattle ...... 185 per Sire From 1939 to 1979...... 176 Chapter 9 Advances in Reproductive Biology and Their Effects on Animal Improvement

Background

During the past 30 years, new technologies as from the use of antibiotics in livestock have led to a fundamental shift in the way the feed; United States produces meat and livestock. One environmental concerns, such as the prob- set of these technologies-the subject of this lems of waste removal, especially near fac- section-uses knowledge of the reproductive tory farms; process in farm animals to increase production. the growth of knowledge, in-e. g., the re- ‘l-he impacts of existing breeding technologies productive processes of farm animals and have been great, and much progress is still pos- the accuracy of evaluating the genetic sible through their continued use. The develop- merit of breeding animals; and ment of new technologies is inevitable as well. complementary technologies such as re- frigerated storage and transportation. in a market economy like that of the United States, the factor that most influences the adop- New technologies, from breeding to food de- tion of technology is economics. New technolo- livery systems, have reshaped the traditional gies in reproductive physiology will be used American farm into a modern production sys- widely only if they increase the efficiency of tem that is increasingly specialized, capitalized, breeding programs—i.e., only if they provide and integrated with off-farm services. Applied greater control over breeding than present genetics in animal production has been one of methods do, and only if the economic advan- the forces behind these changes. The technolo- tages of the increased control can be recov- gies that have sprung from it include not only ered. * the new, esoteric techniques for cellular manip- But economic factors are not the only ones ulation discussed in other parts of this report, but also more well-known technologies, like ar- that influence technological change—e.g., poul- tificial insemination. * try and livestock production have influenced and have been influenced by: “’1’e(.tlll(}l(jgi(>s selected !(w discussion in this part of the report in~’oh’(? dirf~rl lllillliplllill ion of S(’,X rells. }Iore spwulati\f> twhnol- ● Government regulation such as meat grad- ogies tol” lllilllil)lllilt ions iit t 11(’ SLlt)(’f>lllllii 1’ 1(’1”~1 il 1’(’ ii SS(>SSP(i }1(>1’[> ing standarcls; iis I\’(?ll. N() (’t’fort t\riis llliid~ [[) (sO\r(’l$ iill te(’hnologies with l)ol~nt iiil . increased awareness of health effects, such for improving the genetic qualities ofIivestock-e.g., management techniques like estrus detection and pregnancy diagnosis were ● .\\ (Iis(.ilss(’(1 ill il])j). III-B, \ (’l’)’ (’ill’IJ’ il(lO})lP1’S of il 1(’(’tllloI()~.V omitted, as wehe various other methods for improving reproduc- ( )1’1(’11 (lo $(1 I 01” ()111(’1’ I Ililll (’(’0110111” ic l’(’il Sl)ll S. tion efficiency.

The scientific era in livestock production

Producing purebred beef livestock has been fenced-in and the longhorn was replaced with the dominant breeding objective throughout new breeds by the turn of the century—the be- most of the 20th century. The open range of the ginning of the “purebred” era. American West and Southwest—the “romantic” era in beef cattle production—lasted until about Pedigree records and visual comparison of 1890. (See figure 30,) Then the range was conformation to breed type were the basic tools

167 168 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

Figure 30.— Eras in U.S. Beef Production Ch. 9—Advances in Reproductive Biology and Their Effects on Animal Improvement ● 169

Table 30.—Heritability Estimates of Some Like land, equipment, and cash, breeding Economically Important Traits stock represents capital available to the commercial farmer. Because all inputs must be used Trait Heritability efficiently, modern herd or flock managers can- Calving interval (fertility)...... 10% Birth weight ...... 40 not afford to leave reproduction to chance Weaning weight...... 30 mating in the pen or on the range. These pres- Cow maternal ability ...... 40 sures for efficient production have been de- Feedlot gain ...... 45 4 Pasture gain ...... 30 scribed as follows: Efficiency of gain...... 40 Final feedlot weight ...... 60 Where dairymen are judged by the number of Conformation score: cows milked in an hour, there is no place for the Weaning ...... 25 slow milking cow or the man who will patiently Slaughter ...... 40 milk her out. There is no place for the time-con- Carcass traits: Carcass grade ...... 40 suming hurdle flock of sheep, for the small flock Ribeye area...... 70 of chickens maintained under extensive condi- Tenderness ...... 60 tions, or for the sow that must be watched Fatthickness ...... 45 while she farrows. By degrees all classes of Retail product (percent) ...... 30 stock are being subjected to selection which Retail product (pounds) ...... 65 Susceptibitity to cancer eye ...... 30 favors animals that need a minimum of individual attention. SOURCE: LarryV. Cundiffand KeithE. Gregory, Beet Caft/e Breeding, USDA, Agriculture Information Bulletin No.2B6, revised November1977, p.9. The scientific basis for modern breeding has developed slowly over the last century. Applied 3 genetics—one part of today’s programs-has may also have economic value, but they are much harder to measure. helped modernize livestock and poultry breeding by elaborating on the variation of continu- The extent to which important economic or ously distributed traits in a population; carrying performance traits are genetically determined over what was known about rapidly reproduc- and heritable varies from trait to trait and from ing laboratory species, like fruit flies or mice, to animal to animal. (See table 30.) Heritability is the much slower reproduction of large farm defined as the percentage of the difference animals; and developing the statistical tech- among animals in performance traits passed niques for predicting breeding values or merit from parent to offspring*—e.g~ bulls and and analyzing breeding programs. s heifers with superior weight at weaning might average 5 pounds (lb) more than their herd- Two examples show the power of breeding mates. Because weaning weight has an average tools and the increased efficiency and produc- heritability estimate of 30 percent, the offspring tivity of today’s breeders’ stocks. of these top performing animals can be ex- ● Over the past 30 years, the average milk pected to average 1.5 1b heavieratweaningthan yield of cows in the United States has more their contemporaries (0.30 X 5 = 1.5). This than doubled. At the same time, the num- improvement can normally be expected to be ber of dairy cows in the United States has permanent and cumulative as it is passed onto been reduced by more than 50 percent. the next generation. The improvement accumu- (See figure 31.) Of this increase in output lates like compound interest in a savings ac- and efficiency, more than one-fourth can count; gains made in each generation are com- be attributed to permanent genetic change pounded on the gains of previous generations. for at least one breed (Holsteins) participating in the Dairy Herd Improvement Pro- 3Nlirhael [. [.erner and H. P. Oonald, &foc/ern Developments in gram. (See figure 32.) Anirr?a/ Breeding [New’ }’ork: Academic Press, 1966). ● Poultry production in the United States has * Heri[ahility and genelir association are important in decisions iit)oot indi~ idl]iil mat ings. hlost hreeding programs are concerned become the most intensive industry among ~i’ it h spreading geoet ir gain rapidly throughout a population (Ilt”rd, tlork): thus two other refinements for selection enter the qlhid., p. 20. pirt ur[’-~tvl(>riit ion irlte[’tiil, and selection differential. ~lbid., p. 126. 170 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Figure 31 .—Milk Yield/Cow and Cow Population, - other species of poultry as well, production United States, 1875-1975 processes have become equally efficient. As A. W. Nordskog has noted: Compared with the breeding of other economically important animals, poultry breeding has been the first to leave the farm . . . to become part of a sophisticated breeding industry. On a commercial level, chickens have been the first to be commercially exploited by the application of inbreeding-hybridization techniques, as earlier used in corn, as well as by methods of selective improvement using the principles of quantitative genetics. Thus, the poultry industry, compared to 1875 ’85 ’95 ’05 ’15 ’25 ’35 ’45 ’55 ’65 1975 other animal industries, seems to have been Year the quickest to apply modern methods of genetic improvement, including the employ- SOURCE: J. T. Reid, “Progress in Dairy Cattle Production,” Agricultural and Food Chemistry. Past, Present, and Future, R. Teranishi (cd.) ment of formally trained geneticists to handle (Westport, Corm.: Avi Press, 1978). breeding technology plus the use of computers and other modern business methods.6

Figure 32.—Milk Production per Cow (Holsteins) in 1958=78 (New York and New England) Scientific production

2-year old Holstein cows in DHIA by Al. Sires Farm resources include land, labor, capital, + 4000 and, increasingly, new knowledge. Today, those who innovate recapture the costs of innovating by maintaining output while lowering costs or + 3000 by increasing output while holding costs down. Some results of the drive toward efficiency have included included increasing specializat ion, intensified + 2000 use of capital and land relative to labor and integration of production phases. + 1000 Poultry and livestock operations have slowly become specialized over the past 50 years. The Base farmer who used to do his own breeding, rais- 1958 1962 1966 1970 1974 1978 ing, feeding and slaughtering is disappearing. Year Now, the beef cattle industry in the United

SOURCE: R. H. Foote, Department of Animal Science, Cornell University, States consists of: the purebred breeder who Ithaca, N.Y. from unpublished data of R. W. Everett, Corneil provides breeding stock, the commercial pro- University. ducer, the feeder, the packer, and the retailer. Similar specialization has occurred for most those for farm species. For turkeys, the use other species—e.g., less than 15 primary breed- of Al in breeding for breast meat has been ers maintain the breeding stock that produces so successful that commercial turkeys can the 3.7 billion chickens consumed each year in no longer breed naturally. The big- the United States. The emergence of other spe- breasted male, even when inclined to do so cialized services—such as AI providers, manage- finds it physically impossible to mount the female. ‘A. W. Nordskog, “Success and Failure of Quantitative Genetic As a result, a full 100 percent of the Theory in Poultry” in Prwceedinga of the International Conference commercial turkey flock in the United on Quantitative Genetics, Edward Pollacket, et al. (cd.) (Amers, States is replaced each year using Al. In Iowa: Iowa State University Press, 1977), pp. 47-51. Ch. 9—Advances in Reproductive Biology and The Effects on Animal lmprovement ● 171 ment consultants, equipment manufacturers— the linking of supply industries (feeds, medi- has accelerated the trend toward specialization, cines, breeding stock) with production and then and has given the commercial operator more with marketing services (slaughtering, dressing, time to concentrate on his specific contribution packaging). Entire industries and the Govern- to the chain of production. ment in combination have produced a complex chain of operations that makes use of Govern- Intensification is the increasing use of some inputs to production in comparison to others. ment inspectors, the pharmaceutical industry, Increasing the use of land and capital relative to equipment manufacturers, the transportation labor describes the development of U.S. agricul- industry, and the processed feed industry in ad- ture, including livestock raising, in this century. dition to the traditional commercial farmer. The “factory” farm typifies this trend. Herds Because of this complex linkage, meat grades, and flocks are bred, . born, and raised in en- cuts, and packaging have become fairly stand- closed areas, never seeing a barnyard or the ard in the American supermarket. Shoppers open range. The best examples of land- and cap- have come to expect these standards; consum- ital-intensive systems are those of poultry ers wanting special services have learned to pay (layers, broilers, and turkeys), confined hog pro- more for them. Thus, the American farm has duction, drylot dairy farming, and some veal changed radically over the past 30 years. This 8 production. change has been described as follows: The greater use of land has been encouraged As farming enterprises grow larger, their by several factors, including improved corn pro- management have to equip themselves with induction for confined hog feeding, programs of formation and resort to technologists to help preventive medicine curtailing the spread of them reach decisions and plan for more distant goals. Industrial developments of this kind diseases in close spaces, and environmental con- widen the range of farming activities, since the trol (light, temperature, water, humidity) to in- old style farmer, sensitive to local markets and crease output under closely controlled condi- operating on hunches, remains as a contrast to tions. However, extensive ranching for beef and those for whom farming is rapidly becoming sheep is still common in the United States; the more of a programme than a way of life. difficulties associated with detecting estrus (“heat”) in these species and their relatively slow Resistance to change rates of reproduction have made it uneconomical to invest in them the capital necessary for New technologies in U.S. agriculture and new intensive farming. Furthermore, beef and sheep ways of producing food and fiber have been on extensive systems forage on marginal land both a cause and an effect of the movement that might otherwise have no use. Beeflot feed- from farms to cities in the 20th century. Com- ing, or the fattening of cattle before slaughter at mercial farmers, operating on thin or nonexist- a centralized location, is the only aspect of the ent profits and under extreme competition, beef industry that is land-intensive; in 1977, ap- have had strong reason to innovate. They have been forced by the availability of new technol- proximately7 one-fourth of U.S. beef cattle were fed" ogies either to do so or to watch their potential earnings go to the neighboring farmer. Various Linking phases of production to eliminate policies that have been adopted to soften the im- waste or inefficiencies in the system has pro- pacts of the “technological treadmill,” have gressed with great speed. For some species, somewhat slowed the exodus from the farms. such linkages now extend from breeding to the They may have been adopted for social reasons, supermarket (and, in the case of fast food but they have also become increasingly costly to chains, to the dinner table). Integration includes society. The taxpayer pays for them; the consumer pays as well for every failure to innovate 7Lyle P. Schertz, et al., Another Revolution in U.S. Farming? on the farms. USDA, ESCS, Agricultural Economic Report No. 441, December 1979. Wundiff, et al., op. cit., p. 9. 172 ● Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

Besides a lack of capital or a lack of interest in Societal pressures are one of the many fac- innovating, some farmers have resisted applied tors that influence the introduction of these genetics because efficiency is not their most im- technologies. Several developments around the portant priority. This attitude has been de- world will have a clear impact on innovation in scribed as follows: general and on genetics in particular: It is easy to see why breeders are unreceptive An expanding population, with its growing to the science of genetics. The business of demand for food products of all kinds. breeding pedigree stock for sale is not just a The growth in income for parts of the pop- matter of heredity, perhaps not even predomi- ulation, which may increase the demand nantly so. The devoted grooming, feeding and fitting, the propaganda about pedigrees and for sources of meat protein. wins at fairs and shows, the dramatics of the Increasing competition for the consumer’s auction ring, the trivialities of breed characters, dollar among various sources of protein, and the good company of fellow breeders, con- which could reduce demand for meat. stitute a vocation, not a genetic enterprise. Increasing competition for prime agri- Farmers are traditionally an independent cultural land among agricultural, urban, group. Many believe that they may not directly and industrial interests. Less-than-prime recapture the benefits of participating in a land may also be brought back into produc- breeding program based on genetics; having no tion as demand rises, and the same pres- records on one’s animals is often preferable to sures may cause land prices to rise high . discovering proof that one’s herd is performing enough to encourage greater, or intensi- poorly. On the other hand, one impact of AI has fied, use of land in livestock production. been to demonstrate to farmers the value of Increasing demand for U.S. food and fiber adopting new technologies. Furthermore, the products ‘from abroad, leading to oppor- economic reward of production records has in- tunities for increased profits for successful creased, since AI organizations purchase only producers. dairy sires with extensive records on relatives. Changes like these will strongly affect the way American farmers produce food and fiber Somefuture trends products. The economics of efficiency and a growing world population will continue to place Applied genetics in poultry and livestock pressure on the agricultural sector to innovate. breeding comprise a group of powerful technol- In animals and animal products, efficiencies will ogies that have already strongly influenced be found in all steps of production. Efforts will prices and profits. Nevertheless, the effect of be made to increase the number of live births genetics is only just beginning to be felt; much and to reduce neonatal calf fertility, presently improvement remains to be made in all species. l0 one of the costliest steps—in terms of animals It has been observed that modern genetics: lost–throughout the world. Estimates of the po- . . . provides a verifiable starting point for the tential monetary benefits of the application of development of the complex breeding operation knowledge obtained from prior research in re- that many populations now require . . . (which) productive physiology range as high as $1 bil- are as far removed from simple selection as the lion per year. Another area for great economies motor car is from the bicycle. in production is genetic gain. Much genetic Of these technologies, some are already in progress remains to be made in all species. regular use, some are in the process of being applied, and others must await further research Certain technologies promise to increase the ability of farmers to capitalize on the genetic im- and development before they become generally provement of economically important traits. available. Suppliers of genetic material (semen, embryos) Slbid., p. 170. will focus increased attention on the value of ‘“E. P. Cunningham, “Currem Developments in the Cenetics of [,iwstock Improvement,” in 15th International Conference on Ani- their products for sale both in the United States mal Blood (hwups and Biochemistry, Genelics 7:191, 1976. and abroad. Ch. 9—Advances in Reproductive Biology and Their Effects on Animal improvement ● 173

The development and application of certain research funds, changing consumer tastes, and key technologies will affect related technol- growth of regulations (for instance, stricter con- ogies—e.g., the availability of reliable estrus trols on environmental quality or hormonal detection and estrus synchronization methods treatments). The expansion of an animal rights should increase the use of AI and embryo trans- movement may influence the degree to which fer in beef and dairy cattle, thereby spreading confinement housing, and therefore controlled genetic advantage. Further progress in the breeding, is acceptable. And increased energy freezing of embryos should facilitate the genetic costs may either encourage development of the evaluation of cows and heifers. technologies (through efforts for greater efficiency) or discourage them (through greater use Other trends that may influence techno- of forage and extensive systems). logical change include the shifting availability of

Technologies

Sexual reproduction is a game of chance. Be- duction of technologies that might prove eco- cause sperm and ova each contain only a ran- nomically advantageous. dom half of the genes of each parent, the number of possible combinations that can result is Several observations can be made about the nearly infinite. Some progeny are likely to sur- state of the art for 16 technologies that enhance vive and reproduce; others die either before the inherited traits of animals. (See also app. birth or without producing offspring. II-C.) The great variation achieved through sexual The technologies are at different stages of re- reproduction produces certain animals that search and development. satisfy the needs and desires of the breeder far more than others. On the other hand, the off- The practice of AI in dairy cattle has had the spring of these outstanding animals are usually greatest practical impact of all the genetic tech- less so than their parents, although they are nologies used in the breeding of mammals. In generally still above average. contrast, not a single farm animal has been successfully raised after a combination of in vitro Animal breeders have invested great effort in fertilization and embryo transplant. The use- improving succeeding generations of domestic fulness of several of the technologies for animal animals, both by limiting the differences due to production, such as recombinant DNA (rDNA) the chance associated with sexual reproduction and nuclear transplantation, is purely specu- and by taking advantage of the favorable combi- lative at this writing. nations that occur. Examples of these efforts include keeping records, establishing progeny The usefulness of the technologies differs from testing schemes, amplifying the reproduction of species to species. . outstanding individuals by AI and embryo trans- These differences can often be explained by fer, and establishing inbred lines to capitalize on biological factors-e. g., sperm storage capabil- their more reliable ability to transmit charac- ities are currently limited for swine because teristics to their offspring. freezing kills so many of the sperm. Manage- Because of these efforts, and because dairy ment techniques are important as well; exten- cattle breeders have adopted innovative tech- sive beef-raising systems have in the past made nologies through the years, far more is known estrus detection and synchronization imprac- about reproduction in the cow than in other tical, thereby limiting the use of AL (Fewer than farm animals. The demand for milk and beef 5 percent of the U.S. beef herd are artificially in- has provided an impetus for the speedy intro- seminated, compared with 60 percent of the na- 174 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals tional dairy herd.) And economics can also play Technologies that are presently useful a role; in general, the lower an animal’s value, the less practical the investment in the technol- SPERM STORAGE ogies, some of which are relatively expensive. The sperm of most cattle can be frozen to – 196° C, stored for an indefinite period, and Several technologies are critical to the introduction then used in in vivo fertilization. Although of others. many of the sperm are killed during freezing, A methodology that could reliably induce success rates [or successful conceptions (table estrus synchronization increases the economic 31)] combined with other advantages of the feasibility of AI and embryo transfer. Likewise, technologies are enough to ensure widespread the refinement of embryo storage and other use of the technology. Short-term sperm storage freezing techniques would advance the develop- (for one day or so) is also well-developed and ment of those technologies still being developed, widely used. like sex selection and embryo transfer. Ad- vances in in vitro fertilization will be especially The major advantages of storing sperm are useful to a better understanding of basic repro- the increased use of desirable sires in breeding ductive processes and therefore to the devel- (see figure 34), the ease of transport and spread opment and application of the more speculative of desirable germplasm throughout the country technologies. and the world, and the savings from slaughtering the bull after enough sperm has been col- The technologies interrelate. lected. The sperm can also be tested for vene- All the technologies combined make possible real and other diseases before it is used. There- almost total control of the reproductive process fore, the use of sperm banks is expected to in- of the farm animal: a cow embryo donor may be crease. Little change is anticipated in semen superovulated and artificially inseminated with processing, other than the continued refine- stored, frozen sperm; the embryos may be rement of freezing protocols, which differ for covered, then stored frozen or transferred di- each species. rectly to several recipient cows whose estrous ARTIFICIAL INSEMINATION cycles have been synchronized with that of the The manual placement of sperm into the donor to insure continued embryonic develop- uterus has played a central role in the dissemi- ment. Before the transfer, a few cells may be nation of valuable germplasm throughout the taken for identification of male or female chro- world’s herds and flocks. Virtually all farm spe- mosomes as a basis for sex selection. Finally, cies can be artificially inseminated, although use two embryos may be transferred to each recip- of the technology varies widely for different ient in an effort to obtain twins. (See figure 33.) species—e.g., 100 percent of the Nation’s domes- Techniques not yet commercially applicable tic turkeys are produced via AI compared with all require embryo transfer in order to be useless than 5 percent of beef cattle. Even honey- ful. They include in vitro fertilization, parthenogenesis, production of identical twins, cloning, Table 31.—Results of Superovulation in Farm Animals cell fusion, chimeras, and rDNA technology. The technologies described in this section are Average number ovulations normally Number of ovulations designed to increase the reproductive efficiency expected with superovulation of farm animals, to improve their genetic merit, cow ...... 1 6-8 and to enhance general knowledge of the repro- Sheep...... 1.5 9-11 ductive process for a variety of reasons, includ- Goat...... 1.5 Pig ...... 13 30 ing concern with specific human medical prob- Horse...... 1 1 lems, such as fertility regulation and better SOURCE: George Seidel, Animal Reproduction Laboratory, Colorado State Uni- treatments for infertility. versity, Fort Collins, Colo. Ch. 9—Advances in Reproductive Biology and Their Effects on Animal Improvement ● 175

Figure 33. —The Way the Reproductive Technologies Interrelate

Sperm Artificial insemination

Recovered embryos Bull Superovulated cow Sexed?

Sexed?

Frozen?

Calves

Recipient herd: synchronized estrus Embryo transfer Each get two for twinning

SOURCE: Office of Technology Assessment 176 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Figure 34.—Change in the Potential Number of Federal regulations that limit the use of pros- Progeny per Sire per Year From 1939 to 1979 taglandins 01* pro#?sto#?ns to inc]uce s~nchro- nized estrus in horses and nonlactating cows are the ma jot’ barrier to more 1%’idespread use of exist ing technology. SUPEROVULATION SL1~]el*()\~llliitioll is the hormonal stimulation of the female, resulting in the release from the Okr; it’y of a kit>ger number of ova than nornla]. (St?e tiil]]e 31. ) (k)mhined with AI and WNIII*YO transt’er of’ the i“(?rtilized ok’;i into surro#ite mothers, sL]l)eI*ol’l]liite(i ova c;iI I result in the production of normal of fsl)ring with the sanle rates of succc?ss as thos(? toilowi )g Ilol”lllal o\’u- kit ion.

‘1’he greatest Imrrier to sll~ei’(]il]l:itiotl is that the degree of success cannot be predicted for an inciividual aninuil. other barriers incluc]e widely Varvillg q~l;~]itv of hormone batches fOI’ Ol’Ul~- Year tiot; treatmeni, l~()()d and Drug Administration (FDA) restrictions, and lack of ckita from which SOURCE: R. H. Foote, Department of Animal Science, Cornell University, to judge the effects of repeated sllpel’ot~t]llltioll. Ithaca, N. Y., unpublished data. In the future, increaseci L]tl(le].stiillciitlg of t)asic physiological mechanisms will facilitate et’- bees and fish can now be artificially insemi- forts to imprm~e the technology. It has addi- nated. tional commercial potential for sheep and cattle It permits the widespread use of germplasm hustmnclry, and much current effort is directed from genetically superior sires. It saves the tmvarcls det~eloping and testing a commercial farmer the cost of maintaining his own sires and procedure. is valuable in disease control, especially when EMBRYO RECOVERY germplasm, rather than animals, is imported or The ability to collect fertilized ova from the exported. An important barrier to the wider use oviducts or uterus is a necessary step for em- of Al, especially in producing beef cattle, is the t)ryo transfer or storage and for many experi- need for application of reliable estrus detection ments in reproductive biology. The technology and estrus synchronization technologies. is especiall.v important for research into produc- An expanded role for Al in the future will de- ing identical twins, performing embryo biopsies pend on the availability of accurate information for sex (ietel*llli]]~itiotl, and other projects. Conl- about the genetic value of sperm available for I)ining superovulation, art i ficial insemination, insemination. A nationwide information system and embryo rec(n’ery makes it possihle to col- for evaluating germplasm presently exists for lect embryos from a young heifer before reach- only one species, dairy cattle. ing puberty. When some disorder has damaged the oviducts or uterus, embryo recovery from a ESTRUS SYNCHRONIZATION Va]uilt)]e l~ninlal makes procreation possible. Estrus, or “heat,” is the period during which the female will allow the male to mate with her. Both surgical and nonsurgical methods are The synchronization of estrus in a herd, using currentl.v in use. Surgical recovery is necessary various drug treatments, greatly enhances Al for sheep, goats, and pigs; such operations are and other reproduction programs. limited by the development of scar tissue. Non- Ch. 9—Advances in Reproductive Biology and Their Effects on Animal improvement ● 177 surgical embryo recovery is preferred for the Adequate culture systems exist for short-term cow and the single ovulation of the horse. The storage of embryos. They hai’~? been d[?~~(?kq)ed approach is especially important in dairy cattle, by tl*ial-iil]d-el.l’01* and are not optimally defin[?d since it can be performed on the farm without for farm species at present. Nmfertheless, COJ%r interrupting milk production. embryos ha~’e been stored for 3 cki.vs in the tied oviduct of a I’al)l]it. No significant advances can be predicted for the immediate future. Long-term storage, or freezing of enlbr.yes, exists, but protoco]s need to be improkfed. As EMBRYO TRANSFER many as two-thirds of the stored embryos die Embryos can be removed from one animal with present methods. However, for some uses and implanted into the oviduct or uterus of embryo freezing is already profitable. another. Both surgical and nonsurgical methods are currently in use, though success rates of the In the future, the de~’elopnwnt of precise enl- latter are much lower. br~o culture technology tvoL]ld help the deJwl- opment of all technologies in~~olkting the pro- The technology can obtain offspring from fe- longed manipulation of gametes and embryos males unable to support a pregnancy, increas- outside the reproducti~w tract. Event uall.v, as ing the number of offspring from valuable fe- freezing technology imprm’es, nearly all em- males and introducing new genes into patho- bryos taken from cattle in North America will gen-free herds. Because more offspring can be be stored, rather than transferred inln]ediately. obtained from the donor, undesirable recessive It appears that embryos successfully stored ~vi]l traits can be rapidly detected. The technology is survive for several centuries and possibly for also used, along with short- or long-term storage millenia. of the embryos, as a means of transporting germplasm rather than the whole animal. Cur- SEX SELECTION rent barriers to its further use are the costs in The ability to determine the sex of the Lln- personnel and equipment, especially for surgi- born, or of sperm at t’ertilization, will ha\’e nu- cal procedures, and the provision of suitable merous practical and experimental applications. recipients for a successful transfer. The most reliable method is karyotyping, by means of which nearly tm’o-thirds of embryos The use of embryo transfer should increase can be sexed. Another method, which tries to in the future, especially with animals of high identif~v sex-specific products of certain genes, value. Nonsurgical methods will increasingly is under development. A reliable method for replace surgical ones, especially for cows and separating ma]e-producing sperm from female- horses. A role for embryo transfer can also be producing sperm has not been achieved, though predicted in progeny testing of females, obtain- several patents are held on ~’arious tests of this ing twins in beef cows, obtaining progeny from type. prepubertal females, and in combination with in vitro fertilization and a variety of manipula- Before any method has any practical effect on tive treatments (production of identical twins, the production of farm animals it must become selfing or combining ova from the same animal, simple, fast, inexpensive, re]iable, and harmless genetic engineering). to the embryo. The present state of the art is largely a consequence of research in male fertil- EMBRYO STORAGE ity and in sperm survival after frozen storage. The ability to store embryos increases the advantages of embryo transfer procedures, low- TWINNING ers the cost of transporting animal germplasm, Twins can be artificially induced by using and reduces the need to synchronize estrus in either embryo transfer or hormonal treatments. recipients. It will also be important in the study The first approach is more effective. Selection and control of genetic drift in animals. among female sheep for natural twin produc- 178 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

tion has been very rewarding, while selection PARTHENOGENESIS for twinning in other species has not received Parthenogenesis, or “virgin birth, ” is the ini- much attention. tiation of development in the absence of sperm. It has not been demonstrated or described for Twinning in nonlitter-bearing species would mammalian species, and the best available infor- greatly improve the feed conversion ratio of mation indicates that the maintenance of par- producing an extra offspring. The most impor- thenogenetic development to produce normal tant barriers, besides the high cost of embryo offspring in mammals is presently impossible. transfer techniques, include extra attention needed for the dam during gestation, parturi- CLONING tion, and lactation. The possibility of producing genetically identical individuals has fascinated both scientists More speculative technologies and the general public. As far as livestock are concerned, there are several ways to obtain IN VITRO FERTILIZATION genetically identical animals. The natural way is The manual joining of egg and sperm outside through identical twins, although these are rare the reproductive tract has, for some species, in species other than cattle, sheep, and pri- been followed by successful development of the mates. For practical purposes, highly inbred embryo through gestation to birth. The species lines of some mammals are already considered include, at this writing, the rabbit, mouse, rat, genetically identical; first generation crosses of and human. Consistent and repeatable success these lines are also considered genetically iden- with in vitro fertilization in farm species has not tical and do not suffer from the depressive ef- yet been accomplished. The cases of reported fect of inbreeding. success of in vitro fertilization, embryo reim- plantation, and normal development in man are Laboratory methods for producing clones in- beginning to be documented in the scientific clude dividing early embryos. The results of re- literature. cent experiments in the production of identical offspring using these techniques are shown in The in vitro work to date has attempted to de- table 32. velop a research tool so that the physiological and biochemical events of fertilization could be Another methodology involves the insertion better understood. Despite the wide public at- of the nucleus of one cell into another, either tention it has received in the recent past, the before or after the original genetic complement of the “receiver” cell is destroyed. Researchers I technology is not perfected and will have little I practical, commercial effect in producing in- have found in certain amphibia that nuclear dividuals of any species in the near future. transplantation from a body cell of an embryo into a zygote can lead to the development of a Practical applications would include: a means sexually mature frog. of assessing the fertility of ovum and sperm; a means of overcoming female infertility by embryo transfer into a recipient animal; and, when Table 32.—Experimental Production coupled with storage and transfer, a means of of Identical Offspring facilitating the union of specific ova and sperm Methodology Result for production of individual animals with pre- Dividing 2-cell embryo in 1 pair identical mouse twins dicted characteristics. half Dividing morulaea in half 8 pairs of identical mouse Many of the practical applications should be- twins come available within the next 10 to 20 years. Dividing 2-cell embryos in 5 pairs of identical sheep Further development, along with the storage of half twins Dividing 4-cell embryos in 1 set identical sheep gametes, should allow fertilization of desired four parts quadruplets crosses. This technology may be combined with aAn embryo with 16 to 50 cells; resembles a mulberry. genetic engineering and sperm sexing in the SOURCE: Benjamin G. Brackett, School of Veterinary Medicine, University of more distant future. Pennsylvania, Kennett Square, Pa. Ch. 9—Advances in Reproductive Biology and Their Effects on Animal improvement . 179

The ideal technique for making genetic copies tion of extra cells to blastocysts. These genetic of any given adult mammal involves inserting components may be from closely related but dif- the nucleus from a body cell (not a sex cell) from ferent species. an adult individual into an ovum. Achieving this Live chimeras between two species of mouse will probably take years, if indeed it is possible have been produced. However, practical appli- at all, since there is some evidence that most cations of chimera technology to livestock are adult body cells are irreversibly differentiated. * not obvious at this stage of development. The Serious technical barriers must be overcome main objective of this research is to provide a before advantages in animal production can be genetic tool for a better understanding of devel- foreseen. opment and maternal-fetal interactions.

CELL FUSION RECOMBINANT DNA AND GENE TRANSFER This technology fuses two mature ova or fer- The mechanics of directly manipulating the tilizes one ovum with another. Combining ova DNA molecules of farm animals have not yet from the same animal is called “selfing.” The been worked out. However, cells from mice combination of ova has resulted in very early have been mixed with pieces of chromosomal development of the transferred embryo, but no DNA, which became stably associated with the further development has been reported. cells’ own DNA. In addition, on September 3, 1980, the successful introduction of foreign Cell fusion technology may someday prove DNA into mouse embryos was announced. The useful for transferring genetic material from a embryos were implanted into surrogate moth- somatic cell into a fertilized single-cell embryo ers who gave birth to mice containing altered for the purpose of cloning. Selfing would rapid- DNA. whether or not the DNA was active is un- ly result in pure genetic (inbred) lines for use as known at this writing. breeding stocks. The technique could also lead to the rapid identification of undesirable reces- Knowledge of the genetics of farm animals sive traits that could be eliminated from the must improve before rDNA or other gene trans- species. fer methods will be of practical benefit in producing meat and livestock products. Before CHIMERAS genes can be altered they must be identified, The production of chimeras requires the fu- and gene loci on chromosomes must be sion of two or more early embryos or the addi- mapped. Work toward this goal has begun only recently and rapid progress cannot be antici-

● Ii) JanLIary 1981, it was reported that body cells from a very pated. Multivariate genetic determinants of [~iii’l,v embryo could art as donors of nuclei for cloned mice. characteristics are anticipated to be the rule.

Genetics and animal breeding

Two characteristics distinguish the reproduc- ments can be made. Reliable information about tion of farm animals from that of single-cell or- the genetic value of particular individuals is the ganisms: animal reproduction is sexual–male key to overcoming limitations, for it can simpli- and female germ cells must be brought together fy specific breeding decisions and spread desir- to initiate pregnancy and produce offspring; able genes throughout the Nations’s herds and and animal reproduction is slower (the genera- flocks. tion interval is longer), thus the economic benefits of specific gene lines may take years to be The use of applied genetics for farm species is captured. These two characteristics limit the indirect. Breeders do not work with individual speed and extent to which genetic improve- genes; rather, they must accept a genetic pack- 180 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

age that inclucies both beneficial and harmful Sire Summary List, ” published biannually. tl’iIitS. i‘ ‘W? bl’eeck?r’s most illlpOI’tiiIlt capital is These summaries are public information. (?mt)odied in the iininliils with which he works. In addition to the official plan, NCDHIP also ‘J-o Llf)~]*iid(? this (;iij)itiil, to ii~creiise the genetic includes several unofficial plans, which have \’iill]~ ot his br[?eding stock, the breeder must less stringent regulations for data collection but Iliit’t? N?liiiblC? illfol’miitioll” 011 the genetic value which offer each dairyman a comparison of his he of the ~(?l’ll~[)liis]ll is considering introducing. herds with other herds across the Nation. The Since iiil incli\’icltiiil tiirnlel. usuiill~ does not results of unofficial plans are not intended to be tlii\e the t’esou]x:es to collect iilld process data used as guidelines for selecting germplasm from on ~]ert’ol*illiill(~e of indi\’iduiils outside his own outside one’s herd. herds, he must turn to outside sources of infor- llli~tioll when deciding which ne\v germplasm to The following characteristics contribute to int reduce. NCDHIP’s success: ‘1’he requirements of such iill information sys- ● It is a cooperative program; no group or in- tem ii]’(? [?xtensitw. Ill the [Jnited States today, dividual is forced to participate. Neverthe- onl.v om? such system exists. The National Co- less, it has successfully brought together ()}]~riitiir[? t)iiir~ HeI*ci Impro\rement Program individuals, State and Federal agencies, (N(II)HIP) is ii illoclel piwgraNl thi~t could be breed associations, and professional and ii(~iipt E?(l to Ot IIC?l’ species where the benefits scientific societies for the pursuit of a com- from ii(l\’iiil[~~(l technok)gies ~’ould be enhanced mon goal. It is almost totally financed by l)y at’;liliit)ility of populiit ioflwide data. the dairymen themselves. In the national coordinating group, all those with an inter- The NationaZ Cooperative Dairy Herd est in the industry have a voice in formu- Improvement Program lating policy for the program. ● It is flexible; a dairyman can use the per- Over the past 50 years, the U.S. dairy indus- formance records from the unofficial plans try has used test records of individual animals to evaluate the animals within his herd or to help in breeding decisions. NCDHIP is a na- he can turn to the official sire summaries tionwide program for collecting, analyzing, and to make comparisons with participating disseminating information on the performance herds throughout the Nation. These data of dairy cattle.12 It is the result of a memoran- are useful both for comparing the perform- dum of understanding among Federal and State ance of one’s herd and breed with others agencies, local dairymen, and industry groups and for selecting new germplasm for in- across the United States. troduction into the herd. ● In NCDHIP, local Dairy Herd Improvement Its data are regarded as impartial; disinterest Association (DHIA) officials go to the dairies to on the part o-f the local DHIA official who collect the performance data on individual ani- collects the data and the high security sur- mals. These data then become part of the @~i- rounding the processed information are cial Dairy Recordkeeping Plans. The data are central to the program’s success. AIPL’s standard for all participating herds across the analyses and sire summaries are respected United States. They are sent to the Animal Im- both nationally and internationally, in no provement Programs Laboratory (AIPL) at small part because of freedom from com- USDA in Beltsvi]le, Md., which analyzes them mercial pressures. and incorporates them into the “USDA-DHIA Approximately 36,000 herds with almost 2.8 11 I%il ip Ililll(ll(’1’, i3iologv and the P“uture qf- Man (New York: Ox- million cows were enrolled in the official plans I’ord t lni~(}rsit}’ Press, 1 $)70), pp. .5.5.5 -557. of NCDHIP in 1979. In each of 18 years recorded IZI:O1. il [.oll)PI(~l(! llistort~ of ~](?l’tol’llliitl(:e tesi ing of dairy cattle between 1961 and 1978, cows enrolled in the in the [ Inil(’({ Stii((~s, sw: ( ;t~l.iild J. King, The Nationa/ Cooperative /)airy Herd Improtwnent Program, [liiir}~ Herd Improvement 1~t- Official Dairy Recordkeeping Plans in NCDHIP tt>r 49, N(), 4, July 1973, IISI)A, /\ KS. have outproduced cows not enrolled by over Ch. 9—Advances in Reproductive Biology and Their Effects on Animal improvement ● 181

4,000 lb of milk per lactation. In the testing year of combining reliable evaluation of germplasm (1977-78), the superiority surpassed 5,000 lb per with the use of reproductive technologies. cow. This 5,()()()-lb superiority represents 52 These technologies are of only academic in- percent more milk per lactation. The increases terest when they are used alone; it is when in production per cow result from improvement superior germplasm can be spread throughout in both management techniques and genetic the Nation that the American consumer producing ability. benefits. Several factors influence the rates of participation in the NCDHIP from State to State, from Other species region to region, and from breed to breed. In Progeny testing schemes for other species are some States, expansion of NCDHIP membership not as developed as they are for dairy cattle. is not a high priority of the State Cooperative There are several reasons for this lack of Extension Service. In some areas, the relative testing: importance of dairying as an enterprise is low; therefore, a strong local DHIA organization ● Difficulty in establishing a selection objective does not exist. Likewise, in areas where dairy- around which to design a testing program. ing is a part-time operation, dairymen have less Milk yield and fat content were obvious time and initiative for participating in the pro- traits for selection in dairy cattle. other gram (although many participate in NCDHIP’s species have no such simple traits for selec- unofficial plans). Where dairymen rely on their tion. It has been observed that, “The lack of own bulls and use little AI in breeding, progeny definition of economic selection objectives testing is extremely limited. No single factor in a precise, soundly based manner is one causes dairymen in some States to take greater of the serious weaknesses of much animal 13 advantage of the superior germplasm available breeding of the past. ” to them. The importance of strong national ● Differences in management systems. Artifi- leadership cannot be overemphasized in ex- cial insemination is essential to the intro- plaining the great differences among breeds in duction of superior germplasm; where it is participation rates. (See table 33.) Farsighted difficult to practice Al, elaborate testing leadership played a large role in developing the schemes are not useful—e.g., in the Na- genetic gain of Holsteins, which represent 90 tion’s beef herds, progeny testing will have percent of the U.S. dairy herd today. to await more widespread use of AI. Though swine are increasingly raised in The genetic gains resulting from NCDHIP are confined housing systems, poor fertility of impressive, suggesting a model for spreading boar sperm after freezing and thawing and genetic superiority throughout the Nation’s heat detection difficulties have limited the other herds. NCDHIP also shows the importance use of AI. ● Conflicting commercial interests. Beef bulls, Table 33.-National Cow-Year and Averages for for example, continue to be sold to some All Official Herd Records, by Breed extent on the basis of fancy pedigrees and May 1, 1978-Apr. 30,1979 lines, with relatively little objective information on their genetic merit. Although Cow-years some genetic improvement programs now lreed (#) Milk (lb) Fat (VO) Fat (lb) exist, the beef breed associations may not ~yrshire ...... 17,135 11,839 3.96’XO 469 iuernsey ...... 57,577 10,858 4.64 504 support interbreed comparisons because Iolstein ...... 2,297,684 15,014 3.64 547 some breeds would show up poorly. ersey...... 89,449 10,231 4.90 501 ● Conflicts between short- and long-term gains. rown swiss. . . . . 24,247 12,368 4.04 500 Iilking shorthorn 2,130 10,451 3.65 381 Cross-breeding for the benefits of hybrid- Iixed and others. 83,139 13,077 3.80 497

)URCE: U.S. Department of Agriculture, Science and Education Administra- ‘31.. E. A. Rowson, ‘Techniques of l.ivestoch lnlpro~renm~t ,“ Ou[- tion, Dairy Herd hnprovement Letter 55, #2, December 1979, pp. 5-6. /ook on Agricuhure 6:108, 1970.

76-565 0 - 81 - 13 182 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

ization is particularly attractive to owners of local and breed groups was formed to try to of commercial herds and flocks who con- consolidate the different systems of the State stantly replace their stocks. This genetic improvement programs. The Federation is now improvement is noncumulative—the im- involved in: 15 provement does not continue from gener- . establishing uniform, accurate records, ation to generation. At present, no strong . assisting member organizations in develop- interest exists for improving the Nation’s ing performance programs, beef herd as a whole, and the individual ● Encouraging cooperation among all seg- breeder cannot effectively evaluate the ments of the industry in using records, germplasm available to him. ● Encouraging education by emphasizing the Swine.—There is no Nationwide testing pro- use of records, gram for hogs in the United States. * However, a ● developing confidence in performance test- study of needed research prepared by the ing throughout the industry. USDA in 1976 noted that the production rate of Despite these efforts, only about 3 percent of approximately 13 pigs marketed per sow per beef cattle nationally are recorded. This rel- year in the United States could be significantly atively low participation rate, when compared improved. The biological potential is at least 20 with NCDHIP, has both a technological and an to 25 pigs per year. Similarly, a successful institutional explanation. Under the largely ex- breeding program, along with other managerial tensive beef raising system in the United States, changes, could reduce the fat and increase the AI is difficult as long as estrus detection lean content of pork by as much as 10 to 15 lb technologies are unavailable. Natural stud serv- per carcass. ice is usually more economical. Institutional bar- The ARS study noted that “. . . an area that riers also prevent the development of a strong warrants particular attention is the develop- genetic evaluation program–e.g., the breed ment of a comprehensive national swine testing associations are not all eager to have their program leading to the identification, selection, breeds consistently compared with others. Like- and use of genetically superior boars, together wise, some owners of bulls for stud service with guidelines for the development and use of would lose business in a strict testing scheme. sow productivity and pig performance in- 14 Goats .—Though little genetic work has been dexes. ” In the case of swine, the increased use done on goats in the past, the dairy goat in- of intensive housing, which allows reproductive dustry has become more visible in the past few control, should increase the impetus for prog- years. The desire of goat breeders to participate eny testing. Likewise, pinpointing areas where in NCDHIP led to the formation of a Coor- considerable improvement remains to be made dinating Sub-Group for Dairy Goats. A review of should lead to the identification of selection the research performed indicated a great need objectives. for research in almost every area of production. Beef.—After World War II, a few breeders As a result, AIPL developed a plan for a genetic became increasingly interested in problems of improvement program. The leadership in the inbreeding and the economic costs of dwarfism. dairy goat industry was convinced that it could By that time, some had been trained in genetics attain genetic improvement faster and at a and some breed associations and State agencies lower cost via NCDHIP than it could for any initiated localized testing programs for these other type of research. traits. In 1967, a “Beef Improvement Federation” In 1979, AIPL received a $15,000 grant from the Small Farms Research Funding to supper *There are several State programs—in Indiana, North Carolina, and Tennessee. Some of these programs may test only growth and the development of genetic evaluation proce not litter size. 14[1 .s. ~epartnlen~ of ~gri~ulture, ~gricultural Research Serv - ISR, [,. Willhanl, ‘i(;~neti(: AN k’itv in the 11. S. Beef [lld Ustl’.v, ire, AILS Na!ional Research Program, .Sn,ine Production, NRP No. JOUIYMI Paper No. J-7923 of the low;a Agricultur:tl itnd Home M 20370,” ()(’t()hw’ 1976. nomk!s I’:xperiment Station, Ames, l~wii, project No. 2?,()()(), n.cj. Ch. 9—Advances in Reproductive Biology and Their Effects on Animal improvement . 183 dures for goats. Genetic evaluations for yield of breeding or producing stock. (DHIA programs dairy goat bucks will be available before the end are the best example of how information might of fiscal year 1980. Because limited genetic im- be distributed.) provement for yield has occurred in dairy goats Resources invested in genetics and in technol- in the past, these evaluations will probably have ogies related to genetics will have high payoffs— a significant impact on the industry. AIPL can 16 virtually guarantee beneficial results because of e.g.) in a classic study of the payoff to research in hybrid corn and in subsequent studies of the data available from NCDHIP, its own exper- other types of genetic improvement, a high tise in genetics, statistics, and computer technology, and the decades of highly effective re- cost/benefit ratio for such research was found. The original study also showed that the absolute search on genetic improvement of dairy cattle market value of a particular product is an im- that can be adapted for the dairy goat industry. portant factor influencing the rate of return on However, funding for the goat testing program a given research expenditure. In general, the remains on a year-to-year basis. greater the aggregate value of the product, the CONCLUSION greater the rate of return on a research expend- 17 NCDHIP has shown how important genetic in- iture. Thus, the large expenditures for meat formation is to the production of meat and dairy and animal products in the United States sug- products. The obstacles to such a program are gest a great payoff in applied genetic research. also formidable, but every failure to capitalize Beef purchases alone account for between 2 and on genetic potential is paid for by American 5 percent of the American consumer dollar, and consumers. It has also shown that where selec- the total market value for beef is more than tion objectives can be identified and agreed on, twice that for corn in the United States. and where conflicting interests can be brought together to develop a program serving all in- DAIRY CATTLE terests, genetic improvement can become a cen- Total milk production has been stable for tral objective in breeding programs across the many years. While milk production per cow has country. Without reliable, evaluative data on gone steadily upward, the number of cows breeding stock, the Nation’s breeders will have during the past 35 years has decreased propor- little interest in adopting new breeding technol- tionately. (See figure 29.) Milk production per ogies as they become available. cow should continue to increase, assuming that no radical changes in present management sys- Impacts on breeding terns occur. The increase in production per cow could continue even if no bulls superior to those An improvement in germplasm, like an in- already available are found, simply as a result of crease in the nutritional content of fertilizer or more farms switching to existing technology new and improved herbicides and pesticides, in- and existing bulls. Moreover, bulls produced creases the quality of the physical capital used from this system are increasing in superiority. on the farm. It is likely that much improvement can still be made in the germplasm of all major The number of dairy cows calved as of Janu- farm animal species using existing technology. ary 1, 1980, was 10,810)000. It has remained relatively stable for the past year, but may de- Selecting for desired characteristics causes a specific qualitative change; it enhances the efficiency of the information contained within each Iszvi ~l.iliches, “Research [;osts and Social Returns: H.vbrid {~01’1) cell. The genetic information in each cell of a and Related Innovations, “Journal of Politica/ Economy 66:419, october 1958. See also R. E. Flrenson, P. E. Waggoner, and \’. W. Rut- farm animal is either more or less desirable or tan, “Economic Benefit From Research: An Example From Agricul- efficient than information in the cells of another ture, ” Science 205: 1101, Sept. 14, 1979. Peterson }’ujino animal, depending on how it performs on im- IW. and Ha.vami, “Technical Change in Agricul- ture, ” Staff Papers series No. DP73-20, Depiirtment of Agrkxdture portant traits. Superior germplasm can be used and Applied klonomics, [ lnitwrsit. v of hlinmsota, St. Paul, Minn., in breeding decisions to upgrade a farmer’s July 1973. 184 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals crease to around 10 million in the next decade if duce sires of cows, and too much emphasis on milk production continues to increase. nonproductive traits of questionable economic value. The progress that has been made has re- Artifical Insemination.—An example of sulted from the increased use of AI, the avail- the interaction between technologies and genet- ability of data through NCDHIP, and the actual ic improvement is shown in table 34. The “pre- use of reliable genetic evaluations. If any of dicted difference” (PD) in milk production rep- these three factors had been missing, far less resents the ability of individual bulls to genet- improvement would have occurred, ically transmit yield—the amount of milk above or below the genetic base that the daughters of Semen Storage.–It is doubtful that major a bull will produce on average due to the genes technological changes in processing semen will they receive. As indicated in table 34, the pre- occur, However, since the rate of conception is dicted difference for milk yield transferred via as important as the genetic merit of a sire to the the bull shows an improvement from 122 to 908 economy of a dairy enterprise, more attention lb for active AI bulls in the United States over will be given to selecting sires of high fertility. the past 13 years. Progress should be made in banking semen by This impressive improvement still lags behind AI studs as a hedge against costs of inflation. In what is theoretically possible. A hypothetical the future, some of the increased costs of hous- breeding program could result in an expected ing and feeding bulls will probably be offset by yearly gain of 220 lb of milk per cow, using AI; semen banking and earlier elimination of many and the biological limits to this rate of gain are bulls. not known. In practice, the observed genetic Sexed Semen.—Sexing of semen to produce trend in the U.S. national dairy herd is about heifer calves (for dairymen) or bull calves (for 100 lb—70 lb from the PDs of bulls plus 30 lb or AI organizations) has been attempted without so from the female, most of which is actually success for many years. carryover effect from the previous use of superior bulls. Perfect determination of the sex of progeny could practically double selection intensity in AI organizations, many of which are coop- two ways—with dams to produce bulls for test- eratively owned by dairymen, have not rigor- ing in AI and dams to produce replacements. If ously applied the principles of AI. Their efforts sexed semen is used with an AI plan, the theo- have been limited by reluctance to break with retical improvement in milk yield would be 33 traditional selection practices, financial con- lb per year, with 23 lb due to selection of dams straints for proper testing of young bulls to pro- for replacements.

Table 34.–Predicted Difference (PD) The value of this additional amount per year of Milk Yield of Active Al Bulls may not seem great for any individual cow, but when it is multiplied by a national herd of 7 Year PD milk (lb) million cows using AI and is accumulated for 10 1967...... 122 years, the economic value, at $0.10/lb, is about 1968...... 198 1969...... 205 $1.1 billion—an average of$110 million per year 1970...... 276 and $231 million during the l0th year. The cost 1971 ...... 301 of sexing semen is not known, since no one has 1972...... 346 1973...... , ...... 348 successfully done it. If a way is found, the cost 1974...... , ...... 338 would have to be under $10 per breeding unit 1975...... 425 for the procedure to be economical. 1976...... 501 1977...... 558 Embryo Transfer.—The transfer of fer- 1978...... 748 1979...... 908 tilized eggs from a cow to obtain progeny has been accomplished with great success, Most SOURCE: Animal Improvement Programs Laboratory, Animal Science Institute, Beltsville Agricultural Research Center, USDA. transfers have involved popular or exotic breed- Ch. 9—Advances in Reproductive Biology and Their Effects on Animal improvement ● 185 ing animals with little regard for genetic poten- gain for milk yield more than two times tial. faster than is occurring today. Improved evaluation of cows, proper economic em- Embryo transfer may never pay for itself in phasis on other traits, and strict adherence terms of milk production of the animals pro- to selection standards are the keys. Bio- duced except indirectly through bulls. Rather, it logical limitations to this rate of genetic im- is used mostly to produce outstanding cows for provement cannot be anticipated in the sale. Other commercial applications for cattle foreseeable future. include obtaining progeny from otherwise in- AI of dairy cattle, with the present intensi- fertile cows, exporting embryos instead of ani- ty of sire selection, should increase the net mals, and testing for recessive genetic traits, worth or profit of animals (increased value Embryo transfer progeny must be worth minus extra costs of the AI program) about $2,500 each to justify the costs and risks. About $10.00/head per year. By 1990, 8 million $1,500 of this represents costs due to embryo dairy cows in AI programs would be worth transfer and $1,000 the costs of producing about $800 million (8 X 106 X 10 X 10 calves normally. If genetic gain from embryo years) more at current market prices as a transfer comes only from dam paths, the ex- result of continued use of AI. pected gain over AI alone is 76 lb/yr. Extra gain Sexing of semen when used with AI may at $0.05/lb above feed cost would have to ac- pay for itself if the cost per breeding unit cumulate for 79 years before added gain would can be kept between $10 and $20. equal even a $300 embryo transfer cost per Embryo transfer is unlikely to pay for itself pregnancy. If less semen is needed (allowing genetically unless the cost is reduced to be- more intensive bull selection), the expected gain tween $50 and $90 per conception. How- of 129 lb/yr must accumulate for 46 years to ever, despite its high costs, it is used to pro- balance an embryo transfer cost of $300 per duce animals of exceptionally high value. pregnancy. (See app. II-C for an explanation of reasons other than genetics why embryo transfer is Embryo transfer and perfect sexing of semen used. ) would combine to improve genetic gain (in milk Estrus synchronization is now available for production) slightly. The use of less semen use with heifers, and should increase the might be possible through application of in vitro use of AI and consequently the genetic im- feasibility based on fertilization. However, provement of dairy cattle. genetic gain would still require holding all costs A secondary benefit of all technologies is down to around $50 to $90 per conception. The the increased number of skilled persons general conclusion is that costs of embryo trans- who can provide technical skills as well as fer must be greatly reduced to be economically educate dairymen in all areas. Also, a feasible if only genetic gain is considered. unique pool of reproductive and genetic Estrus Synchronization.—The availability data has been accumulated. of an effective estrus synchronization method BEEF CATTLE would provide strong impetus for increased use of AI and embryo transfer in dairy cattle. The There is no single trait of overriding ire-’ detection of estrus is an expensive operation; ef- portance (like milk production in dairy cows) to fective control of estrus cycling also requires in- emphasize in the genetic improvement of beef tensive management, adequate handling facil- cattle, the rate of growth is a possibility. * It is ities, and close cooperation between the pro- also difficult to select for several traits at once, ducer, veterinarian, and AI technician. ● Beef and dairy cattle are usually different breeds in the LJnited States. In the literature and in research they are often referred to Summary.— as dit”ferent species. In other countries, notably in Western Europe and in Japan, so-called “dual purpose” cattle are used to produce Proper application of progeny testing with both beef and milk. In the [Jnited States, old dairy cows usually be- selection and AI can increase the genetic come hamburger. 186 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals especially when some are incompatible—e.g., it The standardized measure of weaning weight is desirable to produce large animals to sell, but in beef cattle is the weight at 205 days, adjusted undesirable to have to feed large mothers to for sex of calf and age of dam. In a recent study produce them. There are also other complica- in West Virginia—the Allegheny Highlands Proj- tions. Growth rate has two genetic components, ect—calf weights have averaged an increase of for which one can select—the maternal con- 10 lb per year of participation in the project, via tribution (primarily milk production) and the AI and crossbreeding. Estimates of increased calf’s own growth potential. Other traits of in- value of calves statewide, should the same tests terest are efficiency of growth, carcass quality and AI program be expanded, add up to $3.6 traits (such as tenderness), calving ease, and million per year when calf prices average $50 reproductive traits, such as conception rate to per hundredweight.l9 Rapid adoption of AI first service with AI, could bring about this kind of increase in as little as 40 to 48 months. Genetic improvement programs for beef have two major advantages over those for dairy cat- The costs and returns of AI vary from farm to tle traits such as growth rate and carcass quality farm and with the number of cattle in estrus. In can be measured in both sexes (whereas one general, it becomes more valuable with smaller cannot measure the milk production of bulls); herds, more cows in estrus, higher conception and the traits are more heritable than milk rates, and better bulls. For purebred herds, production. even larger benefits have been estimated—e.g., in a 1969 study, the estimated increase in value Artificial insemination.—Between 3 and per calf when AI was used was $30.02 on pure- 5 percent of the U.S. beef herd is artificially inbred ranches compared to $3.31 on commercial seminated each year. This low rate is due to sev- 20 ranches in Wyoming. eral factors, including management techniques (range v. confined housing), availability of re- A major secondary, or indirect, benefit of the lated technologies (especially, until recently, use of AI is feed saved for other uses. It has estrus synchronization), and the conflicting ob- greatly reduced the number of sires necessary jectives of the individual breeders, ranchers, for stud service and, through radically im- and breed associations. proved milk production, the number of females as well. These reduced requirements together Because little is known about the effectiveness of Al in spreading specific genes through- are equivalent to more than 1 billion bu of corn out the Nation’s beef herds, analysts have con- and other concentrates. This situation will be further enhanced as beef cattle AI expands. centrated on their reproductive performance. Calf losses are heavy throughout the Nation. synchronization of Estrus.—Differences The calf crop-the number of calves alive at in the rates of application of AI between beef weaning as a fraction of total number of females and dairy herds can be explained partly by the exposed to breeding each year—is estimated to differing management systems for the two be between 65 and 81 percent. To put these types of classes of cattle. Dairy herds are kept data in perspective, USDA18 has estimated that a close to the barn for milking and are accus- 5-percent increase in the national calf crop tomed to being approached by humans. In con- would yield a savings of $558 million per year in trast, beef herds may number a few thousand the supply of U.S.-grown beef. Techniques now head on 100,000 acres of arid pasture land. The available can produce such an increase when detection of estrus under these conditions is they are integrated into an adequate manage- difficult. ment program. MB. s, ~iik~l., AI. R. ~’~llsett, P. k:. Lewis, and F.. K. lnskq) “A P,.(,gl.:llll RePolq 011 the Allegheny Highlands Project” Uvlorgan- town, W. \’a.: West t’irginia University, January-December 19791. IM[ I s, [)t,,,i,,.l ,,,(,,11 of ~~gl.i[.llltlll.p, t\gricuttural Research Sel~’- Z(j[). ~l. st[?l,ells and “I’. M~hl’, “Artificial Insemination of Range i(.v, “Beet Production,” /\RS Niitio!lil] Research Pi’Og]siIM Repel’t No. (httle in \V~onling: An Economic Analysis, ” Wyoming Agricultural 20360” (\ Vilshillgtoll, [).(;.: ( ISI)/\, ()(-t(}hel’ 19761. k;xperiment Stiitk)n Bulletin No. 496, 1969. Ch. 9—Advances in Reproductive Biology and Their Effects on Animal Improvement ● 187

It has been predicted that the availability of growth rate; but the programs are much too ex- prostaglindin agents for regulating estrus could pensive to be used over the entire population. increase the number of beef calves born from One problem is that the economic value of the superior bulls by 10 times, and that perhaps 20 product of a beef cow is around 25 percent (or percent of the U.S. beef cow herd could receive even less) of that of a dairy cow. Nevertheless, 2 at least one insemination artificially by 1990. 1 If in populations in which AI is used, embryo this lead to a 50-lb increase in weight for 10 per- transfer was found to be useful for obtaining cent of the calves born, it should be worth $114 more bulls from top cows. The females pro- million to $122 million each year, assuming 80 duced by embryo transfer would be worth mar- or 85 percent net calf crop and $60 per hun- ginally more than females produced conven- dredweight. tionally, but the costs and influence of males The implementation of recently developed could spread over the population through the use of AI. The extent of this use of embryo estrus synchronization technology might in- transfer would be very small; only a few hun- crease the number of beef cows bred artificially dred bulls would be produced per year for very by 4,000,000 in the United States. Such a pro- large populations, and over 99 percent of the gram should be successful in advancing the population would reproduce conventionally. calving date by one week (by decreasing the However, such programs could have consider- calving interval), and in increasing the quality of the calves produced. These new calves could be able economic benefit. Care must be taken to minimize increased inbreeding of the popula- worth about $100 million annually, less about tion with such a breeding scheme. $50 million due to extra costs associated with the synchronization program. Summary.— Sex Control.—Sex control would have a ● AI could substantially improve economical- dramatic effect on the beef industry. In 1971, it ly important traits in beef herds. However, was projected that by 1980 sex control could because of the diversity of traits consid- have an annual potential benefit of $200 mil- ered important by different breed groups lion based on 10 million female calves being re- and the lack of a national beef testing and placed by male calves produced through the recording system comparable to NCDHIP, 22 sexing of semen. At the time of the prediction, economic estimates of its value have not the market value for steers was about $20 more been developed. than for heifers. (Steers wean heavier and gain ● A sexing technology to produce mostly more efficiently. ) Now the margin is much males (they grow faster than heifers) could greater–approximately $50. This potential be of enormous potential benefit to the method of biological control is more attractive beef industry. However, no successful than the use of additives like steroids or im- technique yet exists. plants because of the possible hazards associ- ● Estrus cycle regulation could lead to a sub- ated with them that preclude their use. stantial increase in the number of beef cat- Embryo Transfer.—The possibilities for tle in AI programs. The net benefit of this genetic improvement in beef cattle using em- technology, coupled with AI, may be as bryo transfer have been analyzed. It appears high as $50 million per year. Similarly, the that embryo transfer programs can be devel- availability of reliable progeny records oped to increase the rate of genetic progress for would add to the beneficial impact of AI in beef and would probably contribute significantly to its use in beef cattle. ZIH. c). Hafs, “potential Impact of Prostaglandin on prospects for F’ood f’rom Dairy Cattle,” Proc. L.ufa/vse Symposium, J. w. Lauder- dak and J. H. Sokolowski (eds. ) (Kalamazoo, Mich.: Lfpjohn, 19791, OTHER SPECIES pp. 9-14. Swine.—Much progress has been made in UK. H. F’oot[? ~IMl P. Nli]ler, “what Might Sex Ratio Contro] Mean in the Animal World,’” S.vmposium, Am. Soc. of Animal Science, improving the overall biological efficiency of 1971, pp. 1-1o. pork production in the United States. Improved 188 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

growth rates, feed efficiencies, carcass merit, sheep in the United States until systems for per- and litter sizes have helped keep pork prices formance and progeny testing are implemented down and improve its quality in the Nation’s that will track the number of lambs born and markets. Pork today is leaner and contains more their growth rate, and until routine freezing of high-quality protein calories than it was just a raw semen is achieved.24 few decades ago. Goats.—The research performed on goats is AI in swine production could expand, al- largely designed for application to other ani- though it will be limited by the relatively poor mals. However, interest in goats in the United ability of swine sperm to withstand freezing States and the demand for their products and by the problem of detecting estrus. It will through the world is increasing. be encouraged by the strong trend toward con- NCDHIP has just started providing sire eval- finement housing and integration of all phases uations to goat breeders. These data, along with of hog production. The industry—especially the artifical insemination, should increase milk pro- individual, family-farm type units—would bene- duction. The genetic data might be of particular fit by the establishment of a progeny testing usefulness in the less developed countries scheme to identify superior boars. Publicly where most goat raising occurs. Greater use of available information on genetic merit would all reproductive technologies on valuable Ango- decrease dependence on a few corporate breed- ra goats might be expected. ing organizations. Embryo transfer in swine will be strictly Other technologies limited by difficulties in developing nonsurgical methods of recovery and transfer, and by the The use of any reliable twinning or sex selec- low economic value per animal in comparison to tion technologies will be limited until such pro- cattle and horses. However, embryo transfer is cedures can be made simple, fast, inexpensive, useful in introducing new genetic material into and innocuous. No widespread use of these breeding herds of specific pathogen-free swine technologies should be expected within the next and in transporting genetic material to various decade. regions of the world. The more esoteric techniques for manipu- Sheep. —The processes of selection and of lating sex cells or the germplasm itself will have crossing specific strains, which have been so ef- no impact on the production of animals or fective in poultry and hogs, have been virtually animal products within the next 20 years. In ignored in sheep. Selection of replacement ewes vitro manipulations, including cloning, cell fu- from the fastest growing ewe lambs born as sion, the production of chimeras, and the use of twins and the use of flushing to increase ovula- rDNA techniques, will continue to be of intense tion rates have led to annual increases of 1.8 interest. However, it is unlikely that they will percent in lambing; in one test the market have practical effects on farm production in the weight of lambs ws increased by one lb per year United States in this century. Each technique of cooperation .23 will require more research and refinement. Un- til specific genes can be identified and located, Synchronization of estrus in ewes can be no direct gene manipulation will be practicable. achieved with prostaglandin and many differ- A polygenic basis for most traits of importance ent progestogens. The technique is used exten- can be expected to be the rule rather than the sively in many countries, but no products for exception. this purpose are currently marketed in the United States. Should such techniques become available, limited use for producing breeding stock can be AI rates abroad sometimes approach 100 per- expected. Experience with early users of AI and cent. However, AI will not be used widely on

z3K;. K. Illskeep, personal Lmllllllllllicatioll, 1980. Z41bid. Ch. 9—Advances in Reproductive Biology and Their Effects on Animal improvement ● 189

embryo transfer is strong evidence for the pre- which has been successful, should increase be- dicted use of the technologies, no matter what cause of the savings in transport costs. Although their economic justification. (See app. II-C.) culture systems for mollusks are fairly well- defined, little applied genetics work has been A major, secondary effect of animal research done with these popular marine species. Some in reproductive biology is increased under- success has been reported in selection for standing leading to the possible solution of growth rate and disease resistance of the human problems—e.g., the concept, efficacy, American oyster, and selection for growth rate and safety of the original contraceptive pill was of the slow-growing abalone is underway. The developed and established in animals. It in- crustaceans, of which the Louisiana crayfish is volves the same principle as estrous cycle reg- the largest and most viable industry, are the ulation discussed above. least understood. Successful hybrids of lobsters AQUACULTURE have been developed. Aquiculture is the cultivation of freshwater Aquaculture suffers from an insufficient re- and marine species (the latter is often referred search base on the species of interest. However, to as mariculture). While fish culture is about growing appreciation of and demand for ma- 6,000 years old, scientific understanding of its rine species should result in increased support basic principles is far behind that of agriculture. for basic and developmental work on all aspects Aquiculture is slowly being transformed into a of control, including basic reproductive biology. modern multidisciplinary technology, especially in the industrialized countries. Increasing POULTRY BREEDING awareness of human nutritional needs, over- The quantitative breeding practices of com- fishing of natural commercial fisheries, and ris- mercial breeders have changed very little over ing worldwide demand for fish and fish prod- the last 30 years. Highly heritable traits, such ucts are trends that indicate a growth in inter- as growth rate, body conformation, and egg est in aquiculture as a means to meet the food weight, are perpetuated by mass selection be- needs of the world’s population. cause little advantage is gained from hybrid vigor. Low heritable traits (egg production, fer- As part of the trend toward the high tech- tility, and disease resistance) are perpetuated by nology and dense culturing of intensive aqua- crossbreeding and identified through progeny culture systems in the industrialized countries, and family testing. problems of reproductive control, hatchery technology, feeds technology, disease control, The goals of the industry are to increase egg and systems engineering are all being investi- production of the layers–both in quality and gated. Reproductive control and genetic selec- quantity–and, with broilers and turkeys, to im- tion are important because most commercial prove growth rate, feed efficiency, and yield, as aquiculture operations must now depend on well as to reduce body fat and the incidence of wild seedstocks. Very little information on the defects. animals in culture is available. The technologies of AI and semen preser- With all three of the aquiculture genera (fish, vation have accelerated the advances made mollusks, and crustaceans), selective breeding through quantitative breeding technology. AI is programs have long been established, healthy widely used in commercial turkey breeding be- gene pools are available, and advantageous hy- cause of the inability of modern strains to mate. bridizations have been developed. In fish rais- It makes breeding tests more efficient, steps up ing, culture systems often demand sterile hy- selection pressure on the male line, reduces the brids, especially of carp and tilapia, Selective number of necessary breeder males, and inbreeding of salmon has been limited by political creases the number of females that may be pressures. Very little work has been conducted mated to one male. Semen diluents were intro- with catfish, the largest aquiculture industry in duced to the turkey industry about 10 years ago the United States. The use of frozen sperm, to lower the cost of AI. Currently, a little over 190 . Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

half of the turkeys are inseminated with diluted AI will assume increasing importance. Recent semen. advances in procedures for long-term freezing of chicken semen will allow breeders to extend Preservation of poultry semen by freezing is the use of outstanding sires. The sale of frozen now practiced by several primary breeders. Al- seman may eventually substitute, in part, for though freezing chicken semen causes it to lose the sale of breeder males. some potency, the practice allows increased genetic advancement and the distribution of ge- Dwarf broiler breeders will also assume in- netic material worldwide. creasing importance over the new few years. The dwarf breeder female is approximately 25- The amount of genetic variation available for percent smaller than the standard female, and breeding stock is not expected to diminish in the even though the dwarf’s egg is smaller and the near future. Ceilings for certain traits will even- progeny’s growth rate slightly less than that of tually be reached, but certainly not in the the standard broiler, the lower cost of produc- 1980’s. Advances in breeding laying chickens ing broiler chicks from the dwarf breeder more will be less dramatic than in the past, but efforts than offsets the slight loss in their growth rate. will continue to develop new genetic lines and Dwarf layers and the dwarf breeder hens could to improve reserve lines and crosses to meet fu- reduce production costs by 20 percent and 2 ture needs. percent, respectively. The growth rate of broilers will continue t. There is some interest among poultry breed- increase at 4 percent a year, which suggests ers in cloning, gene transfer, and sex control that birds will be reaching 4.4 lb in 5 weeks by but progress toward successful technologies is the 1990’s. Breeding for stress resistance will be slow. increasingly important, not only because of the increased use of intensive production systems, but also to meet the physiological stresses resulting from faster growth and greater weight.

Issue and Options for Agriculture—Animals

ISSUE: Should the United States in- the impacts of new technology and to rescue the crease support for programs in marginally efficient farmer from bankruptcy. applied genetics for animals and Current projections of U.S. and world popula- animal products? tion growth show increasing demand for all food products. Other predictable trends with Advocates of a strong governmental role in implications for agricultural R&D, include: support of agricultural research and development (R&D) have traditionally referred to the ● growth in income for some populations, small size of the production unit: U.S. farms are which will probably increase the demand too small to support R&D activities. Throughout for sources of meat protein; this century a complicated and extensive net- ● increasing competition among various work of Federal, State, and local agricultural sources of protein for the consumer’s support agencies has been developed to assist dollar; the farmer in applying the new knowledge pro- ● increasing awareness of nutrition issues duced by research institutions. This private/ among U.S. consumers; public sector cooperative network has pro- ● increasing competition for prime agricul- duced an abundant supply of food and fiber, tural land among agricultural, urban, and sometimes in excess of domestic demand. Social- industrial interests; ly oriented policies have been adopted to soften ● increasing demand for U.S. food and fiber Ch. 9—Advances in Reproductive Biology and Their Effects on Animal improvement ● 191

products from abroad, leading to oppor- designed to maintain constant milk supplies tunities for increased profits for successful without large fluctuations in price. producers; and . increasing demands on agricultural prod- The NCDHIP model program for dairy cattle has shown that an effective national program ucts for production of energy. requires the participation by the varied interests in program policymaking in an extension OPTIONS: network, for local collection and validation of A. Governmental participation in, and funding of, data and for education and of expertise in data programs like the National Cooperative Dairy handling and analysis. Also important is a Herd Improvement Program (NCDHIP) could strong leadership role in establishing the pro- be increased. The efforts of the Beef Cattle gram. This option implies that the Federal Gov- Improvement Federation to standardize pro- ernment would play such a role in new procedures could be actively supported, and a grams and expand its role in existing ones. similar information system for swine could be B. Federal funding of basic research in total ani- established. mal improvement could be increased. The fastest, least expensive way to upgrade The option, in contrast with option A, breeding stock in the United States is through assumes that it is necessary to maintain or ex- effective use of information. Computer technol- pand basic R&D to generate new knowledge ogy, along with a network of local represent- that can be applied to the production of im- atives for data collecting, can provide the indi- proved animals and animal products. vidual farmer or breeder with accurate information on the germplasm available, so that he Information presented in this report supports can then make his own breeding decisions. In the conclusion that long-term basic research on this way, the Nation can take advantage of pop- the physiological and biochemical events in ulation genetics and information handling capa- animal development results in increasing the ef- bilities to upgrade one of its most important ficiency of animal production, both in total forms of capital: poultry and livestock. Breed animal numbers and in quality of product. In- associations and large ranchers who sell the creased understanding of the interrelationships semen from their prize bulls based on pedigrees among various systems—including reproduc- rather than on genetic merit may act as barriers tion, nutrition, and genetics—gradually leads to to the effectiveness of such an objective infor- the development of superior animals that effi- mation system. ciently consume food not palatable to humans and are resistant to disease. The benefits of such programs would accrue both to U.S. consumers, in reduced real prices Earlier studies also support the importance of of meat and animal products, and to producers basic research–e.g., the National Research who participate in the programs, in increased Council found in 1977 that”. , . not as much fun- efficiency of production. Consumers spend such damental research on animal problems has been conducted in recent years . . . it should a large part of their incomes on red meat that 25 every increase in efficiency represents millions receive increased funding. ” USDA also found, of dollars saved. Beef producers too, should in a review of various conference proceedings, welcome any assistance in upgrading their congressional hearings, special studies, and stocks. The price of semen has remained rel- other published materials on agricultural R&D atively stable, and semen from bulls rated priorities, strong support for more research on highly on certain economic traits costs only a the basic processes that contribute to reproduc- few dollars more than that from average bulls. tion and performance traits in farm animals: However, efficiency of production is not the 2sN~~t iollill [~f,$(,ill.(,ll (hllll(;il, Worki FIIod aIId Nutrition .stlJd.Vt only value to be upheld in U.S. agriculture—e.g., ‘/’/If; Polfmlia/ (,’onlrilmlions qf’ Rfksfwrch ILVilShill#Oll, l). (:. illlttlol’, in milk production complex policies have been 1977), p. 97. 192 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Specific livestock research areas identified as Regardless of the effectiveness of present having significant potential for increased pro- population control programs or of current duction both in the United States and develop- trends in individual decisions about family size, ing countries include: 1) control of reproductive the output of the Nation’s agricultural activities and respiratory diseases, 2) developing geneti- must increase over the next decades if sufficient cally superior animals, 3) improving nutrition food is to be available for the world’s popula- efficiency, and 4) increasing the reproductive 26 tion. Basic research is the source from which performance of all farm animal species. new applications to increase productivity arise. z611. S. Department of A#ictdture, Science and Education Ad- ministration, Agricuhura/ and Food Research Issues and Priorities (Washington, D. C.: author, 1978), p. xiii. Part III Institutions and Society

Chapter 10. The Question of Risk ...... 197 Chapter 11. Regulation of Genetic Engineering...... , ...... 211 Chapter 12. Patenting Living Organisms...... , ...... 237 Chapter 13. Genetics and Society ...... 257 Chapter 10 The Question of Risk Chapter 10

Page Page Introduction...... 197 Ethical and Moral Concerns. , ...... 207 The Initial Fear of Harm ...... 197 Conclusion ...... 207 Classification of Potential Harm ...... 198 Identification of Possible Harm ...... , . . . . zoo Estimates of Harm: Risk...... 200 The Status of the Current Assessment of Physical Risk ...... 201 Perception of Risk ...... 203 Figures Burden of Proof ...... 203 Other Concerns ...... 204 Figure No. Page Concerns Raised by Industrial Applications .. .204 35. Flow Chart of Possible Consequences of Using Concerns Raised by the Implications of the Genetically Engineered Micro-Organisms , . . 199 Recombinant DNA Controversy for General 36. Flow Chart to Establish Probability of Harm Microbiology...... 204 Caused by the Escape of a Micro-Organism Concerns Raised by the Implications of the Carrying Recombinant DNA ...... 201 Recombinant DNA for Other Genetic 37. Alternative Methods for Transferring DNA From Manipulation ...... 206 One Cell to Another...... 206 Chapter 10 The Question of Risk

Introduction

The perception that the genetic manipulation Yet none of this earlier public concern led to of micro-organisms might give rise to unfore- as great a controversy as has research with re- seen risks is not new. The originators of chem- combinant DNA (rDNA). No doubt it was en- ical mutagenesis in the 1940’s were warned that couraged because scientists themselves raised harmful uncontrolled mutations might be in- questions of potential hazard. The subsequent duced by their techniques. In a letter to the open debates among the scientists strengthened Recombinant DNA Advisory Committee (RAC) of the public’s perception that there was legitimate the National Institutes of Health (NIH) in Decem- cause for concern, This has led to a continuing ber of 1979, a pioneer in genetic transformation attempt to define the potential hazards and the at the Rockefeller University, wrote: “. . . I did chances that they might occur. in 1950, after some deliberation, perform the first drug resistance DNA transformations, and in 1964 and 1965 took part in early warnings against indiscriminate ‘transformations’ that were then being imagined. ”1

‘ Rollin 1). Ho[chkiss, Recombinant DNA Research, vol. 5, NIH publication No. 80-2130, March 1980, p. 484.

The initial fear of harm

For the purposes of this discussion, harm (or judgment, led to a concern that something injury) is defined as any undesirable conse- might go wrong. The dangerous scenario went quence of an act. Such a broad definition is war- as follows: ranted by the broad targets for hypothetical ● SV40 causes cells in tissue culture to be- harm that genetic manipulation presents: injury have like cancer cells, to an individual’s health, to animals, to the en- ● SV40-carrying E. coli might be injected ac- vironment. cidently into humans, The inital concern involved injury to human ● humans would be exposed to SV40 in their health. Specifically, it was feared that combin- intestines, and ing the DNA of simian virus 40, or SV40, with an ● an epidemic of cancer would result. Escherichia coli plasmid would establish a new This chain of connections, while loose, was route for the dissemination of the virus. Al- strong enough to raise questions in at least some though the SV40 is harmless to the monkeys people’s minds. from which it is obtained, it can cause cancer when injected into mice and hamsters. And The virus SV40 has never actually been while it has not been shown to cause cancer in shown to cause cancer in humans; but the po- humans, it does cause human cells to behave tential hazards led the Committee on Recombi- like cancer cells when they are grown in tissue nant DNA Molecules of the National Academy of culture. What effect such viruses might have if Sciences (NAS) to call in 1974 for a deferment of they were inserted into E. coli, a normal in- any experiments that attempted to join the DNA habitant of the human intestine, was unknown. of a cancer-causing or other animal virus to vec- This uncertainty, combined with an intuitive tor DNA. At the same time, other experiments,

197 76-565 0 - 81 - 14 198 ● Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals that were thought to have a potential for harm shown in figure 35. Each letter (A through L) –particularly those that were designed to represents the consequence of a particular com- transfer genes for potent toxins or for resist- bination of events and micro-organisms. For ex- ance to antibiotics into bacteria of a different ample, the letters: species—were also deferred. Finally, one other A C represent the intentional release of micro- type of experiment, in which genes from higher ) organisms known to be harmful to the organisms might have been combined with vec- environment or to man—e.g., in biologi- tors, was to be postponed. The fear was that la- cal warfare or terrorism. tent “cancer-causing genes” might be inadver- B,D represent the inadvertent release of tently passed on to E. coli. micro-organisms known to be harmful to Throughout the moratorium, one point was the environment or to man—e.g., in acci- certain: no evidence existed to show that harm dents at high-containment facilities would come from these experiments. But it was where work is being carried out with a possibility, The scientists who originally raised dangerous micro-organisms. questions wrote in 1975: “. . . few, if any, believe E, I represent the intentional release of micro- that this methodology is free from risk. ”2 It was organisms thought to be safe but which recognized at that time that “. . . estimating the prove harmful—when the safety of orga- risks will be difficult and intuitive at first but nisms has been misjudged. this will improve as we acquire additional F)J represent the intentional release of micro- knowledge. ”3 Hence two principles were to be organisms which prove safe as expected— followed: containment of the micro-organisms e.g., in oil recovery, mining, agriculture, (see table 35, p. 213) was to be an essential part and pollution control. of any experiment; and the level of containment H,L represent the inadvertent release of was to match the estimated risk. These prin- micro-organisms which have no harmful ciples were incorporated into the Guidelines for consequences— e.g., in ordinary accidents Research Involving Recombinant DNA Mole- with harmless micro-organisms. cules, promulgated by NIH in 1976. G)K represent the inadvertent release of micro-organisms thought to be safe but But the original fears surrounding rDNA re- which prove harmful—the most unlikely search progressed beyond concern that humans possible consequence, because both an might be harmed. Ecological harm to plants, ani- accident must occur and a misjudgment mals, and the inanimate world were also consid- about the safety must have been made. ered. And other critics noted the possibility of moral and ethical harm, which might disrupt Discussions of physical harm have recognized both society’s structure and its system of values. the possibility of intentional misuse but have minimized its likelihood. The Convention on the Classification of potential “ Prohibition of the Development, Production, physical harm and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on their Destruction Some combinations of DNA may be harmful which was ratified by both the Senate and the to man or his environment—e.g., if an entire President in 1975, * states that the signatories DNA copy of the poliovirus genetic material is will “never develop . . . biological agents or tox- combined with E. coli plasmid DNA, few would ins . . . that have no justification for prophylac- argue against the need for careful handling of tic, protective, or other peaceful purposes. ” this material. Such a provision clearly includes micro-organisms carrying rDNA molecules or the toxins For practical purposes, the potential harm associated with various micro-organisms is 4[;onvention of the Prohibition of the Development, Production, and Stockpiling of Bacleriolo~ical (Biological) and ‘1’oxin Weapons and on Their Destruction, Washin@on, Umdon, and Moscow, Viecombimm[ DNA Research, vol. 1, DHEW publication No. (NIH) Apr. 10, 1972; entered into force on Mar. 26, 1975 (26 [J. S.”r., 583). 76-1138, August 1976, p. 59. *As of 1980, 80 countries hate ratified the treaty; another 40 31bid. have signed but not ratified. Ch. 10—The Question of Risk . 199

Figure 35.—Flow Chart of Possible Consequences of Using Genetically Engineered Micro-Organisms 1

SOURCE: Office of Technology Assessment. produced by them. It must be assumed that already be obtained—e.g., encephalitis viruses those who signed did so in good faith. or toxin-producing bacteria like C. botulinum or C. tetani. While there is no way to judge the likelihood of developments in this area, the problems that Some discussions have centered around the would accompany any attempt to use pathogen- possibility of accidents caused by a break in con- ic micro-organisms in warfare—difficulties in tainment. Construction of potentially harmful controlling spread, protection of one’s own micro-organisms will probably continue to be troops and population—tend to discourage the prohibited by the Guidelines; exceptions will be use of genetic engineering for this purpose. * made only under the most extraordinary cir- Similarly, the danger that these techniques cumstances. To date, no organism known to be might be used by terrorists is lessened by the more harmful than the organism serving as the scientific sophistication needed to construct a source of DNA has been constructed. more virulent organism than those that can However, the biggest controversy has cen- * A 11 hou~h storkpil in~ of” biological ma rf’are agents is prohibited, tered around unforeseen harm—that micro- l’(’S(’ill’(’tl into 11(W’ il#lllS is Ilot. organisms thought safe might prove harmful. 200 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Discussion of this kind of harm is hindered by discover the one that is most similar and to the difficulty not only of quantifying the prob- observe it often. This process then forms the ability of an occurrence but also of predicting basis for extrapolation. the type of damage that might occur. The different types of damage that can be conjured up are For example, it has been argued that ecologi- limited only by imagination. The scenarios have cal damage can be caused by the introduction of plants, animals, and micro-organisms into new included epidemics of cancer, the spread of oil- eating bacteria, the uncontrolled proliferation environments. Scores of examples from history support this conclusion. The introduction to the of new plant life; and infection with hormone- United States of the Brazilian water hyacinth in producing bacteria. the late 19th century has led to an infestation of The risk of harm refers to the chance of harm the Southern waterways. Uncontrolled spread actually occurring. In the present controversy, of English sparrows originally imported to con- it has been difficult to distinguish the possible trol insects has made eradication programs nec- from the probable. It is, for instance, possible essary. Countless other examples are confirma- that an individual will be killed by a meteor fall- tion that biological organisms may, at times, ing to the ground, but it is not probable. Analog- cause ecological damage when introduced into a ous situations exist in genetic engineering. It is new environment. Yet there is no agreement on in this analysis that debate over genetic engi- whether such analogies are particularly rele- neering has some special elements: the uncer- vant to assessing potential dangers from genet- tainty of what kind of harm could occur, the un- ically engineered organisms. It could be ar- certainty about the magnitude of risk, and the gued-e.g., that a genetically engineered orga- problem of the perception of risk. nism (carrying less than 1 percent new genes) is still over 99 percent the same as the original, Identification of possible harm and is therefore not analogous to the “totally new” organism introduced into an ecosystem. The first step in estimating risk is identifying Some experts emphasize the differences be- the potential harm. It is not very meaningful to tween the situations; others emphasize the simi- ask: How much risk does rDNA pose? The con- larities. cept of risk takes on meaning only when harm is Other analogies have been raised. New identified. The question should be: What is the strains of influenza virus arise regularly. Some likelihood that rDNA will cause a specific dis- can cause epidemics because the population, ease such as in a single individual or in an entire never before exposed to them, carries no pro- population? The magnitude of the possible harm tective antibodies. Yet can this analogy suggest is incorporated in the question of risk, but dif- that relatively harmless strains of E. coli might fers in the two cases. A statement about the risk be transformed into epidemic pathogens? There of death to one person is different than one is disagreement, and debates continue about about the risk of death to a thousand. The right what “could happen” or what is even logically questions must be asked about a specific harm. possible. Since no dangerous accidents are known to have occurred, their types remain conjectural. Estimates of harm: risk Identifying potential harm rests on intuition and Assuming that agreement has been reached arguments based on analogy. Even a so-called on the possibility of a specific harm, what can be risk experiment is an approximation of subse- done to ascertain the probability? What is the quent genetic manipulations. That is why ex- likelihood that damage will occur? perts disagree. No incontestable “scientific method” dictates which analogy is useful or ac- Damage invariably occurs as the result of a ceptable. By their very nature, all analogies series of events, each of which has its own par- share some characteristics with the event under ticular chance of occurring. Flow charts have consideration but differ in others. The goal is to been prepared to identify these steps. A typical Ch. 10—The Question of Risk . 201 analysis determines a probability value for each Although the original charter of RAC under- step-e. g., in figure 36 step II the probability of scored the importance of a risk assessment pro- escape can be estimated based on the historical gram, it was not until 1979 that the details of a record of experiments with micro- organisms. formal program were published. For 5 years, Depending on the degree of containment, the risks were assessed on a case-by-case basis probability varies. It is almost certain that through: 1) experiments carried out under con- experiments on an open bench top, using no tract from NIH, 2) experiments that were de- precautions, will result in some escape to the signed for other purposes but which proved to surrounding environment—a much less likely be relevant to the question of risk, and 3) con- event in maximum containment facilities. (See ferences at which findings were examined. table 35.) From the start, it was difficult to design ex- Two points should be noted. First, each prob- periments that could supply meaningful infor- ability can be minimized by appropriate control mation-e.g., how does one test the possibility measures. Second, the probability that the final that “massive ecological disruptions might event will occur is equal to or less likely than the occur?” Or that a new bacterium with harmful least likely link in the chain. Because the prob- unforseen characteristics will emerge? Still abilities must be multiplied together, if the some experiments were proposed. But because probability of any single step is zero, the prob- these experiments had to be approximations of ability of the final outcome is zero; the chain of the actual situation, the applicability of their events is broken. findings was debated. Here too, experts could and did disagree—not about the findings them- THE STATUS OF THE CURRENT ASSESSMENT OF PHYSICAL RISK selves, but about their interpretation. A successful risk assessment should provide For example, in an important experiment de- information about the likelihood and magnitude signed to test a “worst case situation, ” a tumor of damage that might occur under given cir- virus called polyoma was found to cause no cumstances. It is clear that the more types of tumors in test animals when incorporated into 5 damage that are identified, the more risk assess- E. coli. * Since just a few molecules of the viral ments must be carried out. DNA are known to cause tumors when injected directly into animals, it was concluded that tumor viruses are noninfectious to animals Figure 36.— Flow Chart to Establish Probability of when incorporated into E. coli. If polyoma virus, Harm Caused by the Escape of a Micro-Organism Carrying Recombinant DNA which is the most infective tumor virus known for hamsters, cannot cause tumors in the rDNA Event Probability state in E. coli, it is unlikely that other tumor viruses will do so. This conclusion has had wide- P L Inadvertent incorporation of hazardous gene I spread, but not unanimous, acceptance. It has into micro-organism been argued that there might be “something special” about polyoma that prevents it from 11. Escape of micro-organism into environment P, causing tumors in this altered state; other tumor viruses might still be able to do so. At one

Ill. Multiplication of micro-organism and P meeting of RAC, in fact, it was suggested that establishment in ecological niche 3 experiments with several other viruses be carried out to confirm the generality of the finding. P* But how many more viruses? What is enough?

V. Production of factor to cause disease ‘M. A. Israel, H. W. Chan, W. P. Rowe, and M. A. Martin, “Molec- ular Cloning of Polyoma Virus DNA in Eacherichki Cofi: Plasmid NOTE: P, will always be smaller than any of the other probabilities. Vector Systems,” Science 203:883-887, 1979. *Some combinations of free plasmid and tumor virus DNA did SOURCE: Office of Technology Assessment. cause infections. 202 “ Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

For some, one carefully planned experiment than when it existed freely. * Experts in infec- using the most sensitive tests is sufficient to tious disease have stressed repeatedly that the allay fears. But for others, significant doubt ability of a micro-organism to cause disease about safety remains, regardless of how many depends on a host of factors, all working togeth- viruses are examined. The criteria depend on er. Inserting a piece of DNA into a bacterium is an individual’s perception of risk. unlikely to suddenly transform the organism into a virulent epidemic strain. Many experiments carried out for purposes other than risk assessment have provided Careful calculations can also allay fears about evidence that scenarios of doom or catastrophe the damage a genetically engineered micro-or- are highly unlikely. This is the general consen- ganism might cause. Doomsday scenarios of sus of specialists, not only in molecular biology, escaped E. coli that carry insulin or other but in population genetics, microbiology, infec- hormone-producing genes were recently exam- tious diseases, epidemiology, and public health. ined in another workshop.8 Prior to this workshop, newspaper accounts raised the possibility Experiments have revealed that the structure that an E. coli carrying the gene for human in- of genes from higher organisms (plants and sulin production might colonize humans and animals) differ from those of bacteria. Con- thus upset the hormonal balance of the body. sequently, those genes are unlikely to be expressed accidentally by a bacterium; the original The participants calculated how much insulin fears of “shotgun” experiments have become could be produced. First, it was assumed that a less well-founded. Hence, data gathered to date series of highly unlikely events would occur— have made the accidental construction of a new accidental release, ingestion by humans, stable epidemic strain more unlikely. colonization of the intestine by E. coli K-12. E. coli constitutes approximately 1 percent of the Conference discussions have also contributed intestinal bacterial population, and it was to a better understanding of the risks. At one 6 assumed that all the normal E. coli would be such conference, which was attended by 45 ex- replaced by the insulin-producing E. coli. Insulin perts in infectious diseases and microbiology, it is made in the form of a precursor molecule, was concluded that: proinsulin. It was assumed that 30 percent of all ● E. coli K-12 (the weakened form of E. coli, bacterial protein production would be devoted used in experiments) does not flourish in to this single protein, another highly unlikely the intestinal tract of man; situation. If so, 30 micrograms (ug)-or 0.6 ● the type of plasmid permitted by the Guide- units—would then be made in the intestine. lines has not been shown to spread from E. Although proteins are very poorly absorbed coli K-12 to other E. coli in the gut; and from the intestinal cavity, it was assumed for ● E. coli K-12 cannot be converted to a harm- the sake of argument that 100 percent of the ful strain even after known virulence fac- proinsulin would be absorbed into the circula- tors were transferred to it using standard tion. Thus, 0.6 units of insulin would be added genetic techniques. to the normal daily human production of 25 to 7 30 units—an imperceptible difference. A workshop sponsored by NIH provided a forum for scientists to discuss the risks posed by Calculations like these have been carried out viruses in rDNA experiments. They concluded for several other hormones. Even with the most that the risks were probably fess when a virus implausible series of events, leading to the was placed inside a bacterium in rDNA form greatest opportunity for hormone production,

‘W’orkshop on Studies for Assessment of Potentiul Risks Associ- “(hi the other hiil)d, it hils heen iil’gue[i thiit this has provided iit(?d With Recombinant DNA Kxperimenlal ion, ” k’iiln]outh, Miiss., lriruses with ii new IXILIte for IIissell]illilti(]!l. Netw?rl ht?less, thel’e is June W-21 , 1977. 110 fn’idence thiit viruses Cilll readily escilpe tll)[ll the t)ilcleriil illld 7’(Workshop to Assess Risks for Recomhinilnt DNA Experiments Sul)sequellll.v CilUS(? intection. Itlvoh’ing \’il’ill (k+nornes,” C(MpOIISOIWl h~ Iht? Niltiolliil Institutes “

Other concerns

Concerns raised by industrial Concerns raised by the implications of applications the rDNA controversy for general microbiology Originally concerns involved hazards that might arise in the laboratory. Now that there Questions about the potential harm from are industrial applications of genetic engineer- genetically engineered micro-organisms have ing, the concerns include: led to questions about the efforts currently . risks associated with the laboratory con- employed to protect the public from work being done with micro-organisms known to be hazard- struction of new strains of organisms, . risks associated with industrial production ous. These viruses, bacteria, and fungi are handled daily in laboratory experiments, in the or consumer use of the new strains, and routine isolation of infectious agents from pa- risks associated with the products obtained tients, and in the production of vaccines in the from the new strains. pharmaceutical industry. Many similar considerations apply to the as- Questions have been raised about the efficacy sessment of the first two kinds of risks. Unless of regulations established for these various the organisms used in an industrial production potentially hazardous agents. A full-scale assess- scheme are thoroughly characterized, conjec- ment is not within the scope of this study, but it tured fears about their ability to cause disease is clear that the questions are pertinent. Two will continue. Even with a recombinant orga- conclusions have been reached. nism that has a well-defined sequence of DNA, a break in containment would leave its behavior First, there is a growing belief that the mere in the environment questionable. Experience existence of a classification scheme for hazard- with substances such as asbestos gives rise to ous agents by the Center for Disease Control fears that exposure to the new biological sys- (CDC) is not enough to ensure their safe han- tems might also cause unforseen pathological dling. The Subcommittee on Arbovirus Labora- conditions at some future time. tory Safety was formed recently because of concerns expressed in academic circles. Represent- Hazards associated with products raise dif- atives from universities, the Public Health Serv- ferent questions. The growing consensus in ice, the U. S. Department of Agriculture, and Federal regulatory agencies appears to be that the military, who constituted the subcommit- these products should be assessed like all tee, are preparing a report based on an interna- others—e,g., human growth hormone (hGH) tional survey of laboratory practices and infec- produced by genetically engineered bacteria tions. They found wide variation in the ways should be tested for purity, chemical identity, different agents were handled. Most of their and biological activity just like hGH from human recommendations are identical with those ap- pituitary glands. The possibility of product plicable to rDNA–that appropriate containment variation due to mutation of the bacteria, levels be used with different viruses, that the however, suggests that batch testing and certifi- health of workers be monitored, and that an In- cation might be warranted as well. (For further stitutional Biosafety Committee be appointed to discussion see ch. 11.) serve each institution. Ch. 10—The Question of Risk . 205

Second, little is known about the health ● CDC is preparing a complete revision of its record of workers involved in the fermentation laboratory safety manual, which is widely and vaccine industries. For most industrial used as a starting point by other labora- operations the evidence of harm is almost en- tories. tirely anecdotal. Most industrial fermentations ● The Classification of Etiologic Agents on the are regarded as harmless; representatives of in- Basis of Hazard, which was last revised in dustry characterize it as a “non-problem” that 1974, has been expanded by CDC in collab- has never merited monitoring. Comprehensive oration with NIH into a Proposed Biosafety information on the potential harmful effects Guidelines for Microbiological and Biomedi- associated with research using rDNA-carrying cal Laboratories. These guidelines serve the . micro-organisms will not be available because purpose fulfilled by the Dangerous Patho- the Guidelines consider it the responsibility of gens Advisory Group (DPAG) in the United each institution or company to “determine, in Kingdom, although they lack any regula- connection with each project, the necessity for tory strength. medical surveillance of recombinant-DNA re- ● A comprehensive program in safety, search personnel. ” Hence some institutions health, and environmental protection was might decide to keep records of some or all ac- developed in 1979 by and for NIH. It is ad- tivities; others might not. ministered by the Division of Safety, which includes programs in radiation safety, oc- To be sure, some companies have exceeded cupational safety and health, environmen- the minimal medical standards set by NIH for tal protection, and occupational medicine. fermentation using rDNA-carrying micro-orga- ● The Office of Biohazard Safety, National nisms–e.g., Eli Lilly & Co. requires that all Cancer Institute has just completed a 3- illnesses be reported to supervisors and that any year study of the medical surveillance pro- employees who are ill for more than 5 days grams of its contractors; a report is being must report to a physician before being allowed drafted. to return to work. Any employee taking antibiotics (which might make it easier for bacteria to Although the academic, governmental, and colonize) is restricted from areas where rDNA industrial communities have shown growing in- research is being done until 5 days after the dis- terest in biosafety, * no Federal agency regulates continuance of the antibiotic. At Abbott Labora- the possession or use of micro-organisms except tories, a physician checks into the illness of any for those highly pathogenic to animals and for recombinant worker who is off more than 1 interstate transport. * * Whether such regula- day–a precaution taken only after 5 days off tions are necessary is an issue that extends be- for workers in other areas. Lilly maintains a yond the scope of this study. Nevertheless, computer listing of all workers involved in other countries—for instance the United King- rDNA activities. Lilly, the Upjohn Co., and dom, with its DPAG—have acted on the issue. Merck, Sharp and Dohme have been in the This organization functions specifically to guard process of computerizing the health records of against hazardous micro-organisms, by moni- all their employees over the past several years. toring and licensing university and industrial laboratories and meting out penalties when Work with rDNA has focused attention on necessary. biohazards and medical surveillance—an awareness that had arisen in the past but had not been

sustained. * Consequently, several documents “Curiously, there is no formal society or journal, hut there has on the subject either have been or will be pub- been an annual Biological Safety Conference since 1955, con- lished: ducted on a round-rohin hasis primarily h~ close associates of the late Arnold G. Wedum, M. D.—fornler Director of Industrial Health anci Safet-v at the LI. S. Army Bioiogica] Research Laboratories, Fort ● As of September 1980, the National Institutes of occupational Detrick, Md., who is regarded as the “Father of Microbiological Safety and Health and the Ent’ironnlental Protection Agency were Safety. ” planning to fund assessments of the adequacy of current medical ● “In some States and cities, licensing is required for all facilities surveillance technology, handling pathogenic micro-organisms. 206 ● Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

Concerns raised by the implications of outside the cell. If the DNA is combined within the rDNA controversy for other living cells, the Guidelines do not pertain. Figure genetic manipulation 35 shows several methods that achieve the same goal–transfering genetic material from one cell Altering the hereditary characteristics of an to another, bypassing the normal sexual organism-by using rDNA is just one of the mechanisms of mating. It is particularly signifi- several methods of genetic engineering. The cant that DNA from different species can be definition of rDNA refers specifically to the combined by all these mechanisms, only one of combination of the DNA from two organisms which is rDNA. Different species of bacteria,

Figure 37.-Alternative Methods for Transferring DNA From One Cell to Another

A. The two cells are fused in toto B. A microcell with a fragmented nucleus carries the DNA C. Free DNA can enter the recipient cell in a number of ways: by direct microinjection, through calcium-mediated transformation, or by being coated with a phospholipid membrane in order to fuse with the recipient cell D. The free DNA can be joined to a plasmid and transferred as recombinant DNA

SOURCE: Office of Technology Assessment. Ch. 10—The Question of Risk ● 207 fungi, and higher organisms can all be fused or efficient and successful method of combining manipulated. * genes from very diverse organisms. It is reasonable to ask, however, what would happen if any Opponents of rDNA have stated that combin- of the other methods become equally success- ing genes from different species may disturb an ful. Will a profusion of guidelines appear? Will extremely intricate ecological interaction that is one committee oversee all genetic experiments only dimly understood. Hence, such experiments, it is argued, are unpredictable and therefore hazardous. If so, all the other methods Ethical and moral concerns represented in figure 35 should be included in The perceived risk associated with genetic the Guidelines. Yet they are not. engineering includes ethical and moral hazards The most acceptable explanation for this in- as well as physical ones. It is important to consistency is that rDNA is currently the most recognize that these are part of the general topic of risk. To some, there is just as much risk ● For example, antibiotic resistant plasmids have been trans- to social values and structure as to human ferred from Staphylococcus aureus to Bacillus subtilis across species barriers by transformation, not by rDNA. Foreign genes health and the environment. (For further dis- for the enzyme amylase have also been introduced into B. subtilis. cussion see ch. 13.)

Conclusion

Thus far, no demonstrable harm associated The potential benefits must always be con- with genetic engineering, and particularly sidered along with the risks. Decisions made by rDNA, has been found, But although demonstra- RAC have reflected this view—e.g., when it ble harm is based on evidence that damage has approved the cloning of the genetic material of occurred at one time or another, it does not the foot-and-mouth disease virus. The perceived mean that damage cannot occur. benefits to millions of animals outweighed the potential hazard. Conjectural hazards based on analogies and scenarios have been addressed and most have Recombinant DNA techniques represent just proved less worrisome than previously as- one of several methods to join fragments of sumed. Nevertheless, there is agreement that DNA from different organisms. The current certain experiments, such as the transfer of Guidelines do no extend to these other tech- genes for known toxins or venoms into bacteria, niques, although they share some of the same should still be prohibited because of the real uncertainties. Ignoring the consequences of the likelihood of danger. Still other experiments other technologies might be viewed as an incon- cannot clearly be shown to be hazardous or sistency in policy. readily dismissed as harmless. Hence, a political decision is likely to be required to establish While the initial concerns about the possibili- what constitutes acceptable proof and who ty of hazards at the laboratory level appear to must provide it. have been overstated, other types of potential Given that potential harm can be identified in hazards at different stages of the technology some cases, its probable occurrence and magni- have been identified. Emphasis has shifted tude quantified, and perceived risk taken into somewhat from conjectured hazards that might account, a decision to proceed is usually based arise from research and development to those on society’s willingness to take the risk. This that might be associated with production tech- triad of the physical (actual risk), psychological nologies. As a consequence, there is a clearer (perception of risk), and political (willingness to mandate for existing Federal regulatory agen- take risk) plays a role in all decisions relating to cies to play a role in ensuring safety in industrial genetic engineering. settings. Chapter 11 Regulation of Genetic Engineering Chapter 11

Page [introduction . . , . . . . . , . . . . , . . . . , ...... , 211 State and Local Law ...... , 229 Framework for the Analysis...... 211 Conclusion ...... 230 Current Regulation: the NIH Guidelines...... , 212 Issue and options ...... 230 Substantive Requirements ...... 212 Administration ...... , ...... 212 Provisions for Voluntary Compliance . . , . . .215 Evaluation of the Guidelines . , . . . , . . . . . , . . . 216 Tables The Problem of Risk ...... 216 The Decisionmaking Process...... 221 Table No. Page Conclusion...... 223 35. Containment Recommended by National other Means of Regulation ...... 224 Institutes of Health ...... ,213 Federal Statutes...... 224 36. Statutes That Will Be Most Applicable? to “T’ort Law and Workmen’s Compensation . . , . .227 Commercial Genetic Engineering...... 224 Chapter 11 Regulation of Genetic Engineering

Introduction

Although no evidence exists that any harmful with respect to guidelines for rDNA.) Three organism has been created by molecular genetic other available modes of oversight or regulation techniques, most experts believe that some are current Federal statutes, tort law, and State risk* is associated with genetic engineering. and local law. One kind is relatively certain and quantifiable— Framework for the analysis that of working with known toxins or pathogens. Another is uncertain and hypothetical– In deciding how to address the risks posed by that of the possible creation of a pathogenic or genetic engineering, some of the important otherwise undesirable organism by reshuffling questions that need to be examined are: genes thought to be harmless. These may be . How broadly the scope of the issue (or thought of as physical risks because they con- problem) should be defined. cern human health or the environment. —Who identifies the risks and their mag- Concern has also arisen about the possible nitude? long-range impacts of the techniques—that they —Who proposes the means for addressing may eventually be used on humans in some the problem? morally unacceptable manner or may change ● The nature of the procedural, decisionmak- fundamental views of what it means to be hu- ing mechanism. man. These possibilities may be thought of as —Who decides? cultural risks, since they threaten fundamental —Who will benefit from the proposed ac- beliefs and value systems. 1 tion and who will bear the risk? —Will the risk be borne voluntarily or in- The issue of whether or not to regulate voluntarily? molecular genetic techniques—and if so, to —Who has the burden of proof? what extent—defies a simple solution. Percep- —Should a risk/benefit analysis, or some tions of the nature, magnitude, and acceptabili- other approach, be used? ty of the risks differ drastically. Approximately ● The available solutions and their adequacy. 6 years ago, when the scientific community it- —Should there be full regulation, no reg- self accepted a moratorium on certain classes of ulation, or something in-between? recombinant DNA (rDNA) research, some sci- —What actions and actors should be cov-‘ entists considered the concern unnecessary. To- ered? day, even though the physical risks of rDNA re- —What is the appropriate means for en- search are generally considered to be less than forcing a regulatory decision? originally feared—and the realization of its –Which agency or other group should do benefits much closer–some people would still the regulating? prohibit it. Underlying these questions is the proposition, The Federal Government’s approach to this widely accepted by commentators on science issue has been the promulgation of the Guide- policy, that scientists are qualified to assess lines for Research Involving Recombinant DNA physical risk, since that involves measuring and Molecules (Guidelines), by the National Insti- evaluating technical data. However, a judgment tutes of Health (NIH). (See app. III-C for infor- of safety (the acceptability of that risk) can only mation about what other countries have done be made by society through the political proc-

● ess, since it involves weighing and choosing As used in this chapter, risk means the possibility of harm. ‘I-he z 3 probability ot that harm occurring may he extremely low anckr among values. 456 Scientists are not nec- highly uncertain. ‘H. “Ilistam, ftngelhardt, Jr., ‘Taking Risks: Some Background ‘William W. I,owrance, Of Aceep(able Risk: tkience and the De- Issues in the I)etxite (k)ncerning Recombinant DNA Research, termination of &fety (l,os Altos, (hlit.: wi]lianl Kaufmann, ]n(~., Southern (;al(~ornia Law Review .51:6, pp. 1141-1151, 1978. 1976).

211 212 . impacts of Applied Genetics—Micro-Organisms, Plants, and An/reals essarily considered to be more qualified to make high value they place on unrestricted research decisions concerning social values than other and because of possible conflicts of interest. well-informed persons; they may in fact be less Moreover, according to this view, if society is to qualified when the decision involves possible bear a risk, it should judge the acceptability of 7 restrictions on scientific research because of the that risk and give its informed consent to it. *

((:f)ntilll:~!fl,f}olll p. 211) ‘tAlvin W. Wcinlwrg, “Scienct~ illl[l ‘1’l’illlS-SCit? ll(X?, ” A4inerva, 10:2, April 1972. 7Engelhardt, op.cit.; Lowrance, op.cit.; and Bazelon, op. cit. 4Allan Mazur, ‘(Disputes Between Kxperts, ” Minerva 11:2, April ● [n practice, it may often he difficult to keep the two kinds of 1973. decisions separate, since the values of individual scientists may in- ‘A1.thur Kantrowitz, ‘t’r’he Science Court Experiment, ” Juri- tluence their interpretation of technical data, and since policy- metrics ./ourna/, vol. 17, 1977, p. 332. makers may not have the technical competence to understand the ‘David L. Bazelon, “Risk and Responsibility, ” Science, vol. 205, risks sufficiently.e July 20, 1979, pp. 277-280. ‘Weinberg, op. cit.; and Bazelon, op. cit.

Current regulation: the NIH Guidelines

The Guidelines have been developing in from these prohibitions. Five types of experi- stages over a period of approximately 6 years as ments are completely exempt. scientists and policymakers have grappled with Those experiments that are neither prohib- the risks posed by rDNA techniques. (This his- ited nor exempt must be carried on in ac- tory, discussed in app. III-A, is crucial to under- cordance with physical and biological contain- standing current regulatory issues, and it serves ment levels that relate to the degree of potential as a basis for evaluating the Guidelines.) They hazard. (See table 35.) Physical containment re- represent the only Federal oversight mecha- quires methods and equipment that lessen the nism that specifically addresses genetic engi- chances that a recombinant organism might es- neering. cape. Four levels, designated P1 for the least restrictive through P4 for the most, are defined. Substantive requirements Biological containment requires working with weakened organisms that are unlikely to sur- The Guidelines apply to all research involving vive any escape from the laboratory. Three rDNA molecules in the United States or its ter- levels are specified. Classes of permitted ex- ritories conducted at or sponsored by any in- periments are assigned both physical and bio- stitution receiving any support for rDNA re- logical containment levels. Most experiments search from NIH. Six types of experiments are using Escherichia coli K-12, the standard labora- specifically prohibited: 1) the formation of tory bacterium used in approximately 80 per- rDNA derived from certain pathogenic orga- cent of all experiments covered by the Guide- nisms; 2) the formation of rDNA containing lines, may be performed at the lowest contain- genes that make vertebrate toxins; 3) the use of ment levels. the rDNA techniques to create certain plant pathogens; 4) transference of drug resistance ADMINISTRATION traits to micro-organisms that cause disease in The Guidelines provide an administrative humans, animals, or plants; 5) the deliberate framework for implementation that specifies release of any organism containing rDNA into the roles and responsibilities of the scientists, the environment; and 6) experiments using their institutions, and the Federal Government. more than 10 liters (1) of culture unless the The parties who are crucial to the effective rDNA is “rigorously characterized and the operation of the system are: 1) the Director of absence of harmful sequences established. ” A NIH, 2) the NIH Recombinant DNA Advisory procedure is specified for obtaining exceptions Committee (RAC), 3) the NIH Office of Recombi- Ch. 11—Regulation of Genetic Engineering ● 213

Table 35.–Containment Recommended by proposed actions. Such actions include: 1) National Institutes of Health assigning and changing containment levels for Biological—Any combination of vector and host must be experiments, 2) certifying new host-vector sys- chosen to minimize both the survival of the system tems, 3) maintaining a list of rDNA molecules ex- outside of the laboratory and the transmission of the empt from the Guidelines, 4) permitting excep- vector to nonlaboratory hosts. There are three levels of biological containment: tions to prohibited experiments, and 5) adopting HV1– Requires the use of Escherichia coli K12 or changes in the Guidelines. other weakened strains of micro-organisms that are less able to live outside the laboratory. For other specified actions, the Director need HV2– Requires the use of specially engineered strains only inform RAC, the IBCs, and the public of his that are especially sensitive to ultraviolet light, decision. The most important of these are: 1) detergents, and the absence of certain uncommon chemical compounds. making minor interpretive decisions on contain- HV3– No organism has yet been developed that can ment for certain experiments; 2) authorizing, qualify as HV3. under procedures specified by RAC, large-scale Physical—Special laboratories (P1-P4) work (involving more than 101 of culture) with P1— Good laboratory procedures, trained personnel, rDNA that is rigorously characterized and free wastes decontaminated. P2— Biohazards sign, no public access, autoclave in of harmful sequences; and 3) supporting labora- building, hand-washing facility. tory safety training programs. Every action P3– Negative pressure, filters in vacuum line, class II taken by the Director pursuant to the Guide- safety cabinets. P4– Monolithic construction, air locks, all air lines must present ‘(no significant risk to health decontaminated, autoclave in room, all or the environment. ” experiments in class Ill safety cabinets (glove box), shower room. RAC is an advisory committee to the Director

SOURCE: Office of Technology Assessment. on technical matters. It meets quarterly. Its purpose, as described in its current charter of June 26, 1980 (and unchanged since its inception in October 1974), is as follows: The goal of the Committee is to investigate nant DNA Activities (ORDA), 4) the Federal In- the current state of knowledge and technology teragency Advisory Committee on Recombinant regarding DNA recombinant, their survival in DNA Research (Interagency Committee), 5) the nature, and transferability to other organisms; Institution where the research is conducted, 6) to recommend guidelines for the conduct of the Institutional Biosafety Committee (IBC), 7) recombinant DNA experiments; and to recom- the Principal Investigator (PI), and 8) the Bio- mend programs to assess the possibility of logical Safety Officer. spread of specific DNA recombinant and the possible hazards to public health and to the en- The Director of NIH carries the primary bur- vironment. This Committee is a technical commit- den for the Federal Government’s oversight of tee, established to look at a specfic problem. (Em- rDNA activities, since he is responsible for im- phasis added.) plementing and interpreting the Guidelines, establishing and maintaining RAC (a technical ad- The charter and the Guidelines also assign it certain advisory functions that have changed visory committee) and ORDA (whose functions over time. are purely administrative), and maintaining the Interagency Committee (which coordinates all The RAC is composed of not more than 25 Federal activities relating to rDNA). Under this members. At least eight must specialize in mo- arrangement, all decisions and actions are taken lecular biology or related fields; at least six must by the Director or his staff. For major actions, be authorities from other scientific disciplines; the Director must seek the advice of RAC, and and at least six must be authorities on law, he must provide the public and other Federal public policy, the environment, public or oc- agencies with at least 30 days to comment on cupational health, or related fields. In addition,

76-565 0 - 81 - 15 214 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals representatives from various Federal agencies with the PI, the scientist receiving the funding. serve as nonvoting members. This person is responsible for determining and implementing containment and other safe- ORDA performs administrative functions, guards and training and supervising staff. In ad- which include reviewing and approving IBC dition, the PI must also submit a registration membership and serving as a national center document that contains information about the for information and advice on the Guidelines project to the IBC, and petition NIH for: 1) cer- and rDNA activities. tification of host-vector systems, 2) exceptions The Interagency Committee was established in or exemptions from the Guidelines, 3) and de- October 1976 to advise the Secretary of the then termination of containment levels for experi- Department of Health Education and Welfare ments not covered by the Guidelines. Further- (HEW) [now Health and Human Services more, all of the above have certain reporting re- (DHHS)] and the Director of NIH on the coor- quirements designed so that ORDA is eventually dination of all Federal activities relating to informed of significant problems, accidents, vio- rDNA. It has thus far produced two reports. Its lations, or illnesses.** first, in March 1977, concluded that existing The IBC is designed to provide a quasi-inde- Federal law would not permit the regulation of pendent review of rDNA work done at an in- all rDNA research in the United States to the ex- stitution. It is responsible for: 1) reviewing all tent considered necessary and recommended rDNA research conducted at or sponsored by new legislation, specifying the elements of that the institution and approving those projects in legislation. ’” The second, in November 1977, conformity with the Guidelines; 2) periodically surveyed international activities on regulating reviewing ongoing projects; 3) adopting emer- the research and concluded that, while appro- gency plans for spills and contamination; 4) priate Federal agencies should continue to work lowering containment levels for certain rDNA closely with the various international organiza- and recombinant organisms in which the ab- tions, no formal governmental action was neces- sence of harmful sequences has been estab- sary to produce international control by means 11 lished; and 5) reporting significant problems, of a treaty or convention. It is currently con- violations, illnesses, or accidents to ORDA sidering issues arising from the large-scale in- within 30 days.*** The IBC must be comprised dustrial applications of rDNA techniques. of no fewer than five members who can col- Under the Guidelines, essentially all the re- lectively assess the risks to health or the en- sponsibility for overseeing rDNA “experiments vironment from the experiments. At least 20 lies with those sponsoring or conducting the re- percent of the membership must not be other- search. The Institution must implement general wise affiliated with the institution where the safety policies, * establish an IBC, which meets work is being done, and must represent the in- specified requirements, and appoint a Biological terests of the surrounding community in pro- Safety Officer. The Biological Safety Officer, who tecting health and the environment. Comm- is needed only if the Institution conducts ex- ittee members cannot review a project in periments requiring P3 or P4 containment, (see which they have been, or expect to be, involved table 35) oversees safety standards. The initial or have a direct financial interest. Finally, the responsibility for particular experiments lies Guidelines suggest that IBC meetings be public; minutes of the meetings and submitted docu- ‘Interim Report of the Federal Interagency Committee on Recom- ments must be available to the public on binant DNA Research: Su,gested Elements-for Legislation, Mar. 15, request. 1977, pp. 9-10. IDlbid., pp. 11-15, I I Report of the Federal intet-~ency Committee on Recombinant ● ● The PI is required to report this information within 30 days to DNA ftesearch: International Activities, Novendxx 1977, pp. 13-15. ORDA and his IBC. The Biological Siifet~ officer musl report the “These include conducting imy heid[h surveillance that it deter- same to the Institution and the IBC unless the PI has clone so. The mines to be necessary and ensuring iippr~priiiie tritining for the institution must report within 30 dii~s to ORDA unless the PI or IBC, Biological Safety officers, Principal lnv{?sti~iit~rs, and labora- IB(: has done so. tory staff. *” “It does not have to report if the PI hits done SO. Ch. n-Regulation of Genetic Engineering ● 215

The requirements imposed on an institution may request a presubmission review of the and its scientists are enforced by the authority records needed to register its projects with NIH. of NIH to suspend, terminate, or place other The DHHS Freedom of Information Officer conditions on its funding of the offending proj- makes an informal determination of whether ects or all projects at the institution. Compliance the records would have to be released. If they is monitored through the requirements for noti- are determined to be releasable, the records are fication mentioned above. returned to the submitting company. The Guidelines also require that NIH consult with PROVISIONS FOR VOLUNTARY COMPLIANCE any institution applying for an exemption, Organizations or individuals who do not re- exception, or other approval about the content ceive any NIH funds for rDNA research are not of any public notice to be issued when the ap- covered by the Guidelines. These include other plication involves proprietary information. As a Federal agencies, institutions and individuals matter of practice, such applications are also funded by those agencies, and corporations. considered by RAC in nonpublic sessions. Federal agencies other than NIH that conduct Large-scale experiments (more than 10 1 of or fund rDNA research have proclaimed their culture) with rDNA molecules are prohibited voluntary compliance with the Guidelines. * unless the rDNA is “rigorously characterized Staff scientists have been so informed by memo- and the absence of harmful sequences estab- randa. As for outside investigators, this policy lished.” Such experiments are actually scale-ups has been implemented through the grant appli- of potential industrial processes. Those meeting cation process. Instructions in grants appli- this standard may be approved by the Director cations contain policy statements regarding of NIH under procedures specified by RAC. * At compliance with the Guidelines, and applicants its September 1979 meeting, RAC adopted pro- are sometimes contacted to ascertain their cedures for review that require the applicant to knowledge of the Guidelines. Information has submit information on its laboratory practices been requested for certain experiments, and and containment equipment. Subsequently, rec- IBC membership has been reviewed. From time ommendations were developed for large-scale to time, the agencies have consulted with NIH uses of organisms containing rDNA. These were on matters that need interpretation. published in the Federal Register on April 11, Part VI of the Guidelines is designed to en- 1980. Besides setting large-scale containment courage voluntary compliance by industry. It levels, they require the institution to appoint a creates a parallel system of project review and Biological Safety Officer with specified duties, IBC approval analogous to that required for and to establish a worker health surveillance NIH-funded projects, modified to alleviate in- program for work requiring P3 containment. At dustry’s concerns about protection of pro- its September 1980 meeting, RAC modified its prietary information. review procedures so that the application need only specify the large-scale containment level at The Freedom of Information Act requires which the work would be done, without pro- Federal agencies, with certain exceptions, to viding details on containment equipment. RAC make their records available to the public on re- will continue to review the biological aspects of quest.One of the exceptions is for trade secrets the applications in order to determine that and proprietary information obtained from rDNA is rigorously characterized, that the ab- others. Part VI contains several provisions for sence of harmful sequences is established, and protecting this information. Perhaps the most that the proposed containment is at the ap- important is a process whereby a corporation propriate level. *These agencies are the National Science Foundation, the De- partment of Agriculture, the Department of Energy, the Veterans “It is NIH, not the company proposing the scale-up, that deter- dministration, and the Center for Disease Control. Two other mines if the rDNA to be used is “rigorously characterized and the gencies, which have expressed interest in this research but are absence of harmful sequences established.”. *2 ot currently sponsoring any projects, are the Department of De- Iz(;uide]ines for Research [nvolving Recombinant DNA Mole- ;nse and the National Aeronautics and Space Administration. cules, sec. lk’-E-l-b-(3)-(d). 216 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Evaluation of the Guidelines sight has been substantially reduced to the point where almost none remains. Under the 1976 Two basic issues must be addressed. The first Guidelines, all permitted experiments ultimately is how well the Guidelines confront the risks had to be reviewed by the IBC and ORDA before from genetic engineering, which may not have a they could be started; the 1978 Guidelines no definitive answer in view of the uncertainty longer required preinitiation review of most associated with most of the risks. Consequently, experiments by ORDA, although ORDA con- it is also necessary to consider a second issue— tinued to maintain a registry of experiments whether confidence is warranted in the deci- and to review IBC decisions. Under the sionmaking process responsible for the Guide- November 1980 revision to the Guidelines, there lines. will be no Federal registration or review of ex- THE PROBLEM OF RISK periments for which containment levels are The Guidelines are designed to address the specified in the Guidelines. About 97 percent of risks to public health and the environment from the permitted experiments fall into this either rDNA molecules or organisms and vi- category. ruses containing them. The underlying premise Preinitiation review of experiments by RAC is that research should not be unreasonably has been an important part of the oversight restricted. This is essentially a risk-benefit ap- mechanism. Expert review encourages experi- proach; at the time that the original Guidelines mental design to be well thought out and pro- were drafted, it represented a compromise be- vides a means for catching potential problems, tween the extremes of no regulation and of no e.g., one application reviewed by RAC never research without proof of safety. physical and mentioned that the species to be used as a DNA biological containment levels were established donor was capable of manufacturing a potent for various experiments based on estimated neurotoxin; it was turned down after a RAC degrees of risk. The administrative mechanism member familiar with the species brought this created by the Guidelines is that of a Federal fact to the Committee’s attention.13 agency —NIH—advised by a diverse body of experts— RAC. Scientific advice on the technical The burdens imposed on rDNA activities by aspects of risk assessment is provided by techni- the Guidelines appear to be reasonable in view cal experts on RAC; public input is provided by of continuing concerns about risk. Less than 15 experts in nontechnical subjects and by the percent of permitted experiments require pre- right of the public to comment on major actions, initiation approval by the local IBC’s, which usu- which are published in the Federal Register. ally meet monthly. Preinitiation approval of ex- Compliance is accomplished by a combination periments by NIH is required only for: 1) experi- of local self-regulation and limited Federal over- ments that have not been assigned containment sight, with the ultimate enforcement resting in levels by the Guidelines; 2) experiments using the Federal funding power. new host-vector systems, which must be certified by NIH; 3) certain experiments requiring Since their initial appearance, the Guidelines case-by-case approval; and 4) requests for ex- have evolved. As scientists learned more about ceptions from Guideline requirements. The low- rDNA and molecular genetics, two trends oc- est containment levels place minimal burdens curred. First, containment levels were progres- on the experimenter. (see table 35). For in- sively lowered. Major revisions were made in dustrial applications, NIH approval must be 1978 and 1980; minor revisions were often received not only when the project is scaled-up made quarterly, as proposals were submitted to beyond the 10-1 limit, but also for each addi the RAC at its quarterly meetings, recom- tional scale-up of the same project. Many repre mended by RAC, and accepted by the Director. sentatives of industry consider these subse By now, approximately 85 percent of the permitted experiments can be done at the lowest ‘3R. M. Henig, “Trouble on the RAC—Committee Splits Ove physical and biological containment levels. Se- Downgrading of E. co/i Containment,” Bioscience, vol. 29, pp. 759 cond, the degree of centralized Federal over- 762, December 1979. Ch. n-Regulation of Genetic Engineering ● 217 quent approvals to be unnecessary and burden- between the other two techniques; yet no risk some. assessment has been done, and no Federal oversight exists. Information about whether the Guidelines have been a disadvantage for U.S. companies in Another limitation in the scope of the guide- international competition is scanty. Examples lines—and in the process by which they were include the approximately l-year headstart two formulated—is that long-range cultural risks (as European groups were given while the cloning distinguished from policy issues related to safe- of hepatitis B virus was prohibited, the advan- ty) were never addressed. As noted by the Di- 14 tage some European companies had in using rector of NIH: certain species of bacteria for cloning under NIH has been addressing the policy ques- conditions that were prohibited in the United tions involving the safety of this research, not States, and the delays some pharmaceutical the ‘potential future application . . . to the alter- companies faced because they had to build bet- ing of the genetic character of higher forms of ter containment facilities. life, including man’ . . . The present Guidelines are a comprehensive, Perhaps it was inappropriate to do more. Such flexible, and nonburdensome way of dealing ethical issues might be considered premature in with the physical risks associated with rDNA review of the level of the development of the tech- search while permitting the work to go for- nology. The desire among many molecular bi- ward. That is all they were ever intended to do. ologists to move ahead with the research meant that experiments were being done; therefore The Scope of the Guidelines.—In many the immediate potential for harm was to health respects, the Guidelines do not address the full and the environment. Thus, it was arguably scope of the risks of genetic engineering. They necessary to develop a framework to deal with cover one technique, albeit the most important; the risks based on what was known at the time. they do not address the admittedly uncertain, On the other hand, the broader questions of long-term cultural risks; they are not legally where the research might eventually lead and binding on researchers receiving funds from whether it should be done at all have been agencies other than NIH; and they are not bind- raised in the public debate. They have not been ing on industry. formally considered by the Federal Govern- Other genetic techniques present risks simi- ment. lar to those posed by rDNA, but to a lesser de- Another limitation in the scope of the Guide- gree. Recombinant DNA is the most versatile lines is their nonapplicability to research and efficient technique; it uses the greatest funded or performed by other Federal agencies. variety of genetic material from the widest However, agencies supporting such research number of sources with reasonable assurance are complying with the Guidelines as a matter of of expression by the host cell. Cell fusion of micro-organisms, which also involves the uncer- policy. There appears to be little reason for tain risk of recombining the genetic material of questioning these declarations of general policy. In practice, problems might arise if a mission is different species, is significantly less versatile and efficient than rDNA but mixes more genetic perceived to be at odds with the Guidelines or because of simple bureaucratic defense of terri- material. In addition, the parental cells may con- tory —e.g., when the 1976 Guidelines were pro- tain partial viral genomes that could combine to mulgated, two agencies—the Department of De- form a complete genome when the cells are fense (DOD) and the National Science Founda- fused. Transformation, a technique known for tion (NSF) preserved the right to deviate for rea- decades, similarly involves moving pieces of sons of national security or differing interpreta- DNA between different cells. However, it is significantly less versatile and efficient than cell fusion, and it is generally considered to be virtual- 1443 F.R. 6o103, Dec. 22, 1978, citing 43 F.R. 33067, JuI.v 28, ly risk-free. Thus, cell fusion is in a gray area 1978. 218 ● lmpacts of Applied Genetics—Mlicro-Organisms, Plants, and Animals tions, respectively. * DOD no longer claims an well-documented abuses and a host of Federal exception for national security.l5 NSF took its laws to curtail them. However, at least two con- position when it approved funding for an ex- straints are operating in the case of the bio- periment using a particular species of yeast that technology industry. First, the possibility of tort had not been certified by NIH, relying on an am- lawsuits is an inducement to comply with the biguously worded sectionl6 in the Guidelines to Guidelines, which would probably be accepted assert that it could certify the host. subsequent as the standard of care against which alleged revisions explicitly stated that these hosts had to negligence would be evaluated. (This concept is be certified by the Director of NIH1718 and discussed in greater detail in the section on Tort removed many similar ambiguities. Law and workman’s compensation.) second, the threat of statutory regulation, which the In the final analysis, NIH has indirect leverage companies have sought to avoid, always exists. over the actions of other agencies through its Other factors are also at work. Except for the funding. All non-NIH funded rDNA projects at 10-1 limitation, for which cases-by-case excep- an institution which also receives NIH funds for tions must be sought, the large-scale contain- rDNA work must comply with the Guidelines; ment recommendations of April 11, 1980, are otherwise NIH funds may be suspended or ter- minated. not excessively burdensome, at least for pharmaceutical companies. The requirements are While the procedures of other agencies for similar to measures that must currently be administering compliance are significantly less taken to prevent product contamination. In ad- formal than those created by the Guidelines for dition, the public debate should have made each NIH, they do rely heavily on NIH for help and company aware of the problems and the need advice, and they coordinate their efforts for voluntary compliance before it invested sub- through the Interagency Committee and their stantially in biotechnology; expensive controls nonvoting membership on RAC. So far, this vol- will not have to be retrofitted. However, one untary compliance by the agencies appears to definite concern is that new companies at- be working fairly well. tracted to the field will perceive their interests differently. Because they did not actually expe- The most significant limitation in the scope of rience the period when legislation seemed inevi- the Guidelines is their nonapplicability to in- table and because they will be late entries in the dustrial research or production on other than a race, they may be inclined to take shortcuts. voluntary basis. This lack of legal authority raises concerns not only about compliance but Besides the concern about whether industry also about NIH’s ability to implement a volun- has sufficient incentive to comply, there are a tary program effectively. number of other reasons for questioning the effectiveness of the voluntary program. First, until whether every company working with rDNA very recently no member of RAC was an expert will view voluntary compliance to be in its best in industrial fermentation technology-yet the interest depends on a number of factors. In committee has been considering applications the past, certain short-sighted actions by even a from industry for large-scale production since few companies in a given industry has led to September 1979. * This drawback was demon- *For a statement of the DOD position, see the minutes of the strated at its March 1980 meeting, when the November 23, 1976, meeting of the Federal Interagency Commit- tee. At that time, DOD had no active or planned rDNA projects. committee expressed uncertainty over what NSF’s statement of its intention to “preserve some level of inde- Federal or State safety regulations presently pendence of decision” was expressed in an internal NIH memo- cover standard fermentation technology em- randum dated February 24, 1978, from the Deputy Director for Science, NIH, to the Director, NIH. ● At its September 1980 meeting, RAC passed the following res- lsDr. John H. Moxley, III., Assistant Secretary of Defense for olution, which has been accepted by the Director ofNIH: 19 Health Affairs, personal communication, Nov. 18, 1980. Members should he chosen to provide expertise in fermentation lo{’FUngal or similar Lower Eukaryotic Host-Vector Systems, ” 41 techmlo~y, engineering, tind other wpcts of large-wide production. F.R. 27902, 27920, July 7, 1976. A fermentation technology expert was appointed in January 1743 F.R. 60108,” Dec. 22, 1978, sec. 111.C.5 of the 1978 Guidelines. 1981. 1s45 F*R. 6724, Jan$ 29, 1980, ~~o 111.C.5 of the 1980 Guidelines. ]s45 F.R. 77373, Nov. 21, 1980. Ch. 11-Regulation of Genetic Engineering . 219 ployed by the drug industry. Various members of NIH’s quasi-regulation of industry. A primary expressed concern in the March and June 1980 concern was whether the RAC would be viewed meetings about the Committee’s continuance to as giving a “stamp of approval” to industrial pro- make recommendations on the applications jects, when, in fact, it has neither the authority without a firm knowledge of large-scale produc- nor the ability to do so. One member, lawyer tion. Patricia King, stated:2l second, the provisions in part VI of the Guide- Voluntary compliance is the worst of all possi- lines, which allow prior review of submitted in- ble worlds. . . .You achieve none of the objec- formation by the DHHS Freedom of Information tives of regulation and none of the benefits of Act Officer, give an industrial applicant the op- being unregulated. All you’re saying is ‘I give a tion of withholding potentially important infor- stamp of approval to what I see here before me without any authority to do anything.’ mation on the grounds of trade secrecy, even when DHHS disagrees. Third, because some Most of the speakers expressed the desire that RAC members have been opposed to discussing the various agencies in the Interagency Commit- industrial applications in closed session (needed tee be responsible for such regulation. How- to protect proprietary information), they have ever, the Interagency Committee, which has chosen not to participate in those sessions. been studying the problem since January 1980, Thus, some diversity of opinion and expertise has yet to decide what it can do. Thus, many of has been lost. Fourth, monitoring for compli- its members see RAC as filling a regulatory void ance after the scale-up applications are granted until the traditional agencies take action. is limited. Some early applications were granted Some regulatory agencies have begun to deal on the condition that NIH could inspect facili- with specific problems within their areas of in- ties, and at least one inspection was made. terest. The Occupational Safety and Health Ad- Under procedures adopted at the September ministration will decide its regulatory policy on 1980 meeting, a company’s IBC will be responsi- the basis of a study of potential risks to workers ble for determining whether the facilities meet posed by the industrial use of rDNA techniques the standards for the large-scale containment being conducted by the National Institute of level assigned by RAC. A working group of RAC Occupational Safety and Health (NIOSH). In a may visit the companies and their IBCs from letter to the Director of NIH dated September time-to-time but only for information gathering 24, 1980, Dr. Eula Bingham, then Assistant Sec- purposes, rather than for regulatory actions. retary for Occupational Safety and Health of the Fifth, even if noncompliance were found, no Department of Labor, estimated this process penalties can be imposed. would take approximately 2 years. The Environ- The members of RAC, acutely aware of the mental Protection Agency (EPA) has awarded problems with voluntary compliance by indus- several contracts and grants to assess the risks try, have been deliberating about them for of intentional release of genetically engineered almost 2 years. At a meeting in May 1979, they micro-organisms and plants into the environ- decided, by a vote of nine to six with six absten- ment. And the Food and Drug Administration tions, to support the principle of mandatory (FDA) has begun to develop policy with respect compliance with the Guidelines by non-NIH to products made by processes using genetically funded institutions. However, the Secretary of engineered micro-organisms. (Further details HEW (Joseph Califano) decided to continue with on agency actions are discussed in the section, the development of voluntary compliance provi- Federal Statutes.) 20 sions which were adopted as Part VI of the Compliance. —The primary mechanism in Guidelines in January 1980. Actual RAC review the Guidelines for enforcing compliance is local of submissions from the private sector for large- self-regulation, with very limited Federal over- scale work began in September 1979. At a meeting in June 1980, RAC debated the effectiveness 21 Susan Wright, Recommended NA Policy: Controlling Large- ZORAC minutes of sept. 6-7, 1979, p. 16, in Recombinant DNA Re- Scale Processing,” Environment, vol. 22, September 1980, pp. search, vol. 5, (Wash., D. C.: HEW, 1980), p. 165. 29,32. 220 ● Impacts of Applied Genetlcs—Micro-Organisms, Plants, and Animals sight. Penalties are based on NIH’s power to re- IBCs members must be unaffiliated with the in- strict or terminate its funding. stitution. IBC documents, including minutes of meetings, are publicly available, but meetings The initial responsibility for compliance lies are not required to be held in public. On the with the scientist doing the experiments. A re- other hand, the probable inability of the mem- searcher’s attitude toward the risks of rDNA bers who represent the public to understand techniques and the necessity for the Guidelines the technical matters might limit their effective- appear to be an influential factor in the degree ness. of compliance. A science writer who worked for 3 months in a university lab in 1976 noted slop- How successful has compliance been? Three py procedures and a cavalier attitude, stating: known violations have occurred. In each, no “Among the young graduate students and post- threat to health and the environment existed. In doctorates it seemed almost chic not to know each, there was some confusion as to why the the NIH rules.”22 On the other hand, in the case violations occurred. NIH is presently invest- of a recent violation of the Guidelines, it appears igating the third violation. For the first two, it as if the investigator’s graduate students were accepted explanations of misunderstandings the first to raise questions.23 24 Competitiveness and misinterpretations of the Guidelines. How- 25 is another important factor. Novice scientists ever, a Senate oversight report concluded: must establish reputations, secure tenure in a While undoubtedly most researchers have tight job market, and obtain scarce research observed the guidelines conscientiously, it is funds; established researchers still compete for equally clear that others have substituted their grants and certainly for peer recognition. This own judgments of safety for those of NIH. competitive pressure could provide strong incentives to bend the Guidelines; on the other No firm conclusions can be drawn on the ques- hand, it might be channeled to encourage com- tion of compliance. The reporting of only a few pliance if it is believed that NIH will in fact violations could be evidence that the compliance penalize violations by restricting or terminating mechanism embodied in the Guidelines has funding. been working well. Or it could mean that some violations are not being discovered or reported. The first level of actual oversight occurs at the institution. An argument can be made that The November 1980 amendments to the reliance on the PI and an IBC (that might be Guidelines substantially changed procedures designed to monitor compliance by abolishing a composed mostly of the PI’s colleagues) provides document called a Memorandum of Under- too great an opportunity for lax enforcement or coverups. On the other hand, spreading respon- standing and Agreement (MUA). It had been required for 15 to 20 percent of all experiments, sibility among the institution, the PI, the IBC, those thought to be potentially most risky. The and, in the case of more hazardous experiments, the Biological Safety Officer might re- MUA, which was to be filed with ORDA by an institution, provided information about each ex- duce the chance of violations being overlooked or condoned. This responsibility is enhanced by periment, and it was the institution’s certifica- tion to NIH that the experiment complied with the reporting requirements borne by each of these parties, designed so that ORDA learns of the Guidelines. By having the MUAs, ORDA could monitor for inconsistencies in interpret- “significant” problems, accidents, violations, and illnesses. What is “significant” is not defined. ing the Guidelines, actual noncompliance, and the consistency and quality with which IBCs Public involvement at the local level acts as an functioned nationwide. The amendments con- additional safeguard. Twenty percent of the tinued a trend begun in January 1980, when ap- ~ZJanet L. Hopson, “Recombinant Lab for DNA and MY 95 Days proximately 80 percent of the experiments, in It,” Smithsonian, vol. 8, June 1977, p. 62. Z3D0 Dickson, (, Another Vio]ation of NIH Guide] ines, j) ~ature vol. ‘s’’ Recombinant DNA Research and Its Applications,” Oversight 286, Aug. 14, 1980, p. 649. Report, Subcommittee on Science, Technology, and Space of the i~D. Dickson, “DNA Recombination Forces Resignation, ” ~tlfUJl? Senate Committee on Commerce, Science and Transportation, vol. 287, Sept. 18, 1980, p. 179. Aug. 1978, p. 17. Ch. 11-Regulation of Genetic Engineering . 221 those done with E. coli K-12, were exempted ly to find himself accused of high-handedness, from the MUA requirement. deceit, or cover-up. We simply cannot afford to deal with these vital issues in a manner that in- The abolition of the MUA essentially abol- vites public cynicism and distrust. ished centralized Federal monitoring of rDNA experiments. The only current Guideline provi- The manner in which the Guidelines them- sion that serves this kind of monitoring function selves evolved has been controversial. (For a is the requirement that the institution, the IBC, detailed discussion see app. III-A.) Initially, the or the PI notify ORDA of any significant viola- scope and nature of the problem was defined by tions, accidents, or problems with interpreta- the scientific community; NIH organized RAC tion. Limited monitoring of large-scale activities along the lines suggested by the NAS committee continues. Under NIH procedures (which are letter referred to in app. III-A. One of the goals not part of the Guidelines) for reviewing appli- of RAC was to recommend guidelines for rDNA cations for exemptions from the 10-1 limit, the experiments; it was not charged with consider- application must include a copy of the registra- ing broader ethical or policy issues or the funda- tion document filed with the IBC. The manufac- mental question of whether the research should turing facilities may also be inspected by NIH, have been permitted at all. The original Guide- not for regulatory purposes, but to gather infor- lines were produced by a committee having mation for updating its recommended large- only one nonscientist. scale containment levels; The abolition of the In late 1978, the Secretary of HEW signif- MUA is consistent with traditional views that icantly restructured RAC and modified the Government should not interfere with basic sci- Guidelines in order to increase the system’s entific research. Whether or not it will reduce accountability to the public, to “provide the op- either the incentive to comply with the Guide- portunity for those concerned to raise any lines or the likelihood of discovering violations ethical issues posed by recombinant DNA re- remains to be seen. search”{ and to make RAC “the principal ad- 27 THE DECISIONMAKING PROCESS visory body . . . on recombinant DNA policy. ” However, it has remained in large part a tech- Another way to evaluate the Guidelines be- nically oriented body. Its charter was not sides considering their substantive require- changed in this respect; the Guidelines them- ments is to look at the process by which they selves state that its advice is “primarily scientific were formulated. In a situation where there is and technical, ” and matters presented for its uncertainty and even strong disagreement consideration have continued to be mostly tech- about the nature, scope, and magnitude of the nical. One area where RAC has played a signifi- risks, it is difficult to judge whether or not a cant policy role, however, is in dealing with the proposed solution to a problem will be a good issue of voluntary compliance by industry. one. Society’s confidence in the decisionmaking process and in the decisionmakers then be- It could be argued that the system did provide comes the issue. As David L. Bazelon, Chief for sufficient public input into the formulation Judge of the U. S. Court of Appeals for the Dis- of the problem* and that no other formulation trict of Columbia, has stated:26 was realistic. The two meetings in 1976 and 1977 of the NIH Director’s Advisory Committee When the issues are controversial, any decision may fail to satisfy large portions of the com- and the hearing chaired by the general counsel munity. But those who are dissatisfied with a of HEW in the fall of 1978 provided the oppor- particular decision wil1 be more likely to ac- tunity for public comment on the overall Fed- quiesce in it if they perceive that their views and ZTJoSeph A. Ca]ifano, “Notice of Revised Guidelines—Recombi- interests were given a fair hearing. If the deci- nant DNA Research, ” 43 F.R. 60080-60081, Dec. 22, 1978. sion-maker has frankly laid the competing con- ● The problem was conceived in terms of how to permit there. siderations on the table, so that the public search to be done while limiting the physical risks to an acceptable level. Other formulations were possible, the broadest being how knows the worst as well as the best, he is unlike- to limit all risks, including cultural ones, to an acceptable level. Z@D. L. Bazelon, ‘(coping With Technology Through the Legal Such a formulation could have resulted in a prohibition of the Process, ” 62 Cornell Law Review 817,825, June 1977. research. 222 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals eral approach to the controversy, including the risks had been expressed in terms of poten- whether or not the problem had been too nar- tial hazards to human health and the environ- rowly phrased. similarly, Congress had the ment, the original RAC had no experts in the opportunity in 1977 to reevaluate the entire areas of epidemiology, infectious diseases, bot- institutional response, taking into account any any or plant pathology, or occupational health. moral objections to the research in addition to It did have one expert in enteric organisms, E. those concerning safety. Yet the principal bills coli in particular. were based on the proposition that the research These shortcomings were eventually rem- continue in a regulated fashion. edied by expanding RAC’s membership to allow A related issue is the one of burden of proof. the appointment of other experts, including Should the proponents of a potentially benefi- some from nontechnical fields such as law and cial technology be required to demonstrate ethics. In addition to providing knowledge of minimal or acceptable risk even if that risk is other fields, these members served as disin- uncertain or even hypothetical? Or should its terested advisors, since they had no direct in- opponents be required to demonstrate unac- terest in expediting the research. Thus, the Gov- ceptable risk? If the proposition is accepted that ernment dealt with the problem of conflicts of those who bear the risks, in this case the public interest by offsetting the interested group with as well as the scientists, must judge their ac- other groups. In view of the need for the tech- ceptability, then the burden must be on the pro- nical expertise of the molecular biologists, this ponents. The scientific community clearly ac- approach seems reasonable; nevertheless the cepted this burden. The moratorium proposed matter could probably have been handled more by the NAS committee in July 1974 called for a expeditiously. Although the April 1975 amend- suspension of certain types of rDNA experiment to the RAC charter added experts from ments until the risks could be evaluated and such fields as epidemiology and infectious dis- procedures for adequately dealing with those eases, the charter did not require plant experts risks could be developed. The Guidelines pro- until September 1976 (shortly after the passage hibited some experiments, specified contain- of the original Guidelines) and occupational ment levels for others, and required certifica- health specialists until December 1978. In addition of host-vector systems. All actions approved tion, while two nontechnical members were ad- by the Director of NIH, including the lessening ded in 1976 (one before and one after passage of of the restrictions imposed by the original the Guidelines), their number was not increased Guidelines, have had to meet the requirement of until secretary Califano reconstituted the Com- presenting “no significant risk to health or the mittee in late 1978. environment. ” The present makeup of RAC is fairly diverse. Two other criticisms have been directed As of September 1980, nine of its members spe- against RAC, particularly in its early days. The cialized in molecular biology or related fields, first concerned inherent conflicts of interest. seven were from other scientific disciplines, RAC’s members were drawn from molecular and eight were from the areas of law, public biology and related fields. One of the early policy, the environment, and public or occupa- drafts of the Guidelines was criticized as being tional health.30 Moreover, since December 1978, “tailored to fit particular experiments that are representatives of the interested Federal agen- already on the drawing boards. ”28 However, cies have been sitting as nonvoting members. In only a few of the members were actually work- January 1981, an expert on fermentation was ing with rDNA.29 A more serious criticism was added. the lack of a broad range of expertise. Although

‘N. Wade, “Recombinant DNA: NIH Sets Strict Rules to Launch New Technology,” 190 Science 1175,1179, 1975. zsDr. Elizabeth Kutter, a member of RAC at that time, peI’SOnal sODr. Bernard Talbot, Special Assistant to the Director, NIH, per- communication, Sept. 11, 1980. sonal communication, Sept. 18, 1980. Ch. 11-Regulation of Genetic Engineering ● 223

● One conflict of interest not solved by expand- use the rDNA techniques could have threatened ing the diversity of the RAC’s membership is in- the notion of self-regulation. Today, there ap- stitutional in nature. NIH, the agency having pri- pears to be reasonable opportunity for public mary responsibility for developing and adminis- input through the process of commenting on tering the Guidelines, views its mission as one of proposed actions. promoting biomedical research. Although the Guidelines are not regulations, they contain Conclusion many of the elements of regulations. They set standards, offer a limited means to monitor for The Guidelines are the result of an extraor- compliance, and provide for enforcement, at dinary, conscientious effort by a combination of least for institutions receiving NIH grants to do scientists, the public, and the Federal Govern- rDNA work; thus, they may be considered ment, all operating in an unfamiliar realm. They quasi-regulatory. Regulation is not only foreign appear to be a reasonable solution to the prob- but antithetical to NIH’s mission. The current lem of how to minimize the risks to health and Director stated publicly at the June 1980 RAC the environment posed by rDNA research in an meeting that the role of NIH is not one of a academic setting, while permitting as much of regulator, a role that must be avoided. Under that research as possible to proceed. They do these circumstances, perhaps another agency, not in any way deal with other molecular genet- or another part of DHHS, might be more appro- ic techniques or with the long-term social or priate for overseeing the Guidelines, since the philosophical issues that may be associated with attitudes and priorities of promoters are usually genetic engineering. quite different from that of regulators. The Guidelines have been an evolving docu- If RAC has always been essentially a technical ment. As more has been learned about rDNA advisory body, who then has made the value de- and molecular genetics, containment levels cisions concerning the acceptability of the risks have been significantly lowered. Also, the de- presented by rDNA and the means for dealing gree of Federal oversight has been substantially with them? The final decisionmaker has been lessened. Under the November 1980 Guidelines, the Director of NIH, with the notable exception virtually all responsibility for monitoring comin the case of the 1978 Guidelines, which con- pliance is placed on the IBCs. NIH’s role will in- tained the significant procedural revisions volve primarily: 1) continuing interpretation of needed to meet Secretary Califano’s approval.31 the Guidelines, 2) certifying new host-vector The Director did have access to diverse points systems, 3) serving as a clearinghouse of infor- of view through the Director’s Advisory Com- mation, 4) continuing risk assessment experi- mittee meetings and the public hearings held ments, and 5) coordinating Federal and local ac- before the 1978 Guidelines. (See app. III-A.) In tivities. addition, major actions were always accom- The most significant short-term limitation of panied by a statement discussing the relevant the Guidelines is the way they deal with com- issues and explaining the basis for the decisions; mercial applications and products of rDNA tech- after the 1978 revisions, major actions had to be niques. Although large-scale containment levels proposed for public comment before decisions and related administrative procedures exist, were made. In theory, it may have been prefer- there are several reasons for questioning the ef- able for the public to have been substantially in- fectiveness of the voluntary compliance con- volved in the actual formation of the original cept. The most serious problem has been the Guidelines rather than simply to have reacted to lack of expertise in fermentation technology on a finished product. However, this probably RAC. In addition, since the Guidelines are not would have slowed the process at a time when legally binding upon industry, the NIH lacks en- the, strong desire of the molecular biologists to forcement authority, although there has been no evidence of industrial noncompliance. Final- 3’Califano, op. cit. ly, because of its role as a promoter of bio- 224 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals medical research, NIH cannot be expected to act procedural safeguards designed to accommo- aggressively to fill this regulatory void. date social values and to limit conflicts of interest. The only major criticism is that proce- As a model for societal decisionmaking on dural safeguards and public input were not sig- technological risks, the system created by the nificant factors when the rDNA problem was Guidelines could serve as a valuable precedent. first addressed. It does a reasonable job of combining substantive scientific evaluation of technical issues with other means of regulation

There are three other means available for large extent, the genetic technologies will pro- regulating molecular genetic techniques and duce chemicals, foods, and drugs—as well as their products—current Federal statutes, tort pollutant byproducts—that will clearly come law and. workmen’s compensation, and State within the scope of these laws. ’However, there and local laws. These all may be used to remedy may be limitations in these laws and questions some of the limitations of the Guidelines. of their interpretation that may arise with respect to the manufacturing process, which Federal statutes employs large quantities of organisms, and when there is an intentional release of micro- The question of whether existing Federal organisms into the environment—e.g., for clean- statutes provide adequate regulatory authority ing up pollution. For a list of pertinent laws, see first arose with respect to rDNA research. In table 36.) March 1977, the Interagency Committee concluded that while a number of statutes* could The Federal Food, Drug, and Cosmetic Act provide authority to regulate specific phases of (FEDCA) and section 351 of the Public Health work with rDNA, no single one or combination Service Act (42 U.S.C. 262) give FDA authority would clearly reach all rDNA research to the ex- over foods, drugs, biological products (such as tent deemed necessary by the Committee. Fur- vaccines), medical devices, and veterinary medi- thermore, while some could be broadly inter- cines. This authority will also apply to those preted, the Committee believed that regulatory products when they are made by genetic engi- action taken on the basis of those interpreta- 32 Table 36.—Statutes That Will Be Most Applicable tions would be subject to legal challenge. This to Commercial Genetic Engineering was the basis for their conclusion that specific legislation was needed and was one of the rea- 1. Federal Food, Drug, and Cosmetic Act (21 U.S.C. $301 et. seq.) sons behind the legislative effort discussed in 2. Occupational Safety and Health Act (29 U.S.C. $651 et. app. III-A. seq.) 3. Toxic Substances Control Act (15 U.S.C. $2601 et. With respect to commercial uses and prod- seq.) 4. Marine Protection, Research, and Sanctuaries Act (33 ucts of rDNA and other genetic techniques, a U.S.C. $1401 et. seq.) much more certain basis for regulation exists. 5. Federal Water Pollution Control Act, as amended by Many of the Federal environmental, product the Clean Water Act of 1977 (33 U.S.C. $1251 et. seq.) 6. The Clean Air Act (42 U.S.C. $7401 et. seq.) safety, and public health laws are directed 7. Hazardous Materials Transportation Act (49 U.S.C. toward industrial processes and products. To a $1801 et. seq.) 8. Solid Waste Disposal Act, as amended by the “1’he Committee concentrated on the following statutes: 1) the Resource Conservation and Recovery Act of 1976 (42 Occupational %fety and Health Act (29 IJ.S.C. S651 et. seq.); 2) the U.S.C. $6901 et. seq.) Toxic Sul]stiin~es Control Act (15 IJ.S.(:, $2601 et. seq.); 3) the Haz- 9. Public Health Service Act (42 U.S.C. $301 et. seq.) ardous Miiterials ‘1’ransportation Act (49 LJ. S.C. ~ 1801 et, seq.); and 10. Federal Insecticide, Fungicide, and Rodenticide Act (7 4) sec. 361 of the Public Health Service Act (42 LJ.S.C. $264). - U.S.C. $136 et. seq.) az/nterjm Re/lort Of the Federal Interagency CQnlmittee on Recom binant DNA Research: Suggested Elemenfsfor f.e.gislation, op. cit. SOURCE: Office of Technology Assessment. Ch. 11-Regulation of Genetic Engineering . 225

neering methods. However, interpretive ques- The statute most applicable to worker health tions arising out of the unique nature of the and safety is the Occupational Safety and Health technologies—such as the type of data nec- Act, which grants the Secretary of Labor broad essary to show the safety and efficacy of a new power to require employers to provide a safe drug produced by rDNA techniques–will have workplace for their employees. This power in- to be resolved by the administrative process on cludes the ability to require an employer to a case-by-case basis. modify work practices and to install control technology. The statute creates a general duty FDA has not published any statements of of- on employers to furnish their employees with a ficial policy toward products made by genetic workplace “free from recognized hazards that engineering. Since it has different statutory au- are causing or are likely to cause death or seri- thority for different types of products, it is like- ous physical harm, ” and it requires employers ly that regulation will be on a product-by-prod- to comply with occupational safety and health uct basis through the appropriate FDA bureau. standards set by the Secretary of Labor. Accord- Substances produced by genetic engineering ing to a recent Supreme Court case, a standard will generally be treated as analogous products may be promulgated only on a determination produced by conventional techniques with re- that it is “reasonably necessary and appropriate spect to standards for chemistry, pharmacolo- to remedy a significant risk of material health gy, and clinical protocols; however, quality con- 34 impairment. ” Because these fairly stringent re- trols may have to be modified to assure continu- quirements limit the Act’s applicability to ous control of product purity and identity. In recognized hazards or significant risks, the addition, for the time being, the Bureau of statute could not be used to control manufactur- Drugs and the Bureau of Biologics will require a ing where the genetic techniques presented on- new Notice of Claimed Investigational Exemp- ly hypothetical risks. However, it should be ap- tion for a New Drug and a new New Drug Appli- plicable to large-scale processes using known cation for products made by rDNA technology, human toxins, pathogens, or their DNA. even if identity with the natural substance or with a previously approved drug is shown. This The Secretary of Labor is also directed to ac- policy is based on the position that drugs or count for the “urgency of the need” in es- biologics made by rDNA techniques have not tablishing regulatory priorities. How the De- become generally recognized by experts as safe partment of Labor will view genetic technol- and effective and therefore meet the statutory ogies within its scale of priorities remains to be definition of a “new drug. ’’33* seen. NIOSH, the research organization created by this statute, has been studying rDNA produc- FFDCA also permits regulation of drug, food, tion methods to determine what risks, if any, and device manufacturing. Certain FDA regula- are being faced by workers. It has conducted tions, called Good Manufacturing Practices, are fact-finding inspections of several manufac- designed to assure the quality of these products. turers, and it is planning a joint project with FDA may have to revise these to accommodate EPA to assess the adequacy of current control genetic technologies; it has the authority to do technology. In addition, a group established by so. It probably does not have the authority to the Center for Disease Control (CDC) together use these regulations to address any risks to with NIOSH will be making recommendations workers, the public, or the environment, since on: 1) the medical surveillance of potentially ex- FFDCA is designed to protect the consumer of posed workers, 2) the central collection and the regulated product. analysis of medical data for epidemiological purposes, and 3) the establishment of an emergency ssMinUteS of the Industrial Practices Subcommittee of the Fed- 35 response team. eral Interagency Advisory Committee on Recombinant DNA Re- search, Dec. 16, 1980, p. 3. s4/ndustria/ Union Department, AFL-C1O v. American Rtrolellm *Sec. 201(p) of the FFLX:A (21 IJ.S.(:. $321(p)) defines a new drLIg Institute, 100 S. Ct. 2844,2863, 1980. 3SMinute~ as “any drug . . . the composition d’ which is such that such drLJg of the [ndustria] practices Subcommittee of the Federal is not .gen(?rtil]y re(mgnized, i]n](~[l~ t?xperts (Iuiilitied by scientific Interagency Advisory Committee on Recombinant DNA Research, tl’ilillill~ and experience . . iIS Silf[? illld Pt”t”(?d i~’e . .” Dec. 16, 1980, op. cit., p, 6. 226 . Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

The Toxic Substances Control Act (TSCA) was pretation which I am confident the Congress intended by Congress to fill in the gaps in the neither contemplated nor intended. other environmental laws. It authorizes EPA to If EPA were to take the broader interpreta- acquire information on “chemical substances” in tion, and if that were to survive any legal chal- order to identify and evaluate potential hazards lenge, TSCA would have great potential for reg- and then to regulate the production, use, distri- ulating commercial genetic engineering by reg- bution, and disposal of those substances. ulating the organisms. Under section 4 of this A "chemical substance” is defined under sec- Act, EPA can adopt rules requiring the testing of tion 3(2) of this Act as “any organic or inorganic chemical substances that “may present an un- substance of a particular molecular identity, ” in- reasonable risk”* to health or the environment cluding “any combination of such substances oc- when existing data are insufficient to make a curring in whole or in part as a result of a chem- determination. Under section 5, the manu- ical reaction or occurring in nature.”* This facturer of a new chemical substance is re- would include DNA molecules; however, it is quired to notify EPA 90 days before beginning unclear if the definition would encompass gene- production and to submit any test data available tically engineered organisms. In promulgating on the chemical’s health or environmental ef- its Inventory Reporting Regulations under fects. If EPA decides that the data are insuffi- TSCA on December 23, 1977, EPA took the fol- cient for evaluating the chemical’s effects and lowing position in response to a comment that that it “may present an unreasonable risk” or commercial biological preparations such as will be produced in substantial quantities, the yeasts, bacteria, and fungi should not be con- chemical substance’s manufacture or use can be sidered chemical substances:36 restricted or prohibited. Under section 6, EPA can prohibit or regulate the manufacture or use The Administrator disagrees with this com- of any chemical substance that “presents, or will ment . . . . This definition [of chemical sub- present an unreasonable risk of injury to health stance] does not exclude life forms which may or the environment. ” be manufactured for commercial purposes and nothing in the legislative history would suggest As with the Occupational Safety and Health otherwise. Act, the scientific evidence probably does not However, in a December 9, 1977, letter re- support a finding that most genetically en- sponding to a Senate inquiry, EPA Administra- gineered molecules or organisms present an un- tor Douglas M. Costle stated:37 reasonable risk. On the other hand, the standard in section 5—may present an unreasonable [A]lthougkt there is a general consensus that re- risk—and the requirement for a premanufac- combinant DNA molecules are “chemical sub- turing notice would permit EPA to evaluate stances” within the meaning of section 3 of cases where genetically engineered micro-orga- TSCA, it is not at all clear whether a host organism containing recombined DNA molecules nisms were proposed to be released into the fits—or was intended to fit—that definition . . . . environment. If such organisms are subject to TSCA on the Several other environmental statutes will ap- grounds that they are a “combination of . . . ply, mainly with respect to pollutants, wastes, substances occurring in whole or in part as a or hazardous materials.** The Marine Protec- result of a chemical reaction,” the Agency might logically have to include all living things in the *The term “unreasonable risk” is not defined in the statute. definition of “chemical substance’’—an inter- However, the legislative history indicates that its determination involves balancing the probability that harm will occur and the “Substances subject solely to FFDCA or the Federal Insecticide, magnitude and severity of that harm, against the effect of the pro- Fungicide, and Rodenticide Act are excluded from this definition. posed regulatory action and the availability to society of the bene- 3642 F. R., 64572, 64584, Dec. 23, 1977. fits of the substance.sa Sletter to Adlai E. Stevenson, Chairman, Subcommittee on Sci- 3JJH. Rept. W. 1341, ~dth [:ong., 2d sess. 1976, pp. 13-15. ence, Technology, and Space, U.S. Senate Committee on Com- * *Two consumer protection statutes were considered but were merce, Science, and Transportation, in Overs@t Report, lhxombi- determined to be virtually inapplicable These were: the Federal nant DIVA Reseamh and Its Applications, 95th Cong., 2d sess., Au- Hazardous Sul}stances Act (15 11. S.(;. 31261 et. seq.); and the Con- gust 1978, p.88. sumer Product Safet.v Act [15 IJ. S.C. S2051 et. seq.). Ch. 11-Regulation of Genetic Engineering . 227

tion, Research, and Sanctuaries Act prohibits human disease. In any event, HEW declined to ocean dumping without an EPA permit of any promulgate any regulations. material that would “unreasonably degrade or The following conclusions can therefore be endanger human health, welfare, or amenities, made on the applicability of existing statutes. or the marine environment, ecological systems, 39 First, the products of genetic technologies— or economic potentialities. ” “Material” is de- such as drugs, chemicals, pesticides,** and fined as “matter of any kind or description, in- foods—would clearly be covered by statutes cluding. . . biological and laboratory waste 40 already covering these generic categories of . . . and industrial . . . and other waste. ” The materials. Second, uncertainty exists for regu- Federal Water Pollution Control Act regulates lating either production methods using en- the discharge of pollutants (which include bio- gineered micro-organisms or their intentional logical materials) into U.S. waters, and the Solid release into the environment, when risk has not Waste Disposal Act regulates hazardous wastes. been clearly demonstrated. Third, the regu- The Clean Air Act regulates the discharge of air latory agencies have begun to study the situa- pollutants, which includes biological materials. tion but, have not promulgated specific regu- Especially applicable is section 112 (42 U.S.C. lations. Fourth, since regulation will be dis- 7412), which allows EPA to set emission stand- persed throughout several agencies, there may ards for hazardous air pollutants—those for be conflicting interpretations unless active ef- which standards have not been set under other forts are made by the Federal Interagency Com- sections of the Act and which “may reasonably mittee to develop a comprehensive, coordinated be anticipated to result in an increase in mortali- approach. ty or an increase in serious irreversible, or in- capacitating reversible, illness. ” The Hazardous Materials Transportation Act covers the inter- Tort law and workmen’s compensation state transportation of dangerous articles, in- Statutes and regulations are usually directed cluding etiologic (disease-causing) agents. The at preventing certain types of conduct. While Secretary of Transportation may designate as tort law strives for the same goal, its primary hazardous any material that he finds “may pose purpose is to compensate injuries. (A tort is a an unreasonable risk to health and safety or civil wrong, other than breach of contract, for property” when transported in commerce in a 41 which a court awards damages or other relief. ) particular quantity and form. By its nature, tort law is quite flexible, since it Section 361 of the Public Health Service Act has been developed primarily by the courts on a (42 U.S.C. $264) authorizes the Secretary of case-by-case basis. Its basic principles can easily HEW (now DHHS) to “. . . make and enforce be applied to cases where injuries have been such regulations as in his judgment are neces- caused by a genetically engineered organism, sary to prevent the introduction, transmission, product, or process. It therefore can be applied or spread of communicable diseases . . . .“ Be- to cases involving genetic technologies as a cause of the broad discretion given to the Sec- means of compensating injuries and as an incen- retary, it has been argued that this section pro- tive for safety-conscious conduct. The most ap- vides sufficient authority to control all rDNA ac- plicable concepts of tort law are negligence and tivities. * Others have argued that its purpose is strict liability. (A related body of law—work- to protect only human health; for regulations to men’s compensation—is also pertinent. ) be valid, there would have to be a supportable Negligence is defined as conduct (an act or an finding of a connection between rDNA and omission) that involves an unreasonable risk of

39 harm to another person. For the injured party 33 U.s.c. $1412. to 33 U.S.CO ~ 1402(c). to be compensated, he must prove in court that: 4149 U.S.C. ~ 1803. 1) the defendant’s conduct was negligent, 2) the “On Nov. 11, 1976, the Natural Resources Defense Council and the Environmental Defense Fund petitioned the Secretary of HEW ● ● Pesticides iire sut}ject to the ti’ederal Insecticide Fungicide, to promulgate regulations concerningrDNA under this Act. iind Rodt)tlt icidr A(Y, 7 L r. S.(; . ~ 136 et. S(>(l.. 228 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals defendant’s actions in fact caused the injury, precautions or if culpable human action was in- and 3) the injury was not one for which com- volved. On the other hand, if a micro-organism pensation should be denied or limited because or toxin is identified, it may be so unique of overriding policy reasons. because of its engineering that it can be readily associated with a company known to produce it Because of the newness of genetic technol- or with a scientist known to be working with ogy, legal standards of conduct (e.g., what con- it. * * stitutes unreasonable risk) have not been articulated by the courts. If a case were to arise, a The law recognizes that not every negligent court would undoubtedly look first to the act or omission that causes harm should result Guidelines. Even if a technique other than rDNA in liability and compensation—e.g., the concept were involved, they would provide a general of “foreseeable” harm serves to limit a de- conceptual framework for good laboratory and fendant’s liability. The underlying social policy industrial techniques. Other sources for stand- is that the defendant should not be liable for in- ards of conduct include: 1) CDC’s guidelines for juries so random or unlikely as to be not rea- working with hazardous agents; 2) specific Fed- sonably foreseeable. This determination is made eral laws or regulations, such as those under the by the court. In the case of a genetically en- Public Health Service Act covering the inter- gineered organism, extensive harm would prob- state transportation of biologic products and ably be foreseeable because of the organism’s etiologic agents; and 3) industrial or profes- ability to reproduce; how that harm could occur sional codes or customary practices, such as might not be foreseeable. generally accepted containment practices in the Unlike negligence, strict liability does not re- pharmaceutical industry or in a microbiology quire a finding that the defendant breached laboratory. Compliance with these standards, some duty of care owed to the injured person; however, does not foreclose a finding of neg- the fact that the injury was caused by the de- ligence, since the courts make the ultimate judg- fendant’s conduct is enough to impose liability ment of what constitutes proper conduct. In regardless of how carefully the activity was several cases, courts have decided that an entire done. For this doctrine to apply, the activity industry or profession has lagged behind the must be characterized as “abnormally dan- level of safe practices demanded by society. * gerous.” To determine this, a court would look Conversely, noncompliance with existing stand- at the following six factors, no one of which is ards almost surely will result in a finding of 42 determinative: negligence, if the other elements are also present. 1. existence of a high risk of harm, Causation may be difficult to prove in a case 2. great gravity of the harm if it occurs, involving a genetically engineered product or 3. inability to eliminate the risk by exercising reasonable care, organism. In the case of injury caused by a pathogenic micro-organism—e.g., it may be difficult ● “If several companies were working with the micro-organism, to isolate and identify the micro-organism and it could be impossible to prove which company produced the par- virtually impossible to trace its origin, especially ticular ones that caused the harm. A recent California Supreme if it had only established a transitory ecological Court case, Sindell v. Abbott Laboratories, 26 CaL3d 588, 1980, could provide a way around this problem if the new theory of niche. In addition, it might be difficult to liability that it establishes becomes widely accepted by courts in reconstruct the original situation to determine other jurisdictions. The Court ruled that women whose mothers if the micro-organism simply escaped despite hiid taken diethy]stilbest rol, a drug that allegedly caused cancer in their daughters, could proceed to trial against manufacturers of the drug, even though most of the plaintiffs would not be able to ‘For example, see: The T. J. Hooper, 60 F. 2d 737 (2d Cir. 1932), show which particular manufacturers produced the drug. The concerning tugboats; and He/hhg v. Carey, 519 P. 2d 981 (1974), Court said that when the defendant manufacturers had a substan- where the court held that the general practice among ophthalmo- tial share of the product market, liability, if found, would be ap- logists of not performing glaucoma tests onasymptomatic patients portioned among the defendants on the basis of their market under 40 [because they had only a one in 25,000 chance of having share. A particular defendant could escape liability only by the disease) would not prevent a finding of negligence when such proving it could not have made the drug. a patient developed the disease. dz~es.tatement &cond) of Torts $520 (1976). Ch. 11—Regulation of Genetic Engineering . 229

4. extent to which the activity is not common, terrent effect of compensation is less efficient 5. inappropriateness of the activity to the than direct regulation—e.g., the threat of law- place where it is done, and suits will not necessarily discourage high-risk 6. the activity’s value to the community. activities where problems of proof make re- Given the current consensus about the risks covery unlikely, where the harm may be small and widespread (as with mild illness suffered by of genetic techniques, it would be difficult to a large number of people), or where profits are argue that the doctrine of strict liability should less than the cost of prevention but greater than apply. However, in the extremely unlikely event that a serious, widespread injury does occur, expected damage awards and legal costs. that alone would probably support a court’s de- Tort law has two other limitations. First, tort termination that the activity was abnormally litigation involves high costs to the plaintiff, and dangerous, regardless of its probability. In such indirectly to society. Second, it cannot adequate- cases, the courts have generally relied on the ly compensate the victims of a catastrophic sit- principle of “enterprise liability ’’–that those en- uation where liability would bankrupt the gaged in an enterprise should bear its costs, in- defendant. cluding the costs of injuries to others.43 For either negligence or strict liability, the State and local law person causing the harm is liable. Under the Under the 10th amendment to the Constitu- legal principle of respondent superior, liability is tion, all powers not delegated to the Federal also imputed from the original actor to people Government are reserved for the States or the or entities who have a special relationship with people. One of those is the power of the States him—e.g., employers. Thus, a corporation can and municipalities to protect the health, safety, be liable for the torts of its scientists or produc- and welfare of their citizens. Thus, they can tion workers. Similarly, a university, an IBC, a regulate genetic engineering. Biological Safety Officer, and a PI would probably be liable for the torts of scientists and stu- The reasons espoused in favor of local regula- dents under their direction. tion are based on the traditional concept of local autonomy; those most likely to suffer any Another body of law designed to compensate adverse affects of genetic engineering should injuries deserves brief mention. Workmen’s control it. Also, local and State governments are compensation is a statutory scheme adopted by usually more accessible to public input than the the States and—for specific occupations or cir- Federal Government. Consequently, judgments cumstances—by the Federal Government to on the acceptability of the risks will more compensate injuries without a need for showing precisely reflect the will of the segment of the fault. The employee need only show that the in- public most directly affected. jury was job-related. He is then compensated by the employer or the employer’s insurance com- A number of arguments have been made pany. It would clearly apply to genetic engineer- against local as opposed to Federal regulation. ing. The primary one is that regulation by States and communities would give rise to a random patch- Tort law and workmen’s compensation will work of confusing and conflicting controls. In be available to compensate any injuries re- addition, States and especially localities may not sulting from the use of molecular genetic tech- have the same access as the Federal Govern- niques, especially from their commercial appli- ment to the expertise that should be used in the cation. Tort law may also indirectly prevent formulation of rational controls. Finally, any potentially hazardous actions, although the de- risks associated with rDNA or other techniques are not limited by geographic boundaries; 43R. DWOrkln, “giocatastrophe and the Law: Legal Aspects of Re- therefore, they ought to be dealt with national- combinant DNA Research, ” in The Recombinant DNA Debate, Jackson and Stitch (eds.) (Englewood Cliffs, N. J.: Prentice-Hal], Inc. ly. The above arguments reflect the position 1979), pp. 219, 223. that regulation of genetic technologies is a na-

76-565 0 - 81 - 16 230 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

tional issue that can be handled most effectively land and New York.4445 Currently, there is little, at the Federal level. if any, effort on the State or local level to pass laws or ordinances covering rDNA or similar A few jurisdictions have used their authority genetic techniques, and there is little activity in the case of rDNA, * The most comprehensive under the existing laws. regulation was created by the States of Mary- ● Cambridge, Mass., established a citizens’ study group that rec. ommended that researchers be subject to some additional re. striations beyond those of the Guidelines. These were embodied in an ordinance passed by the City Council on Feb. 7, 1977. Serkeley, Calif., passed an ordinance requiring private research to conform 4aAnnotated Code of Maryland, art. 43 ~~ 898-910 (SUpp. 19781. to the Guidelines. Similar ordinances or resolutions were passed 45Mc~jnney’s C“onsoljdated Laws of fVew York, Public Health ~w, by Princeton, N.J., Amherst, Mass., and Emeryville, Calif. art. 32-A S$3220-3223 (supp. 1980)

Conclusion

The initial question with respect to regulating Guidelines, Federal statutes protecting health genetic engineering is how to define the scope and the environment, some State or local laws, of the problem. This will depend largely on and the judicially created law of torts, which is what groups are involved in that process and available to compensate injuries after they oc- how they view the nature, magnitude, and cur. In most cases, this system appears adequate acceptability of the risks. Similarly, the means to deal with the risks to health and the environ- of addressing the problem will be determined ment. However, there is some concern regard- by how it is defined and who is involved in the ing commercial applications for the following actual decisionmaking process. For these rea- reasons: 1) the voluntary applicability of the sons, it is important that regulatory mechanisms Guidelines to industry, 2) RAC’s insufficient ex- combine scientific expertise with procedures to pertise in fermentation technology, 3) the po- accommodate the values of those bearing the tential interpretive problems in applying ex- risk so that society may have confidence in isting law to the workplace and to situations those mechanisms. where micro-organisms are intentionally released into the enviornment, and 4) the absence Currently, genetic techniques and their prod- of a definitive regulatory posture by the ucts are regulated by a combination of the agencies.

Issue and Options

ISSUE: How could Congress address the OPTIONS: risks associated with genetic en- A: Congress could maintain the status quo by let- gineering? ting NIH and the regulatory agencies set the A number of options are available, ranging Federal policy. from deregulation through comprehensive new This option requires Congress to determine regulation. An underlying issue for most of that legislation to remedy the limitations in cur- these options is: What are the constitutional rent Federal oversight would result in unneces- constraints placed on congressional regulation sary and burdensome regulation. No known of molecular genetic techniques, particularly harm to health or the environment has oc- when they are used in research? (This is dis- curred under the current system, and the agen- cussed in app. III-B.) cies generally have significant legal authority Ch. 11-Regulation of Genetic Engineering . 231 and expertise that should permit them to adapt elude the following: 1) a section prepared by to most new problems posed by genetic engi- each agency that assesses its statutory authority neering. The agencies have been consulting and articulates what activities and products will with each other through the Interagency Com- be considered to come within its jurisdiction, 2) mittee, and the three agencies that will play the a summary section that evaluates the adequacy most important role in regulating large-scale of existing Federal statutes and regulations as a commercial activities—FDA, OSHA, and EPA— whole with respect to commercial genetic en- have been studying the situation. gineering, and 3) a section proposing any specific legislation considered to be necessary. The disadvantages of this option are the lack of a centralized, uniform Federal response to The principal disadvantages of this option are the problem, and the possibility that risks that it maybe unnecessary and impractical. The associated with commercial applications will not agencies are studying the situation, which must be adequately addressed. Certain applications, be done before they can act. Also, it is often such as the use of micro-organisms for oil re- easier and more efficient to act on each case as covery are not unequivocally regulated by cur- it arises, rather than on a hypothetical basis rent statutes; broad interpretations of statutory before the fact. language in order to reach these situations may be overturned in court. Conflicting or redun- C: Congress could require Federal monitoring of dant regulations of different agencies would all rDNA activity for a limited number of years. result in unnecessary burdens on those regulated. In addition, some commercial activity is This option represents a “wait and see” posi- now at the pilot plant stage, but the responsible tion by Congress and the middle ground be- agencies have yet to establish official policy and tween the status quo and full regulation. It rec- to devise a coordinated plan of action. ognizes and balances the following factors: 1) B: Congress could require that the Federal Inter- the absence of demonstrated harm to human agency Advisory Committee on Recombinant health or the environment from genetic en- DNA Research prepare a comprehensive re- gineering; 2) the continuing concern that genet- port on its members’ collective authority to ic engineering presents risks; 3) the lack of suf- regulate rDNA and their regulatory intentions. ficient knowledge from which to make a final judgment; 4) the existence of an oversight mech- The Industrial Practices Subcommittee of this anism that seems to be working well, but that Committee has been studying agency authority has clear limitations with respect to commercial over commercial rDNA activities. Presently, activities; 5) the virtual abolition of Federal there is little official guidance on regulatory re- monitoring of rDNA activities by the recent quirements for companies that may soon mar- amendments to the Guidelines; and 6) the ex- ket products made by rDNA methods. -e.g., pected increase in commercial genetic engineer- companies are building fermentation plants ing activities. without knowing what design or other requirements OSHA may mandate for worker safety. Monitoring involves the collection and eval- As was stated by former OSHA head, Dr. Eula uation of information about an activity in order Bingham, it will take at least 2 years for OSHA to to know what is occurring, to determine the set standards, if the current NIOSH study shows need for other action, and to be able to act if a need for them.46 necessary. More specifically, this option would provide a data base that could be used for: 1) de- A congressionally mandated report would termining the effectiveness of voluntary compli- assure full consideration of these issues by the ance with the Guidelines by industry and man- agencies and expedite the process. It could in- datory compliance by Federal grantees, 2) determining the quality and consistency of IBC de- 46[,etter from Dr, ~llla Bingham, Assistant %?cretary for ~ccupa- tional Safety and Health, to Dr. Donald Fredrickson, Director,NIH, cisions and other actions, 3) continuing a formal Sept. 24, 1980. risk assessment program, 4) identifying vague 232 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

or conflicting provisions of the Guidelines for pare an annual report to Congress on the effec- revision, S) identifying emerging trends or prob- tiveness of Federal oversight. lems, and 6) tracing any long-term adverse im- The choice of an agency to administer the pacts on health or the environment back to statute would be important. The selection of their sources. NIH would permit the use of an existing admin- The obvious disadvantages of this option are istrative structure and body of expertise and ex- the increased paperwork and effort by scien- perience. On the other hand, one of the regu- tists, universities, corporations, and the Federal latory agencies may take a more active moni- Government. Those working with rDNA would toring role and be more experienced with have to gather the required information peri- handling proprietary information. odically and prepare reports, which would be This approach is similar to a bill introduced in filed by the sponsoring institution with a the 96th Congress, S. 2234, but broader in designated existing Federal agency. A wide- scope. The latter covered only institutions not range of information would be required for funded by NIH, and did not contain provisions each project. The agency would have to process for requiring amendments to the reports or for the reports and take other actions, such as pre- notifying other agencies of possible noncom- paring an annual report to Congress, to imple- pliance. The bill was broader in one respect ment the underlying purposes of this option. because it would have required information Additional manpower would most likely be about prospective experiments. This provision needed by that agency. had been criticized because of the difficulty of A statute implementing this option could in- projecting in advance the course that scientific clude the following elements: 1) periodic collec- inquiry will take. The goals of a monitoring pro- tion of information in the form of reports from gram can be substantially reached by monitor- all institutions in the United States that sponsor ing ongoing and completed work. any work with rDNA, 2) active evaluation of D. Congress could make the NIH Guidelines ap- that information by the collecting agency, 3) an- plicable to all rDNA work done in the United nual reports to Congress, and 4) a sunset clause. States. Important information would include: 1) the sponsoring institution’s name; 2) all places The purpose of this option is to alleviate any I where it sponsors the research; and 3) a tabular concerns about the effectiveness of voluntary or other summary that discloses for each proj- compliance. RAC itself has gone on record as ect continuing or completed during the report- supporting mandatory compliance with the ing period: the culture volume, the source and Guidelines by non-NIH funded institutions, in- identity of the DNA and the host-vector system, cluding private companies. the containment levels, and other information This option has the advantages of using an ex- deemed necessary to effect the purposes of the isting oversight mechanism, which would sim- act. The statute could also require employers to ply be extended to industry and to academic re- institute and report on a worker health sur- search funded by agencies other than NIH. Spe- veillance program. cific requirements on technical questions such For this option to work, the monitoring agen- as containment levels, host-vector systems, and cy would have to take an active role in eval- laboratory practices would continue to be set by uating the data. It should have the authority to NIH in order to accommodate new information require amendments to the reports when any expeditiously; the statute would simply codify part is vague, incomplete, or inconsistent with the responsibilities and procedures of the cur- another part. It could also be required to notify rent system. There would be few transitional the appropriate Federal funding agency of ap- administrative problems, since the expertise parent cases of noncompliance with the Guide- and experience already exist at NIH. However, it lines by their grantees. Finally, it should pre- would be necessary to appoint several experts Ch. 11-Regulation of Genetic Engineering ● 233 in fermentation and other industrial technolo- vent this type of harm through regulation gies to RAC if production, as well as research, is rather than to compensate for injuries through to be adequately covered. In addition, the rec- lawsuits. Another possible disadvantage of the ommendations for large-scale containment pro- present system is that approval maybe granted cedures would have to be made part of the on a finding that the release would present “no Guidelines. significant risk to health or the environment;” a tougher or more specific standard than this may The major changes would have to be made be desirable. with respect to enforcement. Present penalties for noncompliance—suspension or termination A required study of the possible consequen- of research funds—are obviously inapplicable to ces following the release of a genetically industry. In addition, procedures for monitor- engineered organism, especially a micro-orga- ing compliance could be strengthened. Some of nism, would be an important step in ensuring the elements of option C could be used. An safety. This option could be implemented by re- added or alternative approach would be to in- quiring those proposing to release the organism spect facilities. to file an impact statement with an agency such as NIH or EPA, which would then grant or deny The main disadvantage of this option is that permission to release the organism. A disad- NIH is not a regulatory agency. Since NIH has vantage of this option is that companies and in- traditionally viewed its mission as promoting dividuals might be discouraged from developing biomedical research, it would have a conflict of useful organisms if this process became too interest between regulation and promotion. burdensome and costly. One of the regulatory agencies could be given the authority to enforce the Guidelines and to F. Congress could pass legislation regulating all adopt changes therein. NIH could then continue types and phases of genetic engineering from in a scientific advisory role. research through commercial production. E. Congress could require an environmental im- The main advantage of this option would be pact statement and agency approval before to deal comprehensively and directly with the any genetically engineered organism is inten- risks of novel molecular genetic techniques, tionally released into the environment. rather than relying on the current patchwork system. A specific statute would eliminate the There have been numerous cases where an uncertainties over the extent to which present animal or plant species has been introduced into law covers particular applications of genetic en- a new environment and has spread in an uncon- gineering, such as pollution control, and any trolled and undesirable fashion. One of the concerns about the effectiveness of voluntary early fears about rDNA was that a new path- compliance with the Guidelines. ogenic or otherwise undesirable micro-organism could establish an environmental niche. Other molecular genetic techniques, while Yet in pollution control, mineral leaching, and not as widely used and effective as rDNA, raise enhanced oil recovery, it might be desirable to similar concerns. Of the current techniques, cell release large numbers of engineered micro-or- fusion is the prime candidate for being treated ganisms into the environment. like rDNA in any regulatory framework. It permits the recombination of chromosomes of The Guidelines currently prohibit deliberate species that do not recombine naturally, and it release of any organism containing rDNA with- may permit the DNA of latent viruses in the cells out approval by the Director of NIH on advice of to recombine into harmful viruses. No risk as- RAC. The obvious disadvantage of this prohibi- sessment of this technique has been done, and tion is that it lacks the force of law. The release no Federal oversight exists. of such an organism without NIH approval would be a prima facie case of negligence, if the The principal arguments against this option organism caused harm. However, it may be are that the current system appears to be work- more desirable social policy to attempt to pre- ing fairly well, and that the limited risks of the 234 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals techniques may not warrant the significantly in- spent have not been estimated. Very few people creased regulatory burden and costs that would work full-time on administering or complying result from such legislation. Congress will have with the Guidelines. NIH employs only six peo- to decide if that system will remain adequate as ple full-time for this purpose, and some institu- commercial activity grows. tions employ full-time biological safety personnel. However, over 1,000 people nationally If Congress were to decide on this option, the devote some effort to implementing the legislation could incorporate some or all of op- Guidelines—members of the IBCs and the scien- tions C, D, and E. The present mechanism tists conducting the rDNA experiments who created by the Guidelines could be appropriate- must take necessary steps to comply. ly modified to provide the regulatory framework. The modifications could include a regis- There are several reasons for retaining the tration and licensing system to provide infor- Guidelines. First, sufficient scientific concern mation on what work was actually being done about risks exists for the Guidelines to prohibit and a means for continuous oversight. One certain experiments and require containment important type of information would be health for others. Second, they are not particularly and safety statistics gathered by monitoring burdensome, since an estimated 80 to 85 per- workers involved in the production of products cent of all experiments can be done at the from genetically engineered organisms. Anoth- lowest containment levels and an estimated 97 er modification could be a sliding scale of percent will not require NIH approval. Third, penalties for violations, ranging from monetary NIH will continue to serve an important role in fines through revocation of operating licenses continuing risk assessments, in evaluating new to criminal penalties for extreme cases. host-vector systems, in collecting and dispersing It would not be necessary to create a new information, and in interpreting the Guidelines. agency, which would duplicate some of the re- Fourth, if the Guidelines were abolished, regu- sponsibilities of existing agencies. Instead, Con- latory activity at the State and local levels could gress could give these agencies clear regulatory again become active. Finally, the oversight sys- authority by amending the appropriate statutes. tem has been flexible enough in the past to lib- Designating a lead agency would assure a more eralize restrictions as evidence indicated lower uniform interpretation and application of the risk. laws. H. Congress could consider the need for G. Congress could require NIH to rescind the regulating work with all hazardous micro- Guidelines. organisms and viruses, whether or not they are genetically engineered. This option requires Congress to determine that the risks of rDNA techniques are so insig- Micro-organisms carrying rDNA, according to nificant that no control or oversight is nec- an increasingly accepted view, represent just a essary. Deregulation would have the advantage subset of micro-organisms and viruses, which, of allowing funds and personnel currently in- in general, pose risks. CDC has published guide- volved in implementing the Guidelines at the lines for working with hazardous agents such as Federal and local levels to be used for other pur- polio virus. However, such work is not cur- poses. In fiscal year 1980, NIH spent approxi- rently subject to legally enforceable Federal reg- mately $500,000 in administering the Guide- ulations. It was not within the scope of this lines; figures are not available for the analogous study to examine this issue, but it is an emerging cost to academia and industry. Personnel hours one that Congress may wish to consider. Chapter 12 Patenting Living Organisms Chapter 12

Page A Landmark Decision . . , . , ...... , ...... 237 Patent v. Trade Secret Protection ...... 245 Legal Protection of Inventions, , ...... , .. 237 Impact of the Court’s Decision on the Trade Secrets...... 237 Biotechnology Industry ...... 246 Patents...... , ...... , ...... 238 Impacts of the Court’s Decision on the Patent Living organisms. , . . , ...... 239 Law and the Patent and Trademark Office ..246 The Chakrabarty Case...... , ...... 240 Impact of the Court’s Decision on Academic Potential Impacts of the Decision and Research...... 248 Related Policy Issues...... 242 Impacts of the Court’s Decision on Genetic lmpacts on Industry...... 242 Diversity and the Food Supply...... 249 The Relationship Between Patents and The Morality of Patenting Living Organisms. . .250 Innovation...... 242 Private Ownership of Inventions From The Advantages of Patenting Living Publicly Funded Research...... 250 Organisms...... , ...... 243 Issue and Options ...... 252 Chapter 12 Patenting Living Organisms

A landmark decision

In a 5 to 4 decision (Diamond v. Chakrabarty, Congress may want to reconsider the issue of June 16, 1980), the Supreme Court ruled that a whether and to what extent it should specifi- manmade mice-organism is patentable under cally provide for or prohibit the patentability of the current patent statutes. This decision was living organisms. While the judiciary operates alternately hailed as having “assured this coun- on a case-by-case basis, Congress can consider try’s technology future”l and denounced as cre- all the issues related to patentability at the same ating “the Brave New World that Aldous Huxley time, gathering all relevant data and taking tes- warned of. ”2 However, the Court clearly stated timony from the interested parties. The issues that it was undertaking only the narrow task of involved go beyond the narrow ones of scien- determining whether or not Congress, in enact- tific capabilities and the legal interpretations of ing the patent statutes, had intended a man- statutory wording. They require broader deci- made micro-organism to be excluded from pat- sions based on public policy and social values; entability solely because it was alive. Moreover, Congress has the constitutional authority to the opinion invited Congress to overturn the make those decisions for society. It can act to re- decision if it disagreed with the Court’s inter- solve the questions left unanswered by the pretation. Court, overrule the decision, or develop a comprehensive statutory approach, if necessary. ‘Prepared Statement of C,enentech, Inc., cited in “Science May Most importantly, Congress can draw lines; it Patent New Forms of Life, Justices Rule, 5 to 4,” The New York can specifically decide which organisms, if any, ‘rimes, June 17, 1980, p. 1. ‘Prepared statement of the Peoples’ Business Commission, cited should be patentable. in “Science May Patent New Forms of Life, Justices Rule, 5 to 4,” The New York Times, June 17, 1980, p. 1.

Legal protection of inventions

The inherent “right” of the originator of a both. The separate laws covering trade secrets new idea to that idea is generally recognized, at and patents are the mean by which this is done. least to the extent of deserving credit for it when used by others. At the same time, it is also Trade secrets believed that worthwhile ideas benefit society when they are widely available. Similarly, when The body of law governing trade secrets rec- an idea is embodied in a tangible form, such as ognizes that harm has been done to one person in a machine or industrial process, the inventor if another improperly obtains a trade secret and has the “right” to its exclusive posession and use then uses it personally or discloses it to others. simply by keeping it secret. However, if he may A trade secret is anything—device, formula, or be induced to disclose the invention’s details, information—which when used in a business society benefits from the new ideas embodied provides an advantage over competitors ig- therein, since others may build upon the new norant of it—e.g., improper acquisition includes knowledge. The legal system has long recog- a breach of confidence, a breach of a specific nized the competing interests of the inventor promise not to disclose, or an outright theft. and the public, and has attempted to protect Trade secrecy is derived from the common law,

237 238 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

as opposed to being specifically created by the United States without his consent for 17 statute; the State courts recognize and protect it years. In return, the inventor must make full as a form of property. The underlying policy is public disclosure of his invention. The policy be- one of preventing unfair competition or unjust hind the law is twofold. First, by rewarding suc- benefits. The protection lasts indefinitely. Two cessful efforts, a patent provides the inventor well-known examples of long-time trade secrets and those who support him with the incentive are the formulas for Coca Cola and for Smith to risk time and money in research and develop- Brothers’ black cough drops; the latter is sup- ment. Second, and more importantly, the patent posedly over 100 years old. system encourages public disclosure of technical information, which may otherwise have re- A company relying on trade secrecy to pro- mained secret, so others may use the knowl- tect an important invention must take several edge. The inducement in both cases is the po- steps to effect that protection. These include: tential for economic gain through exploitation permitting only key personnel to have access, of the limited monopoly. Of course, there are requiring such people to sign complex contracts many reasons why this potential may not be involving limitations on subsequent employ- realized, including the existence of competing ment, and monitoring employees and com- products. petitors for possible breaches of security. Even so, there are practical limitations to what can be To qualify for patent protection, an invention done and what can be proved to the satisfaction must meet three statutory requirements: it of a court. Moreover, independent discovery of must be capable of being classified as a process, the secret by a competitor is not improper, in- machine, manufacture, or composition of mat- cluding the discovery of a secret process by an ter; it must be new, useful, and not obvious; and examination of the commercially marketed it must be disclosed to the public in sufficient product. Most importantly, once a trade secret detail to enable a person skilled in the same or becomes public through whatever means, it can the most closely related area of technology to never be recaptured. Thus, reliance on trade construct and operate it. Plants that reproduce secrecy for protecting inventions can be risky. asexually may also be patented, but slightly different criteria are used.

! Patents I Although the categories in the first require- I In contrast to the common law development ment are quite broad, they are not unlimited. In of trade secrecy, patent law is a creation of Con- fact, the courts have held such things as scien- gress. The Federal patent statutes (title 35 of the tific principles, mathematical formulas, and United States Code) are derived from article I, products of nature to be unpatentable on the section 8, of the Constitution, which states: grounds that they are only discoveries of pre- existing things—not the result of the inventive, The Congress shall have Power . . . To pro- creative action of man, which is what the patent mote the Progress of Science and useful Arts, by securing for limited Times to Authors and In- laws are designed to encourage. This concept ventors the exclusive Right to their respective was reaffirmed in the Chakrabarty opinion. Writings and Discoveries. The requirement that an invention be useful, This clause grants Congress the power to cre- new, and- not obvious further narrows the ate a Federal statutory body of law designed to range of patentable inventions. Utility exists if encourage invention by granting inventors a the invention works and would have some bene- lawful monopoly for a limited period of time. fit to society; the degree is not important. Novel- Under the current statutory arrangement, ty signifies that the invention must differ from which is conceptually similar to the first patent the “prior art” (publicly known inventions or statutes promulgated in 1790, a patent gives the knowledge). Novelty is not considered to exist, inventor the right to exclude all others from —e.g., if: 1) the applicant for a patent is not making, using, or selling his invention within the inventor, 2) the invention was previously Ch. 12—Patenting Living Organisms ● 239 known or used publicly by others in the United Any organism that both meets the broad defini- States, or 3) the invention was previously de- tion of a trade secret and may be lawfully scribed in a U.S. or foreign patent or publica- owned by a private person or entity can be pro- tion. The inability to meet the novelty require- tected by that body of law, including micro- ment is another reason why products of nature organisms, plants, animals, and insects. In addi- are unpatentable. Nonobviousness refers to the tion, plants are covered specifically by two Fed- degree of difference between the invention and eral statutes, the Plant Patent Act of 1930 and the prior art. If the invention would have been the Plant Variety Protection Act of 1970. Fur- obvious at the time it was made to a person with thermore, the Supreme Court has now ruled ordinary skill in that field of technology, then it that manmade micro-organisms are covered by is not patentable. The policy behind the dual the patent statutes. Its determination of con- criteria of novelty and nonobviousness is that a gressional intent in the Chakrabarty case was patent should not take from the public some- based significantly on an analysis of the two thing which it already enjoys or potentially plant protection statutes. enjoys as an obvious extension of current Patent protection for plants was not available knowledge. until Congress passed the Plant Patent Act of The final requirement—for adequate public 1930, recognizing that not all plants were prod- disclosure of an invention—is known as the en- ucts of nature because new varieties could be ablement requirement. It is designed to ensure created by man. This Act covered new and dis- that the public receives the full ,benefit of the tinct asexually reproduced varieties other than new knowledge in return for granting a limited tuber-propagated plants or those found in na- monopoly. As a public document, the patent ture. * The requirement for asexual reproduc- must contain a sufficiently detailed description tion was based on the belief that sexually of the invention so that others in that field of reproduced varieties could not be reproduced technology can build and use it. At the end of true-to-type and that it would be senseless to try this description are the claims, which define the to protect a variety that would change in the boundaries of the invention protected by the next generation. To deal with the fact that or- patent. ganisms reproduce, the Act conferred the right to exclude others from asexually reproducing The differences between trade secrets and the plant or from using or selling any plants so patents, therefore, center on the categories of reproduced. It also liberalized the description inventions protected, the term and degree of requirement for plants. Because of the impos- protection, and the disclosure required. Only sibility of describing plants with the same de- those inventions meeting the statutory require- gree of specificity as machines, their description ments outlined above qualify for patents and need only be as complete as is “reasonably possi- then only for a limited time, whereas anything ble.” giving an advantage over business competitors qualifies as a trade secret for an unlimited time. By 1970, plant breeding technology had ad- A patent requires full public disclosure, while vanced to where new, stable, and uniform vari- trade secrecy requires an explicit and often eties could be sexually reproduced. As a result, costly effort to withhold information. The pat- Congress provided patent-like protection to ent law provides rights of exclusion against novel varieties of plants that reproduced sexu- everyone, even subsequent independent inven- ally by passing the. Plant Variety Protection Act tors, while the trade secrecy law protects only of 1970. Fungi, bacteria, and first-generation against wrongful appropriation of the secret. hybrids were excluded. * * Hybrids have a built- ● Approximately 4,500 plant patents have been issued to date, most for roses, apples, peaches, and chrysanthemums. Living organisms ● ● Originally, six vegetables—okra, celery, peppers, tomatoes, carrots, and cucumbers—were also excluded. On Dec. 22, 1980, Although the law for protecting inventions is President Carter signed legislation (H.R. 999) amending the Plant Variety Protection Act to include these vegetables, to extend the usually thought of as applying to inanimate ob- term of protection to 18 years, and to make certain technical jects, it also applies to certain living organisms. changes. 240 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

in protection, since the breeder can control the others from selling, offering for sale, reproduc- inbred, parental stocks and the same hybrid ing (sexually or asexually), importing, or export- cannot be reproduced from hybrid seed. ing the protected variety. In addition, others cannot use it to produce a hybrid or a different The 1970 Act, administered by the Office of variety for sale. However, saving seed for crop Plant Variety Protection within the U.S. Depart- production and for the use and reproduction of ment of Agriculture (USDA), parallels the patent protected varieties for research is expressly statutes to a large degree. Certificates of Plant permitted. The term of protection is 18 years. Variety Protection allow the breeder to exclude

The Chakrabarty case

In 1972, Ananda M. Chakrabarty, then a re- ‘(Whoever invents or discovers any new and search scientist for the General Electric Co., de- useful process, machine, manufacture, or com- veloped a strain of bacteria that would degrade position of matter, or any new and useful im- four of the major components of crude oil. He provement thereof, may obtain a patent there- did this by taking plasmids from several dif- for, subject to the conditions and requirements ferent strains, each of which gave the original of this title. ” strain a natural ability to degrade one of the Specifically, we must determine whether re- crude oil components, and putting them into a spondent’s micro-organism constitutes a “manu- single strain. The new bacterium was designed facture” or “composition of matter” within the to be placed on an oil spill to break down the oil meaning of the statute. into harmless products by using it for food, and After evaluating the words of the statute, the then to disappear when the oil was gone. Be- policy behind the patent laws, and the legis- cause anyone could take and reproduce the mi- lative history of section 101 of the patent crobe once it was used, Chakrabarty applied for statutes and of the two plant protection Acts, a patent on his invention. The U.S. Patent and the Court ruled that Congress had not intended Trademark Office granted a patent on the proc- to distinguish between unpatentable and pat- ess by which the bacterium was developed and entable subject matter on the basis of living ver- on a combination of a carrier (such as straw) sus nonliving, but on the basis of “products of and the bacteria. It refused to grant patent pro- nature, whether living or not, and human-made tection on the bacterium itself, contending that inventions. ”4 Therefore, the majority ruled, living organisms other than plant were not “[t]he patentee has produced a new bacterium patentable under existing law. On appeal, the with markedly different characteristics from Court of Customs and Patent Appeals held that any found in nature and one having potential the inventor of a genetically engineered micro- for significant utility. His discovery is not na- organism whose invention otherwise met the ture’s handiwork, but his own; accordingly it is legal requirements for obtaining a patent could patentable subject matter under $l01.”5 The not be denied a patent solely because the inven- majority did not see their decision as extending tion was alive. The Supreme Court affirmed. the limits of patentability beyond those set by The majority opinion characterized the issue Congress. as follows:3 The Court found that, in choosing such ex- The question before us in this case is a nar- pansive terms as “manufacture” and “com- row one of statutory interpretation requiring us position of matter”—words that have been in every patent statute since 1793—Congress plain- ly intended the patent laws to have a wide

41bid, p, 2,210. 3Diamond v. Chakrabarfy, 100 S. Ct. 2204, 2207 (1980). Slbid, p. 2,208. Ch. 12—Patenting Living Organisms ● 241 scope. Moreover, when these laws were last re- branches of Government, and not the courts, modified in 1952, the congressional committee are competent to make. It further recognized reports affirmed the intent of congress that pat- that Congress could amend section 101 to spe- entable subject matter “include anything under cifically exclude genetically engineered orga- the sun that is made by man. ”6 The Court nisms or could write a statute specifically de- acknowledged that not everything is patentable; signed for them. laws of nature, physical phenomena, and abstract ideas are not. The dissenting Justices agreed that the issue was one of statutory interpretation, but inter- The Court found the Government’s argu- preted section 101 differently. They saw the ments unpersuasive. Specifically, that passing two plant protection Acts as strong evidence of the Plant Patent Act of 1930 and the Plant Varie- congressional intent that section 101 not cover ty Protection Act of 1970, which excluded bac- living organisms. In view of this, the dissenters teria, was evidence of congressional under- maintained that the majority opinion was ac- standing that section 101 did not apply to living tually extending the scope of the patent laws organisms; otherwise; these statutes would beyond the limit set by Congress. have been unnecessary. In disagreeing, the The stated narrowness of the Court’s decision Court stated that the 1930 Act was necessary to may limit its impact as precedent in subsequent overcome the belief that even artificially bred plants were unpatentable products of nature cases that raise similar issues, although not nec- and to relax the written description require- essarily. Certainly, the decision applies to any genetically engineered micro-organism. It is a ment, permitting a description as complete as is “reasonably possible. ” As for the 1970 Act, the technical distinction without legal significance that most of the work being done on such orga- Court stated that it had been passed to extend nisms involves recombinant DNA (rDNA) tech- patent-like protection to new sexually reproduc- niques, which Chakrabarty did not use. The real ing varieties, which, in 1930, were believed to question is whether or not it would permit the be incapable of reproducing in a stable, uniform patenting of other genetically engineered or- manner. The 1970 Act’s exclusion of bacteria, ganisms, such as plants, animals, and insects. which indicated to the Government that Con- Any fears that the decision might serve as a gress had not intended bacteria to be pat- legal precedent for the patenting of human be- entable, was considered insignificant for a num- ings in the distant future are totally groundless. ber of reasons. Under our legal system, the ownership of hu- The Government had also argued that Con- mans is absolutely prohibited by the 13th gress could not have intended section 101 to amendment to the Constitution. cover genetically engineered micro-organisms, Although the Chakrabarty case involved a since the technology was unforeseen at the micro-organism, there is no reason that its ra- time. The majority responded that the very pur- tionale could not be applied to other organisms. pose of the patent law was to encourage new, In the majority’s view, the crucial test for pat- unforeseen inventions, which was why section entability concerned whether or not the micro- 101 was so broadly worded. Furthermore, as 7 organism was manmade. Conceptually, there is for the “gruesome parade of horribles” that nothing in this test that limits it to micro- might possibly be associated with genetic engi- organisms. The operative distinction is between neering, the Court stated that the denial of a humanmade and naturally occurring “things,” patent on a micro-organism might slow the sci- regardless of what they are. Thus, the Chakra- entific work but certainly would not stop it; and barty the consideration of such issues involves policy opinion could be read as precedent for in- judgments that the legislative and executive cluding any genetically engineered organism (except humans) within the scope of section 101. es, R@, N~,, 1979, 82d (:[]ng, , 2d $j~ss. , P. 5, 19,52; H . R. Rq)t . No. Whether a court in a subsequent case will inter- 1923, ml IIong., 2d Sess. j p. 6, 1952, cited in Diam~~~ v. Chakrabarfy, 100” S. Ct. 2204, 2207 [ 1980). pret Chakrabarty broadly or narrowly cannot be 7Diamond t’. Chakrabarty, 100 S. Ct. 2204, 2211 (1980). predicted. 242 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Even if section 101 were interpreted as cover- may be theoretically possible for animals and ining other genetically engineered organisms, sects, it may be logistically impractical. How- they probably could not be patented for failure ever, if tissue culture techniques advance to the to meet another requirement of the patent point where genetically engineered organisms laws—the enablement requirement. It is gener- can be made from single cells and stored indefi- ally impossible to describe a living organism in nitely in that form, there appears to be no rea- writing with enough detail so that it can be son to treat them any differently than micro- made on the basis of that description. Relaxing organisms, in the absence of a specific statute this requirement for plants was one reason prohibiting their patentability. behind the Plant Patent Act of 1930. For micro- organisms, the problem is solved by depositing a publicly available culture with a recognized national repository and referring to the accession number in the patent. * While such an approach ● This procedure was accepted by the Court of Customs and Pat- ent Appeals (CCPA) in upholding a patent on a process using micro- organisms. Application of Argoudelis,” 434 F.Zd 1390 (CCPA 1970). This procedure should also be acceptable for patents on micro- organisms themselves.

Potential impacts of the decision and related policy issues

During the 8-year history of the Chakrabarty THE RELATIONSHIP BETWEEN PATENTS AND INNOVATION case and the surrounding public debate, numerous assertions were made about the poten- The patent system is supposed to stimulate in- tial impacts of permitting patents on genetically novation—the process by which an invention is engineered organisms. They ranged from more brought into commercial use—because the in- immediate effects on the biotechnology indus- ventor does not receive financial rewards until try, the patent system, and academic research the invention is used commercially. The Con- to the long-term impacts on genetic diversity stitution itself presumes this, as do the statutes and the food supply. In addition, two major pol- enacted pursuant to the patent clause in article icy issues that have been raised are the morality I, section 8. Attempts have been made to subject of patenting living organisms; and the propriety this presumption to empirical analysis; but in- of permitting private ownership of inventions novation is extraordinarily complex and in- from publicly funded research. volves interacting factors that are difficult to separate. In addition, the existence of patents and trade secrets as alternative means for pro- Impacts on industry tection makes it almost impossible to study the effects of patents alone on invention and in- The basic question for industry is the extent novation. * to which permitting patents on genetically engineered organisms will stimulate both their de- ● A major reuson for the lack of empirical studies has been the velopment and the growth of the industries em- lack of appropriate data. The information available on the number ploying them. To ascertain this requires first an of patents applied for and issued does not indicate the importance, economic benefits, or economic costs of inventions (whether pat- examination of the theory and social policies ented or unpatented) that may not have existed at all or may have underlying the patent system. been created more slowly if not for the patent system.S In Presi- Ch. 12—Patenting Living Organisms . 243

Several reasonable arguments have been pre- Sir Alexander Fleming had discovered a prom- sented to support the presumption that the pat- ising weapon against bacterial infection, it took ent system stimulates innovation. First, the po- him, over 10 years to get the money and facilities tential for the exclusive commercialization of a he needed to purify and produce penicillin in new product or process creates the incentive to bulk. Only World War II and an international ef- undertake the long, risky, and expensive proc- fort finally accomplished that task. Sir Howard ess from research through development to mar- Florey, who shared the Nobel prize with Flem- keting. At every stage of innovation—from de- ing for developing penicillin, attributed the fining priorities and making initial estimates of delay to their not having patented the drug, an invention’s value to advertising the finished which he termed “a cardinal error. “10 product—the inventor and his backers must Some have claimed that the monopoly power spend time, money, and effort, not only to de- of a patent can be used to retard innovation. A velop a product but to convince others of its corporation can legally refuse to license a pat- worth. Only a small percentage of new ideas or ent on a basic invention to holders of patents on inventions survive. If a competitor, particularly improvements, thus protecting its product from a larger firm with a well-developed marketing becoming less attractive or obsolete. On the capability, were free to copy a product at this point, smaller firms would have little incentive other hand, unless the corporation can satisfy the market for its product, it is usually in its to undertake the process of innovation. economic interest to engage in cross-licensing Second, the information and new knowledge arrangements with holders of improvement pat- disclosed by the patent allows others to develop ents; it receives royalties and all parties can competing, and presumably better, products by market the improved product. Cross-licensing improving on the patented product or “in- has been misused several times by a few domi- venting around” it. Third, patents may reduce nant firms in an attempt to exclude innovative unnecessary costs to individual firms, thereby new firms from their markets. Such arrange- freeing resources for further innovation, Once ments violate the antitrust laws. Whether or not a Patent is issued, competitors can redirect that body of law adequately prevents patent research and development (R&D) funds into misuse is beyond the scope of this report. other areas. For the firm holding the patent, THE ADVANTAGES OF PATENTING maintaining control over the technology is LIVING ORGANISMS theoretically less expensive, since the costs of Given the presumed connection between trade secret protection are no longer patents and innovation, the next question is required. * * whether patenting a living organism would add Anecdotal accounts support the proposition significant protection for the patent holder, or that patents stimulate innovation; probably the whether alternative approaches would be suffi- best known is the story of penicillin. Although cient. In this context, it is necessary to focus on the present industrial applications—which involve only micro-organisms—to examine alter- dent Carter’s recent report on industrial innovation, the patent native forms of patent coverage and to compare policy committee, composed of industry representatives having the protection offered by trade secrecy with long experience with the patent system, recommended ways of that offered by patents. enhancing innovation by improving the patent system, including the patenting of industrially important living organisms. However, Opinions vary widely among spokesmen for they pro~’ided no hard economic data to support their recommen- the genetic engineering companies on the value dation.’ 11 ‘Carole Kitti, and Charles L. Trozzo, T’he Effects of Patent and of patenting micro-organisms. Spokesmen for Antifrusl Laws, fiegulations, and Practices on innovation, vol. II (Arl- Genentech, Inc., have stated numerous times ington, t’a., Institute for Defense Aniilyses, 1976), pp. 2,9. ‘U.S. Department of Commerce, Advisory Committee on lndus- Iria/ Innovation: Final Report, September 1Y79, pp. 148-149. ‘“Ibid, pp. 170-171. ● *Patent rights can be very expensive to enforce against an in- I ID. Dickson, “patenting Living organisms: HOW to Beat the Bug- !’ringer, however, should litigation be necessary. Rustlers, ” Nature, vol. 283, Jan. 10, 1980, pp. 128-129. 244 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals that such patents are crucial to the development the existence of such protection, the question is of the industry, while others have stated their what the actual advantages are to patenting the preference for trade secrecy. micro-organisms as well. Genentech’s friend-of-the-court brief filed in One advantage results from the ability of a the Chakrabarty case stated, “The patent incen- living organism to reproduce itself. Developing tive did, and doubtless elsewhere it will, prove a new microbe for a specific purpose, such as to be an important if not indispensable factor in the production of human insulin, can be a long, attracting private support for life-giving re- difficult, and costly procedure. Yet once it is search. ”l2 Genentech has also supported in- developed, it reproduces endlessly, and any- creased patent protection because, to attract body acquiring a culture would have the benefit top scientists to the company, it had to give of the development process at little or no cost assurances that they would be able to publish unless the organism were patented. freely .13 This severely curtails any reliance on Often, a company is able to keep the microbe trade secrets. a trade secret, since only the product is sold. The rationale behind the contrary position is However, where the microbe is the product— based on the belief that the industry is moving such as with Chakrabarty’s oil-eating bacteri- so quickly that today’s frontrunner is not nec- um—patenting the organism is the best means essarily tomorrow’s, and that unique knowledge of protection. Moreover, even when a microbe translates into competitive advantage. Thus, in a itself can be kept under lock and key, a com- strategy similar to that of the advanced micro- pany desiring to patent the process in which it is electronics industry, firms may prefer to rely used must place a sample culture in a public on trade secrets even for patentable inventions, repository to meet the enablement require- coupled with an intense marketing effort once ment. an invention has reached the commercial stage. A competitor could legally obtain the micro- The idea is to get the jump on competitors and organism. If the competitor were to use it to to stay in front. 14 make the product for commercial purposes, the The uncertainty about whether micro-orga- company might suspect infringement but have nisms could be patented before the Supreme difficulty proving it, especially when the prod- Court’s decision does not appear to have hin- uct is not patented, The infringing activity dered the development of the industry. Clearly, would take place entirely within the confines of companies did not have any difficulty raising the competitor’s plant. Mere suspicion is not suf- capital—e.g., before the decision, Cetus Corp. ficient legal grounds for inspecting the com- had a paper value of $250 million without hold- petitor’s plant for evidence of infringement ing a single patent on a genetically engineered when the unpatented product could theoret- organism. Moreover, products such as insulin, ically be made by many different methods human growth hormone, and interferon were besides the one patented. * being made, albeit in small quantities, by un- A second, but less certain, advantage pro- patented, genetically modified organisms. (See vided by patenting the micro-organism is that ch. 4.) even uses and products of the organism not dis- Before the decision, companies relied either covered by the inventor would be protected in- entirely on trade secrecy for protection, or on a directly. That is, while new uses and products combination of patents on the microbiological could be patented by their inventors, those pat- process and the product and trade secret pro- ents would be “dominated” by the micro-orga- tection of the mice-organism itself. Considering nism patent. Royalties would have to be paid

iZBrief f~r (;ellelltech as Amicus (hriae, p. ~. “Some would answer this assertion by saying that ii lawsuit ‘Whonlas Kiley, t’ice President and (;eneral [kmnsel for coutd he stiirt~d even on I he hasis of little et’idence; the suing com- Genentech, personal communication, Apr. 15, 1980. Piit)~ t~ould ]WIY oil the discovery }) IWC[?SS, which is Iil]eral and j4Di~ks~n, op cit., p. 128. }$’iCi(?-l’illlgillg, to provide iill~ existing evid(?nce of infringenlent. Ch. 12—Patenting Living Organisms ● 245

whenever the micro-organism was used for ● whether the organism itself or the sub- commercial purposes. Whether this would be a stance that it makes will be the commercial significant advantage in practice is uncertain. product, Usually, only one product is optimally produced ● whether there is any significant doubt of by a given micro-organism and only one micro- its meeting the legal requirements for organism is best for a given process. Pre- patenting, sumably, the micro-organism’s inventor would ● whether there is the likelihood of others also have discovered and patented its best use discovering it independently, and product. ● whether it is a pioneer invention, ● what its projected commercial life is and Another alternative to patenting a man-made how readily others could improve on it if it micro-organism, besides trade secrecy, is to pat- were disclosed in a patent, ent its manmade components. Examples of ● whether there are any plans for scientific these include a plasmid containing the cloned publication, and gene, a sequence of DNA, or a synthetic gene ● what the costs of patenting are versus re- made by the reverse transcriptase process. liance on trade secrecy. These components, which are nothing more than strings of inanimate chemicals, would not The first two factors make the decision easy. be unpatentable products of nature if they were Obviously, an organism like Chakrabarty’s can made in the laboratory and were not identical to best be protected by a patent. In most instances, the natural material. Patenting them would not the substance made by the organism is the com- be equivalent to patenting the entire organism, mercial product, In that case, if there are sig- since their function would be affected in vary- nificant doubts that the organism can meet all ing degrees by the internal environment of their the legal requirements for patentability, the host. Nevertheless, the inventor of a partic- company would probably decide to rely on ularly useful component, such as an efficient trade secrecy. and stable plasmid, might want to patent it re- The next three factors require difficult de- gardless of whether or not the organism could cisions to be made on the basis of the charac- be patented, since it could be used in an in- teristics of the new organism, its product, and definite number of different micro-organisms. the competitive environment. If research to de- Thus, if Congress were to prohibit patenting velop a particular product is widespread and in- of micro-organisms because they are alive, intense (as is the case with interferon), the risk of dustry could compensate to a large degree by a competitor developing the invention inde- patenting inanimate components. On the other pendently provides a significant incentive for hand, if Congress allows the Supreme Court’s patenting. On the other hand, reverse engineer- decision to stand, certain components will un- ing (examination of a product by experts to dis- doubtedly still be patented. In fact, such patents cover the process by which it was made) by nay become more important than patents for competitors is virtually impossible for products micro-organisms, since the components are the of micro-organisms because of the variability critical elements of genetic engineering. and biochemical complexity of microbiological processes. PATENT V. TRADE SECRET PROTECTION Even with the advantages provided by pat- Thus, greater protection may often lie in enting a micro-organism, a company could still keeping a process secret, even if the microbe decide to rely on trade secrecy. In choosing be- and the process could be patented. This is es- tween these two options, it would evaluate the pecially true for a process that is only a minor following factors: 15 improvement in the state of the art or that produces an unpatentable product already made by many competitors. The commercial life of the “K. Saliw’atl(’tlik, “hli(’l>ol)iologi( ’ill lmwltiot)s: Prolwt 1).v l)iit(’llt- ~ 01” hlililllilill ilS il ‘1’l’il(lt’ S(>(’l’t’ll’” f}(:l’t’/f)/Jnft:r?l.s in Indms(rial process might be limited if it were patented be- icrobiolo

76-565 0 - 81 - 17 246 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals and not worth the time and money to prosecute Conversely, if the Court had reached the op- Reliance on trade secrecy might then extend its posite decision, the industry would have been commercial life. held back only moderately because of reason- Most companies would patent truly pioneer ably effective alternative means of protection. inventions, which often provide the opportunity for developing large markets. Moreover, pat- Impacts of the Court's decision on the ents of this sort tend to have long commercial patent law and the Patent lives, since it is difficult to circumvent a pioneer and Trademark Office invention and since any improvements are still subject to the pioneer patent. Furthermore, in- The key rationale supporting the Court’s fringement is easy to detect because of the in- holding Chakrabarty’s microbe to be patentable vention’s trailblazing nature. was the fact that it was manmade; its status as a living organism was irrelevant. The Patent Of- The last two factors involve considerations fice interprets this decision as also permitting secondary to a product and its market. Ob- patents on micro-organisms found in nature but viously, any publication of the experiments whose useful properties depend on human in- leading to an invention foreclose the option of tervention other than genetic engineering,l7 trade secrecy. Also, company must evaluate the e.g., if the isolation of a pure culture of a options of protection via either patenting or microbial strain induces it to produce an an- trade secrecy in terms of their respective cost tibiotic, that pure culture would be patentable effectiveness. subject matter. IMPACT OF THE COURT’S DECISION Because of the complexity, reproducibility, ON THE BIOTECHNOLOGY INDUSTRY and mutability of living organisms, the decision The Chakrabarty decision will add some pro- may cause some problems for a body of law detection for microbiological inventions by pro- signed more for inanimate objects than for liv- viding companies with an additional incentive ing organisms. It raises questions about the for* the commercial development of their inven- proper interpretation and application of the re- tions, particularly in marginal cases, by lower- quirements for novelty, nonobviousness, and ing uncertainty and risk. A greater effect will enablement. In addition, it raises questions result from the new information disclosed in about how broad the scope of patent coverage patents on inventions that otherwise might have on important micro-organisms should be and been kept secret indefinitely. Competitors and about the continuing need for the two plant pro- academicians will gain new knowledge as well tection Acts. These uncertainties could result in as a new organism upon which to build. The increased litigation, making it more difficult and Patent office had deferred action on about 150 costly for owners of patents on living organism! applications, while awaiting the Court’s deci- to enforce their rights. sion; as of December 1980, it was processing ap- The complexity of living matter will make i proximately 200 applications on micro-orga- 16 difficult for anyone examining the invention to nisms. * determine if it meets the requirements for nov Depending on the eventual number and im- elty, nonobviousness, and enablement. Micro- portance of patented inventions that would organisms can have different characteristics in have otherwise been kept as trade secrets, the different environments.’ Moreover, microbic ultimate effect of the decision on innovation in taxonomists often differ on the precise classifi- the biotechnology industry could be significant. cation of microbial strains. Even after expensive tests, uncertainty may still exist about whether 16R[+11[) ‘l’[~~llll~v~l,, ,\SSISlillll (:t)llllllissi[)ll(![’ 1’01” PillPlltS, [ 1.S. Piil - a specific micro-organism is distinct from othter (1111 illl(l ‘l’1.iidolliii~.k otl’i(x?, }](~t’sotliil (’otllllltllli($iit iou, Jilt]. 8, I !]& 1. known strains; scientists do not have complete “1’11[’S(’ ill)pli(’iltiol)S i 1l(’lll(lt> ill)()(lt I ()() 011 ~[gllf]t i(’illl}r (’11- gineerd mi(wtws illl(l iil)ollt 100” 011 (“llltlll’[~S ot” stl-;iil]s is(]lilt(~(l 1’1’0111 Ililt(ll’(’. ‘‘1 hid, Ji~ll. 7, 19S 1. Ch. 12—Patenting Living Organisms ● 247

knowledge of any single organism’s biophysical made do not overlap with any “prior art” or ob- and biochemical mechanisms. Consequently, vious extensions thereof—e.g., a person who there may be cases where it is difficult to know developed a particular strain of Escherichia coli the prior art precisely enough to make a deter- that produced human insulin through a geneti- mination of novelty. cally modified plasmid could be entitled to a patent covering all strains of E. coli that produce Similarly, microbial complexity raises prob- the insulin in the same way. Chakrabarty’s pat- lems in determining nonobviousness because ent application—e.g., claimed “a bacterium from there are so many different ways of engineering the genus pseudomonas containing therein at a new organism with a desired trait—e.g., a least two stable energy generating plasmids, gene could be inserted into a given plasmid at each of said plasmids providing a separate several different positions. If a microbe with the hydrocarbon degradative pathway.” Several gene at one position in the plasmid were species and hundreds of strains of Pseudomonas patented, could a patent be denied to an other- fit this description. A patent limited to a par- wise structurally identical organism with the ticular microbial strain is not particularly gene at a different position because the second valuable because it can easily be circumvented was obvious? Perhaps not. The second organism by applying the inventive concept to a sister would probably not be an obvious invention if it strain; on the other hand, a patent covering a provided significantly more of the product, a whole genus of micro-organism (or several) may better quality product under similar fermenting retard competition. This problem will probably conditions, or the same product under cheaper be resolved by the Patent Office and the courts operating conditions. on a case-by-case basis. As to enablement, the major problem has Another aspect of the same problem is been discussed previously; placing a culture of whether a patent on an organism would cover the micro-organism into a repository is the ac- mutants. It would not if the mutation occurred cepted solution. One problem with repositories, spontaneously and sufficiently altered the however, is their potential misuse. In a case in- claimed properties. However, if a new organism volving alleged price fixing and unfair competi- were made in a laboratory with a patented tion—e.g., the Federal Trade Commission found organism as a starting point, the situation would that micro-organisms placed in a public reposi- be analogous to one where an inventor can pat- tory pursuant to process and product patents ent an improved version of a machine but must on the antibiotic Aureomycin did not produce come to terms with the holder of the “domi- the antibiotic in commercially significant nant” patent before marketing it. amounts; in actual practice, other strains were being used for production, and the company in- The Chakrabarty decision also raises ques- volved was able to benefit from a patent, while, tions about the scope of section 101 and its rela- in effect, retaining the crucial micro-organism tion to the plant protection Acts—e.g., plant as a trade secret. 18* tissue culture is, in effect, a collection of micro- organisms; should it be viewed as coming under Complexity also raises questions about the ap- section 101 instead of either of the plant pro- propriate scope of patent coverage. In a patent, tection Acts? Could plants excluded under these the inventor is permitted to claim his invention Acts—such as tuber-propagated plants or first- as broadly as possible, so long as the claims generation hybrids—be patented under section l#Anlerican (:vanamid (:0., ef. al., 6~J k“I’(; 1747, 1905 n. 14 ( 1963), 101? Could any plants or seeds be patented vara[cd and remanded, 363 *’. zcl 7.57 (6th (:ir. 1966), readopted 72 under section 101, and if so, is there still a need E-[’(: 623 ( 1967), affirmed 401 F.2d 574 f6th (;ir. 1968), cerf. denied, 394 [ 1.s. 920 ( 1 969). for the plant protection Acts? If there is a need,

“’1’he rompany had maintained that sec. I I z simply required it would the Acts be administered better by only to deposit ii strain that r(mfnrnwci to the des(x’iption of the one one agency? The Senate Committee on Appro- found in the paterlt application. ti(n~,eier, it is often the ciise with I)ii[’teriii thiit IWiIly striiins ot” it spw’ies will (wniorm to eten th~ priations has directed the Departments of Com- most prcrisel)’ written description. merce and Agriculture to submit a report 248 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals within 120 days of the Chakrabarty decision on information on genetic engineering applications the advisability of shifting the examining func- is not available.20 It may take examiners longer tion to USDA. 19 As of December 1980, this issue to process applications on micro-organisms than was still under study. These questions could be for those covering only microbiological proc- resolved by the courts, but they are probably esses or products because of the interpretive more amenable to a statutory solution. problems mentioned. Moreover, the Patent Of- fice will have to develop greater expertise in Another effect of the decision could be on molecular genetics—a frontier scientific field patent enforcement. The various uncertainties that has only recently been the subject of patent discussed above may have to be resolved applications. On the other hand, the Office through costly litigation. Moreover, in specific generally faces this problem for any new area cases, the problems associated with describing a of technology. micro-organism in sufficient detail may increase the chances that a patent will be declared In terms of increased numbers of applica- invalid. In any event, litigation costs would tions, the decision is not expected to have a sig- probably increase as more expert testimony is nificant effect on the Patent Office operations in needed. the next few years. The Office receives approx- The fact that organisms mutate might intro- imately 100,000 applications a year, and it has about 900 examiners, each processing an aver- duce still another complication into infringement actions. A deposited micro-organism is the age of about 100 applications per year. Figures on the number of applications on genetically standard by which possible infringement would engineered organisms vary, depending on how be judged. If it has mutated with respect to one the category is defined, and precise information of its significant characteristics, a patent holder has not been tabulated by the Patent Office. who is seeking to prove infringement may have Rough estimates indicate that in February 1980 no case. While this problem does not appear to about 50 applications were pending, and by be amenable to a statutory solution, the risk of December 1980, that number had increased to such a mutation is actually quite small. * about 100. Applications are being filed at the Because a living invention reproduces itself, rate of about 5 per month. Also, just over 100 the statutory definition of infringement may are pending on microbes that have been isolated have to be changed. Presently, infringement and purified from natural sources, but have not consists of making, using, or selling a patented been genetically engineered. Four examiners invention without the permission of the patent are working on both categories as well as holder. Theoretically, someone could take part others. Thus, in view of the total operations of of a publicly available micro-organism culture, the office, these applications require only a reproduce it, and give it away. Arguably, this is small part of its resources. over the next few not “making” the invention, and the patent years, the number is expected to increase be- holder would have the burdensome and expen- cause of the decision and developments in the sive task of going after each user. The two plant field but not to a point where more than a few protection statutes deal with this problem by additional examiners will be needed.2l specifically prohibiting unauthorized reproduction of the protected plant. This approach Impact of the Court's decision on may be necessary for other living inventions, academic research How all of these uncertainties will affect the Patent Office’s processing of applications cannot Many academicians have voiced concerns about the effects on research of the Chakrabarty be predicted. Currently, the average processing time for all applications is 22 months; separate decision and the commercialization of molecular biology in general. They claim that the re- j~s. R~P~. No. 96.251, 96th (king. 1s1 Sess., 1979, P. 46. “Most nlicro-or#misms can be stored in ii freeze-dried form, Zot+elle Tegtfllever, person~l c(~lllnllttli~:ati[)ll, Dti:. 1.’, 198(J. which ~ntilils virtually II(J risk (It’ n]tltiiti[)n. Zllt)id, , De[; . 151 1980, iind Jiil). 8, 1981. Ch. 12—Patenting Living Organisms ● 249 suits of rDNA research are not being published are being prepared for publication because of while patent applications are pending, discus- the competitive nature of modern science. sion at scientific meetings is being curtailed, and Essentially, the issue is the effect of the com- novel organisms are less likely to be freely ex- mercialization of research results on the re- changed. A related concern is that scientific search process itself. Even if patents were not papers may not be citing the work of other sci- available for biological inventions, the inventor entists to avoid casting doubt on the novelty or would simply keep his results secret if he were inventiveness of the author’s work, should he interested in commercialization. Viewed from decide to apply for a patent. Finally, there is this perspective, it is difficult to see why the concern that the granting of patents on basic availability of patents should affect the ex- scientific processes used in the research labora- change of scientific information in genetic re- tory will directly impede basic research—e.g., search any more than it does in any other field two scientists have recently been granted a pat- of research with commercial potential. The ent on the most fundamental process of molec- Chakrabarty decision may inhibit the dissemina- ular genetic technology—the transfer of a gene 22 tion of information only if it creates an atmos- in a plasmid using rDNA techniques. The pat- phere that stimulates academic scientists to ent has been transferred to the universities commercialize their findings. However, if it en- where they did their work—Stanford and the courages them to rely on patents rather than on University of California at San Francisco (UCSF). trade secrets, it will ultimately enhance the Although both universities have stated they dissemination of information. would grant low-royalty licenses to anyone who complied with the National Institutes of Health (NIH) Guidelines, subsequent owners of fun- Impacts of the Court’s decision on damental process patents may not be so genetic diversity and the food supply altruistic. Some public interest groups have claimed There are several reasons for believing that that patenting genetically modified organisms these concerns, although genuinely held, are will adversely affect genetic diversity and the somewhat overstated. First, patents on funda- food supply. The claim is based on an analogy to mental scientific processes or organisms should a situation alleged to exist for plants. Briefly, the not directly hinder research. The courts have groups claim that patenting micro-organisms interpreted patent coverage as not applying to will irrevocably lead to patents on animals, research; in other words, the patent covers only which will have the same deleterious effects on the commercial use of the invention.23 Also, it the animal gene pool and the livestock industry would be difficult and prohibitively expensive as the two plant protection Acts have had on the for a patent holder to bring infringement ac- plant gene pool and the plant breeding industry. tions against a large number of geographically The alleged effects are: loss of germplasm re- separated scientists. Second, patents ultimately sources as a result of the elimination of thou- result in full disclosure. If patents were not sands of varieties of plants; the increased risk of available, trade secrecy could be relied on, with widespread crop damage from pests and dis- the result that important information might eases because of the genetic uniformity result- never become publicly available. Third, al- ing from using a single variety; and the increas- though delays occur while a patent application ing concentration of control of the world’s food is pending, they often happen anyway while ex- supply in a few multinational corporations periments are being conducted or while articles through their control of plant breeding com- panics.24

ZZ[ 1s, f~iitt?l]t No. 4,23i’,22LI, issued [](’[’. 2, 198[). z~Kaz ,Man@act[lrjng (1]. v, Chcsehrough-Ponds, Inc., 211 ~’. SUPP. only limited evidence is available, but no con- 815 (S.l). N.Y. 1962) [dictum), +j_irmed 317 F.2d 679 (2cI [:ir. 1963); clusive connection has been demonstrated be- (,’hesterj_ie/d t’. (hi[ed States, 159 h-. Supp. 371 [(It. (:1. 1958); fhgan t’. I,ear A via, 55 F. Supp. 223 (S. [). N.Y. 1944) (dictum); Akro Aga(e zd~l.itlf for the peoples’ Blujincss (;(mmission as Am icus (M-he, (,’o. t. Afas[er Marb/e (,’o., 18 F. Supp. 305 (N.l). JV. \’a. lY37). pp. 6-13, Iliamondv. Chakrabarf-v, 100 S. [1. 2204 (1980). 250 . Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals tween the plant protection laws and the loss of tion—the engineering of people as an industrial genetic diversity, the encouragement of using a enterprise. 25 single variety, and any increased control by a The Chakrabarty opinion was written in nar- few corporations of the food supply. (For a de- row terms. But while its reasoning might be ap- tailed discussion, see ch. 8.) Therefore, any con- plied to a future case involving an animal or in- nection between patenting micro-organisms sect, it simply could not be used to justify the and potential detrimental impacts on the live- patenting of human beings because of the 13th stock industry appears tenuous at best. The amendment to the Constitution, which prohibits Chakrabarty assumptions that the decision will the ownership of humans. inevitably lead to patenting animals, and that the consequences will be the same as those One way to negotiate the slippery slope is to claimed to result from granting limited owner- deal directly with the adverse aspects of the ship rights to varieties of plants, are speculative. technology. Barriers can be erected along the slope; the Constitution already protects The morality of patenting living humans. Congress can erect other barriers by organisms statute, specifically drawing lines as to which organisms can or cannot be patented. The moral issue is difficult to analyze because The third part of the issue is religous or it embodies at least three overlapping questions: philosophical in nature. For many, the patent- whether it is moral to grant exclusive rights of ing of a living organism undermines the awe ownership to a living species; whether patents and deep respect they hold for the unique na- on lower forms of life will inevitably lead to ture of life. Moreover, it raises apprehensions of genetic engineering of humans; and whether an ultimate threat to concepts of the nature of patenting organisms undermines the generally humanity and its place in the universe. To these held belief in the uniqueness and sanctity of life, people, if life can be engineered and patented, especially human life. perhaps it is not special or sacred. If this is true It is difficult to assess the extent of the belief of lower organisms, why would human beings that patenting living organisms is intrinsically be different? (This and other aspects of the immoral, and no such assessment has been morality issue are discussed in greater detail in done. Its extent and intensity will probably be ch. 13.) directly correlated with the complexity of the organism involved. Fewer people will be dis- Private ownership of inventions turbed about patenting micro-organisms than from publicly funded research about patenting cattle. A belief in the immorali- ty of patenting a living organism is a value judg- Much of the basic research in molecular ge- ment to which Congress may wish to give some netics has been funded by Federal grants. Most consideration. of the work leading to the development of rDNA techniques—e.g., was performed at Stanford The second aspect of the moral issue revolves University and UCSF under NIH grants. The around the well-known metaphor of the “slip- scientists involved have received a patent on pery slope”-the fear that the first steps along that fundamental scientific process. Some op- the path of genetic engineering may irrevocably lead to man. Technology, at times, appears to ponents of patenting organisms have argued that private parties should not be permitted to have its own momentum; the aphorism “what own inventions resulting from federally funded can be done, will be done” has been true in the R&D; and in any event, there is something past. Thus, some people fear that patenting micro-organisms may indeed set a dangerous special about molecular genetics that requires the Federal Government to retain ownership of precedent and encourage the technology to progress to the point of the ultimate dehumaniza- Zslhid., p. 25. Ch. 12—Patenting Living Organisms ● 251 federally funded inventions and to make them There is still the question of whether patents generally available through nonexclusive on molecular techniques or genetically en- licenses. gineered micro-organisms are sufficiently different to merit exception from any general pat- Until recently, there had been no comprehen- ent policy decided on by Congress. For some, sive, governmentwide policy regarding ownership of patents on federally funded inventions. the molecular genetic techniques are unique because they are powerful scientific tools that can Some agencies, such as the Department of manipulate the life processes as never before. Health and Human Services (DHHS), permitted However, in a November 1977 report, NIH took nonprofit institutional grantees to own patents the following position with regard to patents on on inventions (subject to conditions deemed rDNA inventions developed under DHHS-NIH necessary to protect the public interest) if they 27 support: * had formal procedures for administering them. However, most agencies generally retained title There are no compelling economic, social, or to such patents, making them available to any- moral reasons to distinguish these inventions one in the private sector for development and from others involving biological substances or possible commercialization through nonex- processes that have been patented, even when clusive licenses. partially or wholly developed with public funds. The rationale behind the policy was simply The report was prompted by the Stanford- that inventions developed by public money UCSF patent application. Even though the appli- should be available to all—including private in- cation was in accord with the funding agree- dustry—on a nonexclusive basis. This arrangements between the institutions and NIH, the ment had been criticized as not providing suf- universities requested a formal NIH opinion on ficient incentive for industry to take the risks to the issue in view of the intense public interest in develop the inventions. Of the more than 28,000 rDNA research. NIH solicited comments from a patents owned by the Government, less than 4 group of approximately 67 individuals, ranging from academic and industrial scientists to percent have been successfully licensed; on the 28 other hand, universities, which do grant ex- students, lawyers, and philosophers. The clusive licenses on patents that they own, have review and analysis of the responses were been able to license 33 percent of their referred to the Federal Interagency Committee patents. 26 on rDNA Research, the Public Health Service, and the Office of the General Counsel of the De- On December 12, 1980, President Carter partment of Health, Education, and Welfare signed the Government Patent Policy Act of (now DHHS). A fairly uniform consensus on the 1980. The Act sets forth congressional policy above-quoted finding developed in this process; that the patent system be used to promote the the one significant dissenter, the Department of utilization of inventions developed under fed- Justice, contended that the Government should erally supported R&D projects by nonprofit retain ownership of any invention resulting organizations and small businesses. To this end, from federally funded rDNA research because the organization or firm may elect to retain title of the great public interest in that research. to those inventions, subject to various condi- zTThe patenting Of n~omlhinant IJVA Research Inventions [le- tions designed to protect the public interest. veloped under DHEW Support: An Anal.vsis b-v the IIirertor, )Vationai Such conditions include retention by the fund- Institutes of Heahh, November 1977, p. 16. ● ing agency of a nonexclusive, irrevocable, paid- “I”he repori further concluded that no change was Iwressary in the hasic NIH policy permitting nonprotil ()]’~iini~ii[i()lls to own up license to use the invention, and the right of ~illelltS 011 intentions developed Uilder (’olltl’ii(”tS (If’ ~1’illllS !1’0111 the Government to act where efforts are not the Department of Health, Edll(:iltion, illld tV’[’lt’ar(’ (n(n~ 1)}{}{ S), being made to commercialize the invention, in subject to several conditions to protect the puhl ic interest. ‘1’he 0111.V rcc’(]llln][~ilcl[!{l (’hii[l~[~ \\Ias I hitt the f(]t.]l]iil i)g]’~?~nl~l)(s IY- cases of health or safety needs, or when the two~]] Nl}l iind the institutions h(> iin~etld[?[l 10 r(quire thitt ii[lv use of the invention is required by Federal reg- licens(~es ot” i[lstitutioniil l)iit[~tlt holders COIIIply with the [.~ntiii]l- ulations. I]l[?nt stiin(iii[-ds 01” th(’ NIH (;ui(l(’lines in iinav production” 01’ LISP of I’I)N/l IIIOIW’LIIPS L] II(iPI> t h(’ I h’ense ii~r[~(~n}otlt. z~s, ~q)t N(). ~~.~~(), 961 h (:ong. 1$1 S~SS, I :)~!], l). z zn[t)i(j ,, a])~). I , I)J) 5-8’ 252 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

Issue and Options

ISSUE: To what extent could Congress tially conflict. Second, the Federal judiciary is provide for or prohibit the pat- not designed to take sufficient account of the entability of living organisms? broader political and social interests involved. In its Chakrabarty opinion, the Supreme B: Congress could pass legislation dealing with Court stated that it was undertaking only the the specific legal issues raised by the Court's narrow task of determining whether or not decision. Congress, in enacting the patent statutes, had Many of the legal questions do not readily intended a manmade micro-organism to be ex- lend themselves to statutory resolution. How- cluded from patentability solely because it was ever, three questions are fairly narrow and alive. Moreover, the opinion specifically invited well-defined and may therefore be better re- Congress to overrule the decision if it disagreed solved by statute: 1) Is there a continuing need with the Court’s interpretation. for the plant protection Acts if plants can be Congress has several options. It can act to re- patented under section 101? 2) If there is a con- solve the questions left unanswered by the tinuing need for these Acts, could they be ad- Court, overrule the decision, or develop a com- ministered better by one agency? 3) Should the prehensive statutory approach. Most important- definition of infringement be clarified by ly, Congress can draw lines; it can decide which amending section 271 of the Federal Patent organisms, if any, should be patentable. Statutes (title 35 U. S. C.) to include reproduction of a patented organism for the purpose of sell- OPTIONS ing it? A: Congress could maintain the status quo. Congressional action to clarify these issues would provide direction for industry and the Congress could choose not to address the Patent Office, and it would obviate the need for issue of patentability and allow the law to be a resolution through costly, time-consuming lit- developed by the courts. The advantage of this igation. Lessening the chances of litigation or option is that issues will be addressed as they the chances of a patent being declared invalid arise in the context of a tangible, nonhypo- will provide some stimulation for innovation by thetical case. Some of the issues raised in the lessening the risks in commercial development. debate on patenting may turn out to be irrel- In addition, Congress could determine that the evant as the technology and the law develop. plant protection Acts could be better admin- Moreover, many of the uncertainties raised by istered by one agency or should be incorporated Chakrabarty the decision regarding provisions under the more general provisions of the patent of the patent law other than section 101 may be law; if so, some administrative expenses prob- incapable of statutory resolution. The complexi- ably could be saved. ty of living organisms and the increase in knowledge of molecular genetics will raise such broad C: Congress could mandate a study of the plant and varied questions that legal interpretations protection Acts. of whether a particular biological invention Two statutes, the Plant Patent Act of 1930 meets the requirements of novelty, nonobvious- and the Plant Variety Protection Act of 1970, ness, and enablement will best be done on a grant ownership rights to plant breeders who case-by-case basis by the Patent Office and the develop new and distinct varieties of plants. Federal courts. They could serve as a model for studying the There are two disadvantages to this option. broader, long-term potential impacts of patent- First, a uniform body of law may take time to ing living organisms. An empirical study of the develop, since judicial decisions about new legal impacts of the plant protection laws has not questions by different Federal courts may ini- been done. Such a study would be timely, not Ch. 12—Patenting Living Organisms . 253 only because of the Chakrabarty decision, but maintaining organisms as trade secrets; patent- also because of allegations that the Acts have en- ing microbiological processes and their prod- couraged the planting of uniform varieties, loss ucts; and patenting the inanimate components of germplasm resources, and increased concen- of a genetically engineered micro-organism, tration in the plant breeding industry. In addi- such as plasmids, which are the crucial ele- tion, information about the Acts’ affect on in- ments of the technique anyway. The develop- novation and competition in the breeding in- ment would be slowed primarily because infor- dustry would be relevant to this aspect of the mation that might otherwise become public biotechnology industry. However, it may be ex- would be kept as trade secrets. A major conse- tremely difficult to isolate the effects of these quence would be that desirable products would laws from the effects of other factors. take longer to reach the market. Also, certain organisms or products that might be marginally D: Congress could prohibit patents on any living profitable yet beneficial to society, such as some organism or on organisms other than those vaccines, would be less likely to be developed. already subject to the plant protection Acts. In such cases, the recovery of development By prohibiting patents on any living orga- costs would be less likely without a patent to nisms, Congress would be accepting the assure exclusive marketing rights. arguments of those who consider ownership Alternatively, Congress could overrule the rights in living organisms to be immoral, or who Chakrabarty decision by amending the patent are concerned about other potentially adverse law to prohibit patents on organisms other than impacts of such patents. Some of the claimed the plants covered by the two statutes men- impacts are: 1) patents would stimulate the de- tioned in option C. This would demonstrate velopment of molecular genetic techniques, congressional intent that living organisms could which will eventually lead to human genetic en- be patented only by specific statute and alleviate gineering; 2) patents contribute to an atmos- concerns of those who fear the “slippery slope. ” phere of increasing interest in commercialization, which will discourage the open exchange E: Congress could pass a comprehensive law of information crucial to scientific research; and covering any or all organisms (except 3) plant patents and protection certificates have humans). encouraged the planting of uniform varieties, This option recognizes the fact that Congress loss of germplasm resources, and increasing can draw lines where it sees fit in this area. It concentration in the plant breeding industry. could specifically limit patenting to micro-orga- Also, by repealing the plant Acts, Congress nisms or encourage the breeding of agricul- would be reversing the policy determination it turally important animals by granting patent made in 1930 and in 1970 that ownership rights rights to breeders of new and distinct breeds. in novel varieties of plants would stimulate Any fears that such patents would eventually plant breeding and agricultural innovation. lead to patents on human beings would be un- A prohibitory statute would have to deal with founded, since the 13th amendment to the constitution, which abolished slavery, prohibits those organisms at the edge of life, such as viruses. Although there are uncertainties and ownership of human life. disagreements in classifying some entities as The statute would have to define included or living or nonliving, Congress could be arbitrary excluded species with precision. Although there in its inclusions and exclusions, so long as it are taxonomic uncertainties in classifying or- clearly dealt with all of the difficult cases. ganisms, Congress could arbitrarily include or exclude borderline cases. This statute by itself would slow but not stop the < development of molecular genetic tech- A statute that permitted patents on several niques and the biotechnology industry because types of organisms could be modeled after the there are several good alternatives for maintain- Plant Variety Protection Act—e.g., it should ing exclusive control of biological inventions: cover organisms that are novel, distinct, and 254 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals uniform in reproduction; such terms would semination of technical information; however, have to be defined, Infringement should include such a statute is not essential to the de- the unauthorized reproduction of the orga- velopment of the biotechnology industry, since nism-although reproduction for research incentives and alternative means for protection should be excluded to allow the development of already exist. The secondary impacts on society new varieties. In fact, consideration should be of the legislation are even harder to assess given to covering in one statute plants and all because of the scarcity of data from which to other organisms that Congress desires to be pat- draw conclusions. The policy judgments will entable. This would provide the advantage of have to be made by Congress after it weighs the comprehensiveness and uniform treatment; it opinions of the various interest groups. could also address the problems discussed Through legislation, Congress has the chance to under option B. balance competing views on this controversial issue and, if necessary, to alleviate the primary The impact of this law cannot be assessed concerns about the long-term impacts of the precisely. A comprehensive statute would stim- decision–that higher organisms will inevitably ulate the development of new organisms and be patented. their products and would encourage dis- Chapter 13 Genetics and Society Chapter 13

Page Genetics and Modern Science ...... 257 The “Public” and “Public Participation” ...... 261 Special Problems Posed by Genetics ...... 258 Issues and Options ...... , ...... 261 science and Society...... 259 Bibliography: Suggested Further Reading , . . . . .26.5 Chapter 13 Genetics and Society

Genetics and modern science

In 1979, the organization for Economic Coop- combinant DNA (rDNA), gene synthesis, eration and Development (OECD)* published a chimeras, fertilization of mammalian em- survey. of mechanisms for settling issues involv- bryos in vitro, and the successful introducing science and technology in its member coun- tion of foreign genes into mammals were 2 tries. I The OECD report noted that: the subjects of science fiction until a few years ago. Now they appear in newspapers Science and technology . . . have a number of distinguishing characteristics which cause spe- and popular magazines. Yet the genera] cial problems or complications. One is ubiquity: public’s understanding of these phenom- they are everywhere. They are at the forefront ena is limited. It is difficult for people to of social change. They not only serve as agents evaluate competing claims about the dan- of change, but provide the tools for analyzing gers and benefits of this new technology. social change. They pose, therefore, special 3. The scale, complexity, and interdependence challenges to any society seeking to shape its among the technologies are greater than own future and not just to react to change or to people suspect. As in other fields, applica- the sometimes undesired effects of change. tions of biological technology often depend After surveying member countries, OECD on parallel developments in areas that pro- identified six factors that distinguish issues in vide critical support systems. Breakdowns science and technology from other public con- in these systems are often as limiting as fail- troversies. ures in the new technology itself. In other parts of this report for example, sophis- 1. The rapidity of change in science and tech- ticated breeding systems in farm animals nology often leads to concern. The science and large-scale fermentation processes for of genetics is one of the most rapidly ex- single-cell cultures are described. Besides panding areas of human knowledge in the the biological technology required to sup- world todayv. And the technology of genetics port these systems, precise computerized is causing quick and fundamental changes operations are required to ensure purity, on a variety of fronts. The news media safety, and process control in fermentation have consistently reported developments and to provide the population statistics in genetics, often with front-page stories. necessary for breeding decisions. Consequently, the public has become in- 4. Some scientific and technological achieve- creasingly aware of developments in genet- ments may be irreversible in their effects. ics and genetic technologies and the speed Because living organisms reproduce, some with which knowledge in the field is gath- fear that it will be impossible to contain ered and applied. and control a genetically altered organism 2. Many issues in today's science and technol- that finds its way into the environment and ogy are entirely new. Protoplasm fusion, reproduces undesirable effects. Scenarios of escaping organisms, pandemics, and care- “’1’he members of’ OE(;D are: Auslra]ia, Austria, Belgium, less researchers are often drawn by critics (;ilni]da, l)enmark, Finland, France, West Germany, Greece, lce- of today’s genetics research. The intention- Iand, lr~liind, Italy, Japan, I,uxemhourg, the Netherlands, New Zeiiliind, Norway, Porttl~i\l, Spain, Sweden, Switzerland, Turkey, al release of recombinant organisms into [he (fnited Kingdom, iitld the LJnited States. the environment is a related issue that will 1( ;uild K. Nichols, ‘1’echnologv on Trial: Public Participation in De- need to be resolved in the future. cision-Makin~ Related to Science and Technology [Paris: organization for kkwnomic (kmperation and Development, 1979). Another example of irreversibility, ‘lt~id., p. 16. brought about by the demands placed on

257 258 ● Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

world resources, is the accelerating loss of sues. The increasing control over the inher- plant and animal species. Concern over this ited characteristics of living things causes depletion of the world’s germplasm arises concern in the minds of some as to how because genetic traits that might meet as widely that control should be exercised yet unknown needs are being lost. and who should be deciding about the 5. There exist strong public sensibilities about kinds of changes that are made. Further- real or imagined threats to human health. more, because genetics is basic to all living Mistrust of experts has been stimulated by organisms, technologies applicable to low- such events as the accident at the Three- er forms of life are theoretically applicable Mile Island nuclear plant and the burial of to higher forms as well, including human toxic chemical wastes in the Love Canal. beings. Some wish to discourage applica- Regardless of the real dangers involved, tions in lower animals because they fear the public’s perception of danger can be a that the use of the technologies will pro- significant factor in decisionmaking. At gress in increments, with more and more present, some perceive genetic technol- complex organisms being altered, until hu- ogies as dangerous. man beings themselves become the object 6. A challenge to deeply held social values is be- of genetic manipulation. ing raised by scientific and technological is-

Special problems posed by genetics

Genetics is just one among several disciplines Others find the idea of directing evolution ex- of the biological sciences in which major ad- citing. They view the development of genetics vances are being made. other areas, such as technologies in a positive light, and see op- neurobiology, behavior modification, and socio- portunities to improve humanity’s condition. biology, arouse similar concerns. They argue that the capability to change things is, in fact, a part of evolution. Genetics differs from the physical sciences and engineering because of its intimate associa- Religious arguments on both sides of this tion with people. The increasing control over challenge have been made. Pope John Paul 11 the characteristics of organisms and the poten- has decried genetic engineering as running tial for altering inheritance in a directed fashion counter to natural law. On the other hand, one is causing many to reevaluate themselves and Catholic philosopher has written:3 their role in the world. For some, this degree of ... We have always said, often without real control is a challenge, for others, a threat, and belief, that we were and are created by God in for still others, it causes a vague unease. Dif- His own image and likeness, “Let us make man ferent groups have different reasons for em- in our image, after our likeness” logically means bracing or fearing the new genetic technologies. that man is by nature a creator, like his Creator. Religious, political, and ethical reasons have Or at least a cocreator in a very real, awesome been advanced to support different viewpoints. manner. Not mere collaborator, nor administrator, nor caretaker. By divine command we The idea that research in genetics may lead are creators. Why, then, should we be shocked some day to the ability to direct human evolu- today to learn that we can now or soon will be tion has caused particularly strong reactions. able to create the man of the future? Why One reason is that such capability brings with it should we be horrified and denounce the sci- responsibility for retaining the genetic integrity of people and of the species as a whole, a re- 3ROheI’t ‘1’. t+all(!O(?U1’, “We [kin-We MUSI: Reflections on the sponsibility formerly entrusted to forces other ‘1’e(~tlll[)l(~~i~ill lnlpentive)” Theological Studies 33:3, September than man. 1972, p. 429 ;Illd ilt toot note 2. Ch. 13—Genetics and Society . 259

entist or physician for daring to “play God?” IS it tures, creatures oriented toward the future because we have forgotten the Semitic (biblical) (here and hereafter), a future in which we are conception of creation as God’s ongoing col- cocreators. laboration with man? Creation is our God-given Genetics thus poses social dilemmas that most role, and our task is the ongoing creation of the other technologies based in the physical sci- yet unfinished, still evolving nature of man. ences do not. Issues such as sex selection, the Man has played God in the past, creating a abortion of a genetically defective fetus, and in whole new artificial world for his comfort and vitro fertilization raise conflicts between in- enjoyment. Obviously we have not always dis- played the necessary wisdom and foresight in dividual rights and social responsibility, and that creation; so it seems to me a waste of time they challenge the religious or moral beliefs of and energy for scientists, ethicists, and laymen many. Furthermore, people sense that genetics alike to beat their breasts today, continually will pose even more difficult dilemmas in the fu- pleading the question of whether or not we ture. Although many cannot fully articulate the have the wisdom to play God with human na- basis for their concern, considerations such as ture and our future. It is obvious we do not, and those discussed in this section are cited. The never will, have all the foresight and prudence strong emotions aroused by genetics and by the we need for our task. But I am also convinced questions of how much and what kind of re- that a good deal of the wisdom we lack could search should be done are at least partly rooted have been in our hands if we had taken serious- in deeply held human values. ly our human vocation as transcendent crea-

Science and society

The public’s increasing concern about the ef- the distribution of benefits in society; it can fects of science and technology has led to de- have unequal impacts, and those who pay or mands for greater participation in decisions on who are most in need are not necessarily always scientific and technological issues, not only in those who benefit. the United States but throughout the world. A national opinion survey of a random sam- The demands imply new challenges to systems ple of 1,679 U.S. adults conducted for the Na- of representative government; in every West- tional Commission for the Protection of Human ern country, new mechanisms have been de- 4 Subjects of Biomedical and Behavioral Research vised for increasing citizen participation. An in- made clear that there is public doubt concern- creasingly informed population, skilled at exert- ing equity. Sixty percent of those polled felt that ing influence over policymakers, seems to be a new tests and treatments deriving from medical strong trend for the future. The media has research are not equally accessible to all Amer- played an important role in this development, icans. Seventy percent felt that those most likely reporting both on breakthroughs in science and to benefit from a new test or treatment of lim- technology and on accidents, pollution, and the ited availability were those who could pay for it side-effects of some technologies. or who knew an important doctor. This should one result has been the growing politiciza- be compared with the 85 percent who felt that a tion of science and technology. While perhaps new test or treatment should be available to misunderstanding the nature of science as a those who apply first or who are most in need. process, the public has become disenchanted by recent accidents associated with technology, by experts who openly disagree with one another, and by the selective use of information by some 4’4S~)[W’iill SIU(IJ’, IIllpli(’iitioll!i 01” ,ll(lt’illl(’t?$i iil f}ioll)fl(li(’ill illl[l B(’- l~il\ iol’ill Ros(’iil’(.ll, ’” R(’l)ol>t iill[l R(’(”otl]tll(’tl( liiliolls” 01” th(~ Niitiolliil scientific supporters to obtain a political objec- ( ‘(mltllissioll t“ol” Illf> l}lx)t(~(liotl ot” Illllllilll Slll)jo(’ls ot” lliolll(~(li(’ill tive. The public has seen that technology affects ilt~(l l\(*ilit\’iol. ii I K(’~f~iit.(’11, I III I“;\\’ I)(ll)li(’iil ion N’(). (OS) 78-()() 15. 260 ● Impacts of Applied Genetics—Micro-organisms, Plants, and Animals

Public concern and demand for involvement corporate research and development efforts. in the policy process is illustrated by the re- The public is also becoming more involved in sponse of communities to plans for laboratories corporate decisionmaking—e. g., through “’pub- that would conduct rDNA research. Perhaps the lic accountability” campaigns by stockholders to best known example is Cambridge, Mass., influence company policies. where plans were announced for construction With the politicization of science, the process of a moderate containment laboratory at Har- of research itself is coming under increasing vard University. Concern over this facility led to public scrutiny–most recently in cases of possi- the formation of the Cambridge Experimenta- ble biohazards, research with human subjects, tion Review Board (CERB). Composed of nine cit- and research on fetuses. Some efforts are un- izens—all laymen with respect to rDNA re- derway to require better treatment of research search—the CERB spent 6 months studying the animals as well. subject and listening to testimony from scientists with opposing points of view. Their final The relationship between science and society, recommendations did not differ substantially between human beings and their tools, is a con- from the NIH Guidelines; but the process was stantly evolving one. The process that has been crucial. CERB demonstrated that citizens could called the “dialogue within science and the dia- acquire enough knowledge about a highly tech- logue between the scientific community and the nical subject to develop realistic criteria and ap- general public"6 will continue to search for ply them. Similar responses to proposed labo- standards of responsibility. It is likely that as ratories have occurred in a number of other long as science remains as dependent on public American communities, including Ann Arbor, funds as it has over the past 40 years, it will be Mich., and Princeton, N.J.5 held accountable to public values. As has been noted: 7 These reactions, and similar phenomena surrounding controversies like nuclear power, in- The technologies of war, industrialization, dicate that the desire for citizen participation is medicine, environmental protection, etc., ap- strong and widespread. Recognizing this, each pear less as the demonstrations of superior Federal agency has its own rules and mech- claims of knowledge and more and more as the anisms for citizen input. Special ad hoc com- symbols of the ethical and political choices un- missions are sometimes formed to collect infor- derlying the distribution of the power of scientific knowledge among competing social val- mation from private citizens before decisions ues . . . . This cultural shift of emphasis from the are made on particular projects. Congressional role of science in the intellectual construction of hearings held around the country and in Wash- reality to the role of science in the ethical con- ington, D. C., are perhaps the best known of struction of society may indicate a profound these inquiries. While these mechanisms some- transformation in the parameters of the social times slow the decisionmaking process, they assessment of science and its relations to the po- help legitimize some decisions, and their role litical order. will probably expand in the future. 6Diitli(d (~iillilhiill, ‘{t:thicid Re$pwlsibili[y in Science in Ihe F’itce In corporate science and technology, public of Llllcel’lilill (~ollse[[llell{;es,’” Ethical and Scientfic Issues Posed by demands are being felt as well. Present regula- Human [Jses qf Mo/ecular Genetics, Mitts~ [.appe and Robert S. Morison” k!ds. ), Atltliils of” the New Y~rsk A(;iidellly of Sciences MS, tions for environmental protection and worker Jiii]. 23, 1976, ~). I(k and product safety have significantly altered ‘Yiit’oil fikf’iihi, “’1’he Politics (Jt’ the !$~~iill Assessment of !$cience” in ‘/’he Socia/ Assessment of Science, b;. Mendelson, D. Nelkin, P. ‘Richi~rd Hulton, Bio-l?evolution.’ DNA and Ihe Ethics of Man- Weit}gilimt (eds. ), (lmlerence Proceedings (Bielefeid, West (;er- Made L;fe [New York: New Atllel.it’iill I,ihrary (Me[~tolT), 1978). ll~iitl~: A&W opilz, 1978), p. 181. Ch. 13—Genetics and Society . 261

The “public’) and ‘(public participation”

These are terms with vastly different mean- Because publics differ with each issue, no def- ings to different people. Some take “the public” inition will be attempted here. It is assumed that to mean an organized public interest group; “the public” is demanding a greater role in de- others consider such groups the “professional” cisions about science and technology, and that it public and feel they have agendas that differ will continue to do so. The different publics that from those of the less organized “general” pub- coalesce around different issues vary widely in lic. As OECD stated:8 their basic interests, their skills, and their ultimate objectives. They are the groups that Public participation is a concept in search of a will be heard in the widening debate about definition. Because it means different things to different people, agreement on what constitutes scientific and technological issues, and are part “the public” and what delineates “participation” of what has been called the “social system of 9 is difficult to achieve. The public is not of course science. ” homogeneous; it is comprised of many hetero- The public has already become involved in geneous elements, interests, and preoccupa- tions. The emergence over the last several dec- the decisionmaking process involving genetic ades of new and sometime vocal special interest research. As the science develops, new issues in groups, each with its own set of competing which the public will demand involvement will claims and demands, attests to the inherent dif- arise. The question is therefore: What is the ficulty of achieving social and political consen- best way to involve the public in decision- sus on policy goals and programmed purporting making? to serve the common interest. “J. Al. Zinl~ln, fublic K/u)w/@e (( ~iitlll)l’idgt~: (~ilt)~l)l.i(l~(l [ ll]i\wr- ‘Nk>hols, op. (’it., p. 7. sil.v PIWS, 1 !)68).

Issues and Options

Three issues are considered. The first is an liberate effort should be made to increase pub- issue of process, concerning public involvement lic knowledge. The last can only be accom- in policymaking; the second is a technical issue; plished by educating the public and increasing and the third reflects the complexity of some its exposure both to the issues and to how peo- issues associated with genetics that may arise in ple may be affected by different decisions. the future. OPTIONS: ISSUE: How should the public be in- A. Congress could specify that the opinion of the volved in determining policy republic must be sought in formulating all major lated to new applications of ge- policies concerning new applications of genetics? netics, including decisions on funding of spe- The question as to whether the public should cific research projects. A “public participation be involved is no longer an issue. Groups de- statement” could be mandated for all such mand to be involved when people feel that their decisions. interests are threatened in ways that cannot be B. Congress could maintain the status quo, allow- resolved by representative democracy, ing the public to participate only when it decides to do so on its own initiative. The more relevant questions are whether current mechanisms are adequate to meet pub- If option A were followed, there would be no lic desires to participate and whether a de- cause for claiming that public involvement was

76-565 0 - 81 - 18 262 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals inadequate (as occurred after the first set of ways to accommodate them need to be iden- Guidelines for Recombinant DNA Research tified. were promulgated). However, option A can be implemented in two ways. In the first, the op- ISSUE: How can the level of public portunity for public involvement is always pro- knowledge concerning genetics vided, but need not be taken if there is no public and its potential be raised? interest in the topic. In the second, public in- If public involvement is expected, an involvement is required. A requirement for public formed public is clearly desirable. Increasing involvement would pose the problem that if the the treatment of the subject, both within and public does not wish to participate in a par- outside the traditional educational system, is the ticular decision, then opinion will sometimes be only way to accomplish this. sought from an uninterested (and therefore probably uninformed) public simply to meet the Within the traditional educational system, at requirement. Option A poses additional prob- least some educators feel that too little time is lems: What is a “major” policy? At what stage spent on genetics. Some, such as members of would public involvement be required—only the Biological Sciences Curriculum Study Pro- when technological development and applica- gram, are considering increasing the share of tion are imminent or at the stage of basic the curriculum devoted to genetics. Because research? Finally, it should be noted that under science and technology cause broad changes in option A, if the public’s contribution significant- society, not only is a clearer perception of ly influences policy, the trend away from deci- genetics in particular needed, but more under- sionmaking by elected representatives (rep- standing of science in general. For about half resentative democracy) and toward decision- the U.S. population, high school biology is their making by the people directly (“participatory” last science course. Educators must focus on democracy) may be accelerated. this course to increase public understanding of science. Because students generally find people Option B would be less cumbersome and more interesting than rats, and because human would permit the establishment of ad hoc mech- genetics is a very popular topic in the high anisms when necessary. On the other hand, by school biology course, educators responsible for the time some issues are raised, strong vested the Biological Sciences Curriculum Study Pro- interests would already be in place. The grow- gram are considering increasing time spent on ing role of single-issue advocates in U.S. politics, its study in hopes of increasing public knowl- and their skill in influencing citizens and policy- edge not only of genetics but of science in gen- makers, might abort certain scientific develop- eral. ments in the future. At the university level, more funds could be Regardless of which option is selected, it provided to develop courses on the relation- would be desirable to encourage different ships between science, technology, and society, forms of structuring public participation and to which could be designed both for students and evaluate the success of each method. Many dif- for the general public. ferent approaches to public participation have been tried in the United States and Western Several sources outside the traditional school Europe in attempts to resolve conflicts over system already work to increase public under- science and technology. Some have worked bet- standing of science and the relationships be- ter than others, but most have had rather tween science and society. Among them are: limited success.l0 Because public demands for ● Three programs developed by the National involvement are not likely to diminish, the best Science Foundation to improve public understanding of and involvement in sci- 1(J[ )i)l.[lll~v N[?l~ill iII)d h~ i[:hiie! Pollil[!k, “}’1’01)1(?111s iIINi 1%’(){:(?- dUI’(W in I}W 11(’~iiiiitioll 01 ‘l’t’(sllllolo~ i(sill” Risk ,“ in SOCield Risk As- ence: Science for the Citizen; Public Under- S~S.WTI~~I, Il. Schwing, illld W. ,III)(YIvJ ((+(1s.1 (N(?w Yot’k: Pl(!I~LII]] standing of Science; and Ethics and Values 1%’ss, 198(1). in Science and Technology. Ch. 13—Genetics and Society ● 263

● Science Centers and similar projects spe- tionships between science, technology, and cifically designed to present science infor- society. mation in an appealing fashion. ● New magazines that offer science informa- Public understanding of science in today’s tion to the lay reader-another indication world is essential, and there is concern about of increasing interest in science. the adequacy of the public’s knowledge. ● Television programs dealing with science and technology. Examples are the two PBS B. Programs could be established to monitor the series, NOVA and Cosmos, and the BBC level of public understanding of genetics and series, Connections. CBS has also begun a of science in general and to determine whether new series called The Universe. public concern with decisionmaking in science ● Television programs dealing with social and technology is increasing. issues and value conflicts. Particularly interesting is the concept behind The Baxters. Selecting this option would indicate that [n this half-hour prime time show, the net- there is need for additional information, and work provides the first half of the show, that Congress is interested in involving the pub- which is a dramatization of a family in con- lic in developing science policy. flict over a social or ethical issue. The second half of the show consists either of a dis- C. The copyright laws could be amended to per- cussion about what has been seen or of mit schools to videotape television programs comments from people who call in. for educational purposes. One interesting possibility would be to combine a series of Baxter-type episodes on genetic Under current copyright law, videotaping issues with audience reaction using the QUBE television programs as they are being broadcast system, a two-way cable television system in may infringe the rights of the program’s owner, Columbus, Ohio (now expanding to other cities). generally its producer. The legal status of such In this system, television viewers are provided tapes is presently the subject of litigation. As a with a simple device that enables them to matter of policy, the Public Broadcasting Serv- answer questions asked over the television. A ice negotiates, with the producers of the pro- computer tabulates the responses, which can grams that it broadcasts, a limited right for either be used by the studio or immediately schools to tape the program for educational transmitted back to the audience. QUBE permits uses. This permits a school to keep the tape for a its viewers to do comparison shopping in dis- given period of time, most often one week, after count stores, take college courses at home, and which it must be erased. otherwise, a school provide opinion to elected officials. It could be must rent or purchase a copy of the videotape effectively combined with a program like The from the owner. Baxters, to study social issues. If several such programs on genetics were shown to QUBE sub- In favor of this option, it should be noted that scribers, audience learning and interest could many of the programs are made at least in part be measured. with public funds. Removing the copyright constraint on schools would make these programs Any efforts to increase public understanding more available for another public good, educa- should, of course, be combined with carefully tion. On the other hand, this option could have designed evaluation studies so that the effec- significant economic consequences to the copy- tiveness of the program can be assessed. right owner, whose market is often limited to education] institutions. An ad hoc committee of OPTIONS: producers, educators, broadcasters, and talent A. Programs could be developed to increase unions is attempting to develop guidelines in public understanding of science and the rela - this area. 264 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

ISSUE: Should Congress begin prepar- B. The life of the President's Commission-for the ing now to resolve issues that Study of” Ethical Problems in Medicine and have not yet aroused much pub- Biomedical and Behavioral Research could be lic debate but that may in the extended for the purpose of addressing these future? issues. As scientific understanding of genetics and The n-member Commission was established the ability to manipulate inherited characteris- by Public Law 95-622 in November 1978 and tics develop, society may face some difficult terminates on December 31, 1982. Its purpose is questions that could involve tradeoffs between to consider ethical and legal issues associated individual freedom and societal need. This will with the protection of human subjects in re- be increasingly the case as genetic technologies search; the definition of death; and voluntary are applied to humans. Developments are oc- testing, counseling, information, and education curring rapidly. Recombinant DNA technology programs for genetic diseases as well as any was developed in the 1970’s. In the spring of other appropriate topics related to medicine 1980, the first application of gene replacement and to biomedical or behavioral research. therapy in mammals succeeded. Resistance to In July and September 1980, the Commission the toxic effect of methotrexate, a drug used in considered how to respond to a statement from cancer chemotherapy, was transferred to sen- the general secretaries of the National Council sitive mice by substituting the gene for resist- of Churches, the Synagogue Council of America, ance for the sensitive gene in tissue-cultured and the United States Catholic Conference that bone marrow cells obtained from the sensitive the Federal Government should consider ethical mice. Transplanted back into the sensitive mice, issues raised by genetic engineering. The re- the bone marrow cells now conferred resist- 11 quest was prompted by the Supreme Court deci- ance to the drug. In the fall of 1980, the first l2 sion allowing patents on “new life forms. ” The gene substitution in humans was attempted. general secretaries stated that “no government Although this study was restricted to non- agency or committee is currently exercising human applications, many people assume from adequate oversight or control, nor addressing the above and other examples that what can be the fundamental ethical questions (of genetic done with lower animals can be done with hu- engineering) in a major way, ” and asked that the mans, and will be. Therefore, some action might President “provide a way for representatives of be taken to better prepare society for decisions a broad spectrum of our society to consider on the application of genetic technologies to these matters and advise the government on its humans. necessary role, ”13 After testimony from various experts, the OPTIONS: Commission found that the Government is al- A. A commission could be established to identify ready exercising adequate oversight of the “bio- central issues, the probable time-frame for ap- hazards” associated with rDNA research and in- plication of various genetic technologies to dustrial production. The Commission decided to humans, and the probable effects on society, prepare a report identifying what are and are and to suggest courses of action. The commis- not realistic problems. It will concentrate on the sion might also consider the related area of ethical and social aspects of genetic technology how participatory democracy might be com- that are most relevant to medicine and bio- bined with representative democracy in deci- medical research. sionmaking. The Commission could be asked to study the areas it identifies and to broaden its coverage to

1‘J(?iill [,. hlil]s~, “(kilt? ‘l’lsitl]sf(?l’ (;h’en ii New Twist,” Science ~()~:~~, ,\pril 1980, p. 386. t~slillell)ellt I)v the gellel.il] s~t:l’~t~l.i~s, (1 .S. ‘C:alholic [k)ll~t?l’- ‘2(;i!]il l\iii’i Kolii[ii illl[{ Ni($holiis Wit(It?, “t{tlt]liiil (kIM ‘l’ls~iill]l~t~t (vM:[?, or”igi[ls, N( I l)()(!tltllelltiilm~ Service, vol. lo, N(.). 7, JLII.V Li, Stirs N(IW I)(II);I[(I, ” Science 2 10:24, octolwr 1980, p. 407. 1 !)/J(). Ch. 13—Genetics and Society . 265 additional areas. This would require that its issues in medicine and biomedical and be- term be extended and that additional funds be havioral research not associated with genetic appropriated. The Commission operated on $1.2 engineering that need review. Whether all million for 9 months of fiscal year 1980 and $1.5 these issues can be addressed by one Commis- million for fiscal year 1981. Given the complexi- sion should be considered. There are obvious ty of the issues involved, the adequacy of this advantages and disadvantages to two Commis- level of funding should be reviewed if additional sions, one for genetic engineering and one for tasks are undertaken. other issues associated with medicine and biomedical and behavioral research. Comments A potential disadvantage of using the existing from the existing Commission would assist in Commission to address societal issues associated reaching a decision on the most appropriate with genetic engineering is that a number of course of action. issues already exist and more are likely to appear in the years ahead. Yet there are also other

Bibliography: suggested further reading

Dobzhansky, Theodosium, Genetic Diversity and Hu- Holton, Gerald, and William A. Blanpeid (eds.), Sci- man Equality (New York: Basic Tools, 1973). ence and Its Public: The Changing Relationship A discussion of conflicts between the findings (Boston: D. Reidel, 1976). of science and democratic social goals. Detailed A collection of essays on the way science and coverage of the scientific basis for present de- the society of which it is a part interact, and how bates about intelligence and the misconceptions that interaction may be changing. often involved in genetic v. environmental determinants of certain human traits. Hutton, Richard, Bio-Revolution: DNA and the Ethics of Man-Made Life (New York: New American Li- Francoeur, Robert T., “We Can - We Must: Reflec- brary (Mentor), 1978). tions on the Technological lmperative, ” Theobgi- Reviews the history of the debate about recom- cal studies 33 (#3): 428-439, 1972, binant DNA, discusses the scientific basis for the Argues that man is a creator by virtue of his new technologies, and discusses the changing special position in nature, and that humans must relationship between science and society. Sug- participate in deciding the course of their evolu- gests how the controversies might be resolved. tion. Monod, Jacques, Chance and Necessity (New York: Goodfield, June, Plaving God: Genetic Engineering and the Manipulation of Life (New York: Harper Col- Alfred Knopf, 1971). ophon Books, 1977). A philosophical essay on biology. Two seem- Discusses the benefits, problems and potential ingly contradictor-v laws of science, the constan- of genetic engineering. Describes the moral cy of inheritance (“necessity”) and spontaneous dilemmas posed by the new technology. Suggests mutation (“chance”) are compared with more that the “social contract” between science and vitalistic and deontological views of the universe. society is being “renegotiated. An affirmation of scientific knowledge as the only “truth” available to man. Harmon, Willis, An Incomplete Guide to the Future (New York: Simon and Schuster, 1976). Nichols, K. Guild, Technology on Trial: Public Participa- Surveys how social attitudes and values have tion in Decision-Making Related to Science and changed throughout history and how they may Technology (Paris: Organization for Economic Co- be changing today. Argues that mankind is in the operation and Development, 1971). midst of a transition to new values that will affect Reviews mechanisms that have been used by our world view as profoundly as did the industri- countries in Europe and North America to settle al revolution in the 19th century. disputes involving science and technology. 266 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Sinsheimer, Robert, “Two Lectures on Recombinant Rationality,” Southern California Law Review 46 DNA Research,” in The Recombinant DNA Debate, (#~): 617-660, June 1973. D. Jackson and Stephen Stich (eds.) (Englewood An essay on the fundamental task facing man- Cliffs, N. J.: Prentice Hall, 1979), pp. 85-99. kind in, the late 20th century: the problem of Argues for proceeding slowly and thoughtfully choice of tools. New knowledge, especially from with genetic engineering, for it potentially has biology, will increasingly offer options for tech- far-reaching consequences. nology, the use of which will cause changes in human values. Tribe, Laurence, “Technology Assessment and the Fourth Discontinuity: The Limits of Instrumental Appendixes

I-A. A Case Study of Acetaminophen Production . . . , ...... 269 I-B. A Timetable for the Commercial Production of Compounds Using Genetically Engineered Micro-Organisms in Biotechnology ...... 275 I-C. Chemical and Biological Processes ...... 292 I-D. The Impact of Genetics on Ethanol—A Case Study ...... , ...... 293 II-A. A Case Study of Wheat. , ...... 304 II-B. Genetics and the Forest Products Industry Case Study...... , ...... 307 H-C. Animal Fertilization Technologies...... , ...... 309 III-A. History of the Recombinant DNA Debate ...... 315 HI-B. Constitutional Constraints on Regulation , ...... , ...... 320 III-C. Information on International Guidelines for Recombinant DNA . . . . . , .. 322 IV. Planning workshop Participants, Other Contractors and Contributors, and Acknowledgments...... , , .329 ——

Appendix I-A A Case Study of Acetaminophen Production

Summary process); and builciing l’e(l~lil’t~ll~t~l~ts (S]}iiCt? nee[is allocate~i accorfi i ng 10 process). ‘1’he ol]jeclil’e ot’ Ihis case slud~ is 10 denmnstriite the [?cononlic t’t?asihilit.v of applying a genetically CONCLUSIONS engineere{i strain to n]ake a chemical product not ● ‘1’he projected cost for illiiilllf~i(:tll]’iflg APAP b-v tlmv produced bv. t’t?i’lll[;ilt:itio[l. means of hatch t“(;l”ttl(;llt[itioll, using a genetically engineered strain, amounts to $ 1.OS/111. This cost is E3ACKGR0UNIl based on a plant pro(iucing 10 million 11] of APAP tl(;[?tiit]~it~()~)ll[~tl (~\P~]II) M’as chosen for the case iill Iltliliicv. Stll[ltV. /1S till ;llliil~f?Si(:, it lii(YliS Sollle of the side ef- ● A S a I}llle (It’ tht]l~ll), tht? ~1’OSS tl)iirgitl ~01’ lllal~l]fa~- fects of aspirin, and is the largest aspirin substitute t~ll’e Of ii (:hellli[:iil SIICI1 ;1S /11]111) ShOUld aJ)Pl’OX- on the market. Around 20 million pounds (lb) are imate 50 percent of SiiieS. ‘1’}1[? gross margin repre- manufactured annually. Mallinckrodt, Inc., produces sents the profit before general ami ilcifllitlistl’;iti~’e, 60 to 70 percent; the remainder is llliillllf’:l{: tlll’e(~ pri- marketing an~i selling, anci research an[i (develop- maril~~ b-v (;l](~ Itltt?l>lliltioll:il iitld Nlo]lsiinto” (1). A PAP ment expenses. ‘I”IIC ~1’OSS margin f“ol” iill of the is sold to l~(?i~ltl~ [;iil’[? (;ol~~l]iitlies, ~i’hich [~~iit’k(?t it to prd~]cts miide by hliillii~(:ki’()[it, th(? largest illii[l- ret a il(?rs. llfilCt U1’el’ of l~~~~p, iilllollfltd to ~~ iiild 37 p(?l’Cellt ‘1’he hfcNeil (k)IlstItl]er Products di~’ision of John- of Siil~S in 1977 itll(i 1978, resfmctil’el. v.z The gross son &, Johnson, itrhich ll]ill’kt~ts APAP Lllldel’ the lllill’~ill fol” NIOilSilIlto, ii lllLICh liil’~t?l’ Colllpiilly tt.ii(!e IIiIIIM?, ‘J~vl(?IIol, h{is th[~ l:it’~~st shaI’~ of the thiin hfiillinckrodt I)llt it sll~iill[?l’ il]iltll]fii~tlll’~1’ of market. (her a dozetl other con~panies in the United ,][JAI}, ill]l{)~lllt(?~i [() 27 illl[l 26 })(?l’C(~llt (If all SaJeS Stilt[?S S[?11 it Lllld(?l’ othel” tl’;id(? Iliill](?!j. in 1976 illl(i 1977, l’t?S~)eCt i\wlv. s It’ ttl(? @’OSS llliil’- one ch[?n] ical l~]ittlllf~i(;tllt’(?l”s bulk selling price for gin f[)r APAP is iis hi@ iis 50- p~?r[:ent of si~les, its ,lp,]p is ;,~-{~~~~~{i $~G~~/]l),l BV th(? tin]e the consumer Clll’1’ellt COSt Of lllilllllfiI(lt Llf’[? ShOUlti iilllOLlllt tO f)l]l’{:hiis[~s it ilt the [Itsug st[)i’e, the markup results in $1.325/11), based On ii bltlk selling price of $2.65/111. ii selling price ot” ill’(llill(i $25 to $50/ltl, (iepen[iing 011 Therefore, its projectmi cost ~then pr(xiuced l~y [iosagf? anci package sixes. ‘1’hus, the? total t’alue of t“ernlentiition is around 20 percent I(n%’er than its ,\Ilt~p to t}l(? ll];lllllf~l(:tlil.f?s k S0111[! $~o Il]i]]bn annu” estimateci cost Whetl pro(iuce(i b-v Cht:llliCiil s.Vn- illl~, while tilt? totill retail \’alU{? fallS in th(? 1’iill#? Of thesis. $500” million to $1 billion. ● If the selling price of APAP protiuce~i hy fel’llletltii- APPROACH ES tion is l~]iil.kd LIp 100 percent, the bulk selling price becomes $2.10/ib. This decrease of $0.55/lb ● A (:olls[;l’~’;lti~~[? approach }i’as taken, in that onley a could be transformed into cost savings of around (}otl\’[?Iltiotl;il” batch fermentation process was con- $5 to $10/lb to the consumer. These economies si(iermi. ● would result in an annual cost saving to the con- \’arial)les were selectwi i]ertaining to the choice of sumer of $100 million to $200 million. the microhial pathway; the nature of the feed- ● (hrrent processes for synthesizing A PAP from stock; con~rersion ei”ficiencies of feedstock to nitrobenzene do not ii~pei~r to pose significant ~1 PA P; anci the finiii yielci of APAP. iilthOU~h ii number Of side ● pollution problems, (;osts were basefi on proprietary processes involv- products are formed anti must he renwved.4 5 G ing startup, large-scale fermentation, and recovery Howe\~er, ii fermentiition process would be even of /1 PA P. ● (k)sts were itemized for materials and supplies; 1 Klilll i 11(’A 1’()(11, I IN’,, ,4/11111;1/ /{ f’/)(Wl, I !)7M. li~t)or tiistributi on; utilities (hroken down by specif- ‘Jll)ll%illllo ( ‘(). , ,,ltl/)t/;l/ Ii f’ixwl, 1 !)77. it{, (;, 1](,1,11(,111 11),.()(,(IS5 [~)1, ))1(,1~:11.illg ,\illilloj~llf)tlol,”” 1 ‘..S. P;ll(’111 ic energy l’e(ll]il’[~t~]t;tlts i~(;~ot’ciitl~ to process and :i,:itKi,4 16, I !)68. equipment ); equipment (groupe[i according to ‘l:. J\. I!ill’oil, 1{. (;. Ii(’11111’l’, illl(l \. l’,. \\’(’illl)(’l.g, “1’111’il’i(’ill iotl of /)-,\lllitlol)ll(ltlol,” “ ( 1,,$. p;~/(,/1( :],(;:)4 ,3(18, I !)72. “l”. \. I}ill’oll itll(l R. ( ; Ik’1111(’l’, ,11(11.if’i(.;l[iol~ (]1’ /).l\ll]illol)ll(,llol,”” ( ‘.s. }’;llrlll :},7 I 7![;so, I !)7:}.

269 270 “ Impacts otApplied Genetics —Micro-Organisms, Plants, and Animals

(~]eiiner. (111].v A PAP W(IU]d iiccL]n~uliit~: ill] ~thel’ t~ltertliitiv~l.v, ilniline could he ii~et.yliited directly llM?tiit)Olitt?S il[’(? lliitlll’illl.’ occurring. Even nlicro- forming i[cetiit~ilide, which in turn would be hydrox- ()]’~iitlit+nls COLIM be c[dl~(:t(d itft~r ~iich biitch iind ~liit~d to A PAP. ‘“ ‘‘ 12 A l~un]her of Streptomyces spe- I)IYXXSSCX1 into ii ciik~ tot” US~ iis ii high protein cies hiiv~ heen f(}u[ld to cotlvei’t iicetitnilide to illl illlill feed. APAP.13 “1’he ~iithwii~ hnrolvhlg p-iiminophenol was chosen simply be(:iiuse the conversion efficiency of Biological parameters it~et ic ii~id to A PAP would l)e slightly higher if acetic iicid entered the ~\’erilll l’~il~t ion i]t the second step MICROBIAL PATHWAY [’iith~[. thii[l iit the first step. A proposed ~iithwii~ for convertin~ aniline to HOST MICRO-ORGANISMS A PAP il~et~liiti(}n of iin viii the intermediilte, p-amino- The most suiti~hle ltlicl>()-()l.gi~tlisn~ for production phend, is shown in figure l-A-l. !’iiI’iOLIS fungi have of APAP in ]iir~e-s(~iil~ fermentiition may not neces- ken identified in which these reiictions occur.7 s 9 Sill’iluV he one thilt 1101’ llliill~ nletilbolizes aniline 01’

7 R. \’. Smith atld J. P. Rosii~~a, “hlk]sobiiil Models of Miill~nli{liiitl ~)-itl~~il~~~llet~ol. While ii hycterium might serve as ii hfetiibolisnl, ’” J. Pharmarxw. Sci. 64: 1737 -17s9, 197s. Sllitiit)k host for insertion iitld expression of the recl- ‘R. t’. Smith iilld J. P. liosit~~a, “M icrol)ial Models of Mammalian uisite genes, it yeiist il~ii~ represent ii hetter choice. [t hl(?tiil)olisn): Aron)iilk: H.vtit.l]xvlatiotl, ” Arch. Biochern. Biol]hvs. . . will Prot]iit)l.v more closeltv resemble the organism 16 1:551-.5.58, 1974. ‘\’. R. klunzner, It. hlutschlwl iind h!. Rummel, “Llher die n)ikro- [IWIN which the genes are isoliited. I)iologische unwandlung N-hitltiger substrate” K:oncerrling the Microbiological “1’1’utlslol’nlatiotl of N- containing Substrate), Plant Fermentation efficiencies Medica 1 s:97- i03, I Y67. CONVERSION EFFICIENCIES Figure l-A-l. Bioconversion of Aniline to APAPa The n]olilr iil]d weight conversion efficiencies for the hioconversion of feedstock to product are projected in tithle 1-A-1. ‘l’he bioconversion of aniline to

NHC10CH3 Iuslllit h, et al., op. d. I 1 hfun~n~r, [?t ill., op. rit . “R. J. ‘1’heriiiult iil~d ‘l’. }1. l,ongtieki, “hlicrohial Conversion of Acetiinilide to 2’-llydl’oxyii(:etal~i]ide and 4’- Hydl.oxya(:etiil] ili[ie, ” Ap/. ,\ficrobio/. 15: 1431-1436, 1967. 131hid.

aceticacid?f I. NH2 Table I=A-l.—Fermentation Efficiencies to Meet the I / Requirements for the Production of Acetaminophen (APAP) From Aniline

Overali molar conversion efficiency of: (a) Aniline to APAP...... 90.25Y0 (b) Acetic Acid to APAP...... 95.0 Overali weight conversion efficiency of: (a) Aniline to APAPa ...... 146.5 NH Ho 2 p-aminophenoi (b) Acetic Acid to APAPa ...... 239.1 I / Utilization of: (a) Aniline in fermentation broth ...... 2.28 lb/gal (b) Acetic acid in fermentation broth . . . 1 .39)gal Production of APAP in broth ...... 3.34 lb/gal Batch volume ...... 33,500 gal Recovery efficiency...... 90.0 % Yield of APAPlbatch ...... 100,701 lb Aniline Number of batches/year ...... 100 Annuat yield of product...... 10,070,100 lb

%erait weight conversion of precursor to APAP = = aApAp = Npacetyl.p.amlnophenol = acetaminophen = p.acetamidophenoi P- molecuiar weight of APAP x molar conversion efficiency hydroxyacetanilide =Tylenol (trade name of McNeii Laboratories). molecular weight of precursor of precursor to APAP SOURCE: Genex Corp. SOURCE: Genex Corp. Appendix l-A—A Case Study of Acetaminophen Production ● 271

~\f}/\ P inlwh’cs two steps. ‘1’he product of the inciivid- Economics II;II I’t?il(;t ions f’ol” [?it(Yll St(?j) l’(?pl’E!St?lltS the ()\’(?I’iill con\wrsion (? fficit?ncv.. tl Illolill” conversion efficiency PROD1JCTION RE[~lJIREMENTS of !)5 l)eITCIII \t’iis iisslltll[![l tot” ~ii(;h step. This i~iill]~ liow the tiiriolts production t’[?c~llil’[?tlletlts would is l)ilS(?({ 011 ii nlultitud[? of l’t?pOl’t S Clelll(lllStl’ilt ill~ I)t? met during th(? Inicl’ohiill tl’illlSfol’lllilti oil” of” illli- Sillliliil’ Illolill ” colli’crsion efficiencies tot” alliilO~OLIS Iine to tlPAP is SLlllllllill’iZ[?d ill tiihlt?s I-A-1 iin(~ 2. Ani- I)io(;ht?llli(:ill l’(?it(:tiollS lllld[?l’ ii(~tllill f“(?l’lll~ll tiltioll Iin(? ittld ii(:(?ti{: ilci[i WOUl(i not i)[? it(i(id to tilt? f[?l’- conditions. I 4 Illt?lltiltioll 1)1’ottl iiii ill ollC(? t)llt l’iitil~l- step-wis[? il(Y- PRODUCT YIELD (x)r(i ing to til(?ir i’iit~s of” coniwrsion. ‘1’he pi;~nt ivoui(i (X)lltilill tlt’() 50, ()()( )-~ili f(?l’lll(?ll t[?l’S, i%’hictl in th[? ‘1’he yield of /\P/lP projected in tiihl~ l-A-l is I]iisd (:0111’S[? of il .Vf?iil’ W’olli(i .vi[?i{i 1() n~iliion lb Of t\ PAP. 011 t;st iil]iitill~ ii rittio of J() pCIYWIIt weight to VOIUtI]~ (i.e., 4(1 II) pt?t’ 10(1 gilllollS (g:Il) Of teI’lllelltiitiO[l bl’Oth) PRODUCTION COSTS I)rior to 90 pcr(x?tll r(?(xn(?r~ . Such ii high yield is per- ‘1’ilt? COStS fO1’ ttl[? illllllliil ~1’OCiLICtiOll iI1’~ SLlllllllii- n]illcd })[?(;iills[? ()[” th(! poor” soluhi]itv of APAP under riz(?ci in till)i~ I-A-3. ‘1’he.v iit’[? I)rok[:t) dO\VIl into their ions. /\s ()~)(?t’iit ing condi[ ii IWSLI]I, h@ IC\r~]S of APAP Illiij(ll’ COllll)oll[+lltS iill{i iil’e (? Xpl’[?SSf3[i i)otil ii!$ iillllllill \\’olll[l llil\’[! 110 il[l\’(?l’S(? t?f’t’(?(;t 011 tht? hOSt llliCl’O-Ol’gil- COStS iilld ii!+ unit COStS. Ik?tiiil[?(i tlUCi#?tS t[)l’ tht? t’ill’i- nism. [ lse of insoilihlc s.vstcms in f“f~t’ill[?lltati(~ll hilS in 011S COSt (X?llteI’S ill’f? ShOWll in tillll~S 1-A-4 till’ oll~il fact been reported in recent years—e.g., in certain l-A-lo. Miiteriiils iiild suppiies iir~ describe(i in tiil)l(? microbial transformations of steroids, yields of 40 l-A-4; iilhol” [iistrihut ion in tiil)]~ l-A-5; utility requir (?- percent may result due to the insolubility of the Il?ellts in till)l[?S l-tl-~ tilrOu~h I-t\-/l; equipnlent ill product. till)le l-/~-9; i~lld sj)ilc~ Isf;(ll]ii>[?ll][+flts in tiil)l~ I-A-IO. ‘1’ilis iiiliil~sis l’[?~~~iils ii unit (;ost Of APAP [?({iiiii to III{ J il)l)O II, ‘I I I II II 11)1)11 Izlt(l((lll s,’” ill ,\/i/l{/;~//{fJ/~(jrl,s (m/.’rrlll(vl- /;i/i(~/t /)l’(l(’(’, 1!)77), ])]) zo.T-2:l:\

Table l-A-2.—Summary of Production Conditions Table 1. A.4.—Materials and Supplies for of APAP Production of APAP

Number of fermenters ...... 2 Materials Cost/batch Cost/year Size of fermenters...... 50,000 gal Fermentation Operating volume ...... 33,500 gal Fishmeal (l.5% @ $0.155) ...... $ 648.68 $ 64,868 7a Cycle...... Glucose (1.5% @ $0.1535)...... 642.40 64,240 Batches ...... 100 Lard oil (2.5% @ $0.325) ...... 2,266.88 226,888 Mineral salts (4,215 lb @ $0.05074) %-day fermentation, l-day turn around. 213.77 21,377 Aniline (76,250 lb@ $0.42)...... 32,027.52 3,202,752 SOURGE: Genex Corp. Acetic acid (46,680 lb@ $0.245) . . 11,436.60 1,143,660 Miscellaneous (1OYO of basic materials)...... 377.17 37,717 Subtotal...... $47,613.02 $4,761,302 Recovery Table l-A.3.—Summary of Costs of Filter aid(0.21b/gal @ $13)...... $ 871.00 $ 87,1OO Other chemicals and supplies. . . . 1,600.00 $ 160,000 Production of APAP Subtotal ...... $2,471.00 $ 247,100 Annual cost Costllb Finishing Packaging (1,255 bag units Materials and supplies ...... $ 6,133,802 $0.6091 at$0.80) ...... $ 1,004.00 $ 100,400 Labor...... 2,012,140 0.1998 Utilities...... 630,200 0.0626 Other (labels, stencils, etc.)...... 1,004.00 $ 100,400 Equipment ...... 1,377,590 0.1368 Subtotal ...... $2,008.00 $ 200,800 Building ...... 439,399 0.0436 General supplies Total ...... $10,593,131 $1.0511b Maintenance (4°Aof capital investment) ...... $ 425,900 Other (laboratory office, plant miscellaneous) . 498,700 Annual production = 10,070,100 lb Total ...... $6,133,802

SOURCE: Genex Corp SOURCE: Genex Corp. 272 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Table l-A-5.-Labor Distribution for Production of APAP

Salary and wage cost Category Man-hours per week Hourly rate $/week $/year Supervision General manager ...... 40 20 $ 800 $ 41,600 Superintendents...... 80 17 1,360 70,720 Managers ...... 80 15 1,200 62,400 Supervisors...... 320 12 3,840 199,680 Hourly rated employees, services Laboratory Level I ...... 80 10 800 41,600 Level II...... 80 8 640 33,280 Level Ill ...... 120 6 720 37.440 Level lV ...... 40 5 200 10,400 Maintenance and engineering Level l ...... 240 10 2,400 124,800 Level lI...... 240 8 1,920 99,840 Level lIl ...... 240 6 1,440 74,880 Level IV ...... 160 5 800 41,600 Hourly rated employees, production Fermentation department Level l ...... 200 10 2,000 104,000 Level II ...... 240 8 1,920 99,840 Level Ill ...... 80 6 480 24,960 Level lV ...... 80 5 400 20,800 Recovery department Level l ...... 320 10 3,200 166,400 Level II ...... 400 8 3,200 166,400 Level Ill ...... 80 6 480 24,960 Level IV ...... 120 5 600 31,200 Subtotal...... , ...... ,...... $1,476,800 Add overtime@ 6% x 1.5 ...... 132,912 Subtotal ...... $1,609,712 Add fringe benefits @25%...... 402,428 Total salaries and wages...... $2,012,140

SOURCE:GenexCorp.

Table l-A-6.-Steam Requirements for Production of APAP

Operation Lb/batch Sterilization, fermenters, and seed tanks: Heating ...... 52,100 Holding ...... 20,000 Sterilization, piping, and equipment (other) . . . 20,000 Heating acetaminophen solution (recovery) ... 163,500 Drying, turbo dryer...... 200,300 General purpose usage ...... 50,000 Total...... 505,900 Cost at $5.00/Mlb: Per fermenter batch =$ 2,530 Per year (100 batches) =$253,000

SOURCE:GenexCorp. Appendix l-A—A Case Study of Acetaminophen Production . 273

Table l-A-7.–Electricity Requirements for Production of APAP

Units/batch Connected load HP kW (hours operation) kWh Fermenters...... 200 149 144 21,456 Seed tanks ...... 47.5 35 24 840 Chillers ...... 580 433 4,763 Air compressor ...... 275 205 86 17,630 Harvest tank...... 100 11 825 Decanter centrifuge...... 120 90 52 4,680 Process tanks...... 300 224 19 4,256 Crystallizing tanks ...... 300 224 2,464 Turbo dryer ...... 30 22 23 Cooling tower...... 40 30 144 4,320 Pumps (est. =6@ 7.5) ...... 45 34 144 4,896 Lighting, instruments and general load...... 25 19 144 2,736 Total kWh ...... 69,372 @O.05/kWh = $3,469 per batch @100 batches/yr= $346,900 per year

SOURCE:GenexCorp.

Table I-A.8.-WaterRequirements for Production of APAP

Gal/batch Fermentation ...... 35,000 Tower makeup ...... 63,000 Process loss ...... 100,000 Chilled water makeup...... 30,000 Direct cooling ...... 50,000 General use...... 25,000 Total ...... 303,000 gal Process Watergate =$1.00/M gals cost = $303/batch 100 batches/yr =$30,300/year

SOURCE:GenexCorp. 274 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

Table l-A-9.—Equipment Costs for Production of APAP

Receiving and batching area 120,000 gal stainless steel side-entering surge 320,000 gal steel aniline storage tanks, tank with agitator ...... $ 56,100 insulated and cooled - @ $47,000 ...... $ 341,000 350,000 gal stainless steel crystallizing tanks, 220,000 gal aluminum acetic acid storage insulated with heavy duty cooling coils and tanks, insulated and cooled - @ $71,300 ...... 342,600 top-mounted agitator - @ $195,000 ...... 585,000 110,000 gal steel nitrogen storage tank with 1 Stainless steel turbo tray dryer...... 653,000 controls and instruments...... 47,000 23,500 ft3 stainless steel hopper bins- 110,000 gal steel lard oil storage tank, @ $66,000 ...... 132,000 insulated and heated ...... 22,300 1 Bagging unit...... 20,000 110,000 gal stainless steel Batch tank with 4 Stainless steel finished product conveyors- programmable controller and agitator...... 59,500 @ $12,000 ...... 48,000 3 21,700 ft stainless steel Hopper bins with Auxiliary equipment conveyors - @ $58,100 ...... 116,200 31,500 c.f.m. reciprocating air compressors - 1 Electric forklift truck ...... 11,400 @ $168,000 ...... 498,000 Fermentation and seed area Laboratory and office equipment , ...... 650,000 1150 gal stainless steel seed vessel, fully Chillers, 500 ton total capacity ...... 575,000 instrumented...... 125,000 1 Cooling tower, 1,500 g.p.m...... 210,000 12,500 gal stainless steel seed vessel, fully 35 Pumps and motors, various sizes...... 104,700 instrumented...... 169,000 2 Dump trucks -$12,000 ...... 24,000 250,000 gal stainless steel fermenters, fully Ventilation, general and spot - @ 7.5% instrumented with central control room - of equipment...... 583,791 @ $399,000 ...... 798,000 Piping, general, materials and installation - Recovery area @ 7.5% of equipment...... 583,790 150,000 gal stainless steel process tank, Miscellaneous equipment (hand tools, etc.) - cooled, agitated and insulated ...... 195,000 @ 5°A of equipment ...... 389,194 13,000 gal steel filter aid slurry tank Total ...... $7,783,875 with agitator ...... 11,300 Annual charge for capital recovery over 10-year 1 Stainless steel continuous decanter period, with 12% interest compounded centrifuge ...... 167,000 annually ($7,783,875 x 0.17698) ...... $1,377,590 2100,000 gal stainless steel process tanks, insulated with external steam injection heater, pump and agitator- @ $333,000 ...... 666,000

SOURCE: Genex Corn.

Table I-A-10.–Buiiding Requirements for Production of APAP

Area Gross space ft2/ft3 Unit valuea cost Central office ...... 940 41.00 b $ 38,540 Laboratories...... 4,500 70.00b 315,000 Warehouse ...... 2,000/36,000 27.00b 54,000 Batching ...... 1,000/30,000 1.75 52,500 Fermentation (including seed) ...... 6,000/320,000 1.75 560,000 Harvest, filter ...... 3,500/169,000 1.75 295,750 Processing, crystallization ...... 8,700/470,000 1.75 822,500 Drying, finishing...... 5,000/270,000 1.75 472,500 Warehouse, finished product...... 11,000/200,000 27.00b 297,000 Auxiliary equipment...... 4,300/154,000 1.75 269,500 Maintenance, engineering...... 11,500/207,000 1.75 362,250 Total...... $3,539,540 Amortization over 30 years@ 12% compound interest $439,399C

aunit values in cubic feet except where noted by “b.” bunit value in square feet. cAmortizatlon = 0.12414 X tOtal. SOURCE: Genex Corp. Appendix I-B A Timetable for the Commercial Production of Compounds Using Genetically Engineered Micro- Organisms in Biotechnology

Objectives quired (of the products and manufacturing processes, not of the rDNA technology per se). ● The estimation of the proportions of various groups of commercial products and processes for which recombinant DNA (rDNA) technology could Sources of information be applicable. While much of the information compiled for this ● The construction of timetables to indicate plausi- report was obtained from published sources, a con- ble sequenccs of commercial developments that siderable amount came from prior proprietary stud- would result from the application of rDNA tech- ies performed by Genex Corp. In the latter case, in- nology. formation is used that is not proprietary, although the sources must remain confidential. In this connec- Approaches tion Genex has had numerous discussions with the technical and corporate management of more than The following five industries were evaluated: 100 large companies (generally multibillion dollar 1. pharmaceutical, companies), concerning research interests, product 2. agricultural, lines, and market trends. Production costs are ex- 3. food, trapolated for four fermentation plants of various 4. chemical, and sizes and capabilities. (See table I-B-1.) 5. energy . A group of Genex scientists, consisting of a bio- The manufacturing processes that would result chemical engineer, two organic chemists, a biochem- from the application of rDNA technology would be ist, and four molecular geneticists rated the feasibili- based on fermentation technology, Therefore, a set ty of devising micro-organisms to produce various of parameters was developed to serve as a guide to chemicals in accordance with the fermentation con- assess the economics of applying fermentation tech- ditions specified in table I-B-1. For those chemicls nology to the manufacture of products currently that appeared to be capable of being produced mi- manufactured by other means. crobiologically, dates were assigned for the times The chemical industry generates a large number when the necessary technology would be achieved in of products that could be attributed to (and is in this the laboratory. By combining both technical and eco- study) the other four industries cited, this particular nomic factors, it then became possible to project a industry was focused on more closely than the timetable for commercial production. (See table others. The following factors were considered in I-B-2.) constructing the timetables showing the applicability It should be emphasized that an extremely con- of rDNA technoloGY: servative approach was taken in considering fermen- ● the current state of the art of genetic engineer- tation economics over the next 10 years. only the rel- ing; atively poor economics of conventional batch fer- ● the current economic limitations of fermentamentation- was considered. immobilized cell proc- t ion technology; esses were projected to be 15 years away, and even ● the length of time to progress from a laboratory then, the incremental cost savings projected (see process to the pilot plant to large-scale produc- table I-B-1) are lower than the incremental cost sav- tion; ings currently obtained with immobilized cell proc- ● the plant construction time; and esses. The assumptions made here, however, did in- ● the Government regulatory agency approval re- clude reasonably high product yields and highly effi-

275 276 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

Table I-B= I.-Unit Cost Assumptions for the Production of Chemicals by Fermentation After Various Intervals of Time

Annual ca Unit cost excluding excluding Complete Earliest date Size of plant Type of Product yield precursor precursor unit cost (year) (lb) fermentation (%) ($ millions) ($/lb) Precursor (W/lb) 5 50 Ordinary batch 12 23.5 0.47 Petrochemical 0.66 10 Ordinary batch 40 24.5 0.25 Petrochemical 0.44 200 Immobilized cells 40 25.5 0.13 Petrochemical 0.32 d 20 200 Immobilized cells 40 25.5 0.13 Carbohydrate 0.24 d aAnnual ~.~t~ fOr ~~di”ary batch fermentation Were estimated from prOpr\@ary data. values obtained for ttre immobilized cell ertarnplea are COrnPutSd at 31.2 PerCent below the comparable valuea for ordinary batch fermentation. bAveraQe cost of petrochemical e~ual~ $0.17/lb. At ~ percent conversion efficiency, cost contribution of petrochemical eqUa[a $0.lQ/lb Of prOdUCt. CAverWe coat of carbohydrate assumed at $o.04/lb of molaaees or $o.02/lb of cellulose-containing pellets from biomass reaidue. For ~ perCent free SUgar COntent Of molasses, cost of sugar equals SO.06/lb. At 70 percent conversion efficiency from the sugar, cost contribution of molasses equals S0.1 I/lb of product. For 50 percent cellulose content in the biomass pellets, coat of cellulose equala $0.04/lb. For 50 percent conversion efficiency to free sugar, followed by 70 percent conversion efficiency from the sugar, coat contribution of the pelleta also equals $0.1 lllb of product. ‘%hese unit costs maybe further reduced to S0.26 and $0.17/lb., respectively, for products whose annual U.S. production currently exceeds 1 billion lb. Asaumptlons include reduction in precursor cost by 20 percent (presumably because manufacturer controls SUPPlY of precursor); reduction in unit cost of immobilized cell process by 13 percent (d) and 42 percent (e), respectively; maximum of 60 percent product yield (e); and a nearly 100 percent bioconversion efficiency from the petrochemical precursor. SOURCE: Genex Corp.

Table l- B-2.-Basis for Estimating the Timetable for microbial origin, which could be effective or superi- Manufacture of Chemicals by Means of Microbial or substitutes for chemically synthesized products Processes that could not he manufactured microbiologically. As examples, dyes of microbial origin, such as pro- And if bulk Assuming If all the selling pricesc unit costsd(in digiosin, might advantageously replace those synthe- Earliest date technologyb (in 1979 1979 dollars) sized chemically, because their toxicity is lower than for commercial is achieved dollars) equal equal or their chemical counterparts. In the case of plastics, a production is: by: or exceed: exceed: new generation of plastics of microbial origin, e.g., 5 years 2 years $1.32/lb $0.66/lb pullulans, would not have to be made from petro- 10 7 0.88 0.44 chemical feedstocks and would be biodegradable. 12 0.64 (0.43) 0.32(0.26) 20 17 0.48 (0.28) 0.24 (0.17) Explanation of tables qt IS ~SUrnad that development of the appropriate manufacturing facilltlm begina at leaat 5 yeare prior to the onset of production. %echnology refers to both genetic and biochemical engineering. Technology Tables l-B-3 through l-B-32 present the compounds would be achieved on demonstrating that the chemical could be biologically produced In the laboratory at commercially desirable yields and reaction effi- from two points of view. Tables l-B-3 to l-B-lo group clenciea. the compounds by industry subgrouped by product Clt \S assurnad that all bulk selllng prices are rnSrkSd up 100 percent from the corresponding unit costs, except for chemicals whose annual U.S. production category. Tables l-B-l 1 to I-B-32 group the corn- currently exceeds 1 billion lb. In those ca$es the bulk seillng prices (numbers pounds by product category irrespective of industry. in parentheses) are assumed to be marked up only 67 pwcent. dunit co8t8 were obtained from table l-B-l. See footnote of table I-B-1 for ex- The tables based on industry present end use data planation of numbera in parentheses. for each compound; e.g., in the pharmaceutical in- SOURCE: Genex Corp. dustry aspirin is listed as an aromatic used as an cient transformations of precursor to product, but analgesic, whereas in the chemical industry aniline is nothing exceptional with respect to current fermen- listed as an aromatic used as a cyclic intermediate. tation technology. Indeed, high product yields and Thus, the similarities and differences between com- highly efficient reactions would be expected with pounds of similar origin, i.e., product category, are ● genetically engineered micro-organisms. revealed. Two points should be stressed that place these The tables based solely on product category are projections on the low side. First, they exclude cer- divided into two types; one type pertaining to market tain groups of products, the end products of which data (tables 1-B-lo, 11, and the subsequent odd num- could not be microbially processed, although their bered ones through table l-B-33), and the other per- basic constituents could be produced microbiologi- taining to technical data (the even numbered tables cally (e.g., monomers of microbial origin could form from I-B-12 through l-B-32.) chemically synthesized polymers). Second, the pro- The market data were obtained both from pub- jections exclude naturally occurring products of lished sources and from prior proprietary studies Appendix l-B—A Timetable for the Commercial Production of Compounds ● 277

Table l-B-3.—Pharmaceuticals: Small Molecules Table l=B=4.—Pharmaceuticais: LargeMoiecuies

Product category End use Product category End use Amino acids Peptide hormones Phenylalanine...... Intravenous solutions Insulin...... Control of diabetes Tryptophan...... Intravenous solutions Endorphins...... Analgesics, narcotics, Arginine ...... Therapeutic: liver disease prophylactics and hyperammonemia Enkephalins ...... Analgesics, narcotics, Cysteine ...... Therapeutic: bronchitis and prophylactics nasal catarrh ACTH a ...... Diagnostic: adrenal instability Vitamins Glucagon ...... Therapeutic: diabetes-induced Vitamin E...... Intravenous solutions, hypoglycemia prophylactic Vasopressin ...... Therapeutic: antidiuretic Human growth hormone. . . Therapeutic: dwarfism Vitamin B12 ...... Intravenous solutions Aromatics Enzymes Aspirin...... Analgesic Glucose oxidase ...... Diagnostic: measurement of p-acetaminophenol ...... Analgesic blood sugar Urokinase ...... Therapeutic: antithrombotic Steroid hormones Asparaginase...... , . Therapeutic: antineoplastic Corticoids Tyrosine hydroxylase . . . . . Therapeutic: Parkinson’s Cortisone ...... Therapeutic: disease anti-inflammatory agent Prednisone...... Therapeutic: Viral antigens ant i-inflammatory agent Hepatitis viruses ...... Vaccine Prednisolone ...... Therapeutic: Influenza viruses ...... Vaccine anti-inflammatory agent Herpes viruses...... Vaccine Aldosterone ...... Therapeutic: control of Varicella virus...... Vaccine electrolyte imbalance Rubella virus...... Vaccine Androgens Reoviruses ...... Vaccine: common cold Testosterone ...... Therapeutic: infertility, Epstein-Barr virus ...... Vaccine: infectious hypogonadism, and mononucleosis, hypopituitarism nasopharyngeal Estrogens carcinoma, Burkitt Estradiol ...... Prophylactic, therapeutic: Iymphoma vaginitis Miscellaneous proteins Antibiotics Interferon ...... Control of infectious diseases Penicillins...... Control of infectious diseases Human serum albumin . . . . Therapeutic: shock and burns Tetracycline ...... Control of infectious diseases Monoclinal antibodies . . . . Diagnostics: hepatitis, cancer, Cephalosporins ...... Control of infectious diseases etc; therapeutics Short peptides Gene preparations Glycirie-Histidine-Lysine. . . Manufacturing processes: Sickle-cell anemia ...... Control of hereditary disorder tissue culture Hemophilias, ...... Control of hereditary disorder Thallasemias ...... Control of hereditary disorder SOURCE: Genex Corp. %drenocofiicotroplc hormone. SOURCE: Genex Corp.

perforlm?d t)y (k?llex. In the hitter case, data are L]st?d termediat[?s with production volumes (which differ that are not ~~roprietat”y ahhough the sources must from market volumes) exceeding 50 million lb were renuiin [;olli”i(l[?t~tiiil. Market values were estimated selected, hut in the case of flavor iimi pertunw n]ate- })ov n~ultiplying the market ~olume (total amount of rials, compounds with production values generally product sold in 1978) by the bulk cost (unit bulk sell- exceeding 1 million lb were selected. In the case of ing price in 1 980). Except for aromatics anci ali- many pharmaceuticals, clinical importance was ~)hatics, iill nmrket data represent worldwide esti- weighed heavily in their selection process. mates. hlarket data for aromatics and aliphatics are The technical data were also obtained both from ix?st rict[?d to the 1 Inited States. Data that could not be published and proprietary sources. \Vith respect to found ~verc marked not available (N/A). Compouncls the timetable for commercial production, the stated \\’it h a high n]arket l’alue were identified, and those Iengt h oft inw is the t i me required to de~elop exist ing [ha! could be pro(iuced biologically were selecte(i for technolo~v (including both genetic and biochemicii] this report. engineering) to the point w’here it ma-v he applied to High market \alues \vcre relati~e to the incjustry ilppropriate man u fact uri ng faci Ii t ies f-or the klr~re- 1111(1 end 11s(? list i tlg of each compound. For example, scale pro(iuct ion of the desired compounds. These \t’il h rcslxx’t to chemicals, normall.v only cyclic irl - time intervilis shoLl]d he sufficient for undertaking

76-565 0 - 81 - 19 278 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

Table l- B-5.-Food Products Table l- B-6.-Agricultural Products

Product category End use Product category End use Amino acids Amino acids Glutamate ...... Food enrichment agent, Lysine...... Feed additive flavoring agent Methionine...... Feed additive Cysteine ...... Food enrichment agent, Threonine...... Feed additive manufacturing processes Tryptophan...... Feed additive Aspartame...... Flavoring agent Vitamins Vitamins Nicotinic acid...... Feed additive

Vitamin C...... ,.. . Food additive, food Riboflavin (B2) ...... Feed additive enrichment agent Vitamin C ...... Feed additive Vitamin D...... Food enrichment agent Aliphatics Aromatics Sorbic acid...... Feed preservative Benzoic acid ...... Food preservative Antibiotics Aliphatics Penicillins ...... Feed additive, prophylactic Propionic acid. . . . . ,.. . Food preservative Erythromycins...... Feed additive, prophylactic Short peptides Peptide hormones Aspartame ...... ,.. . Artificial sweetener Bovine growth hormone...... Growth promoter Enzymes Porcine growth hormone . . . . . Growth promoter Rennin Manufacturing processes Ovine growth hormone...... Growth promoter Amyloglucosidase. . . . . Manufacturing processes Viral antigens a-amylase ...... Food enrichment agent, Foot-and-mouth disease virus. Vaccine manufacturing processes Rous sarcoma virus ...... Vaccine Glucose isomerase. . . . . Manufacturing processes: Avian Ieukemiavirus ...... Vaccine sweetener Avian myeloblastosis virus. . . . Vaccine Nucleotides Enzymes 51-IMP a...... Flavoring agent Papain ...... Feed additive 51-GMP b ...... Flavoring agent Glucose oxidase ...... Feed preservative Pesticides %’-lnosinic acid. b51-guanylic acid. Microbial ...... Insecticide SOURCE: Genex Corp. Aromatic...... Insecticide Inorganic all the R&D starting from the current knowledge Ammonia ...... Fertilizer base necessary to demonstrate that the desired com- SOURCE: Genex Corp. pounds can be biologically produced first in the laboratory and then in the pilot plant at commercially desirable yields and reaction efficiencies. The timetable does not consider delays caused by con- tables from 1-B-12 to I-B-32 project that within 20 struction of new facilities nor delays required to years all these products could be manufactured obtain Government regulatory approval of new using genetically engineered microbial strains on a products. more economical basis than using today’s conven- It should be noted that in the technical data charts, tional technologies. In many cases, the time required when glucose is listed as an alternate precursor by to apply genetically engineered strains in commercial fermentation, other carbohydrates, e.g., cellulose fermentations could be reduced to as little as 5 years. and cornstarch, could be used. Moreover, if glucose The impact of genetic engineering on selected were the precursor of choice, the actual feedstock markets is shown in table I-B-33. only five product would probably be a commodity like molasses as op- categories are considered here, and in one, amino posed to pure glucose. acids, only a few of the compounds comprising it are evaluated. The products represented in the five cate- Summary gories currently have a total market value exceeding $800 million. However, within 20 years this market Over 100 compounds representing 17 different value could rise to over $5 billion (in 1980 dollars) product categories that span the five industries due largely to the application of genetic engineering. under evaluation are represented in table I-B-10. The In a number of cases, the desired products would current market value of all these products exceeds most likely not be available in significant quantities if $27 billion. One particular compound, methane, ac- not for the application of genetic engineering tech- counts for over $12 billion. The even-numbered nology. Appendix l-B—A Timetable for the Commercial Production of Compounds ● 279

Table l- B-7.—Chemicals: Aliphatics Table l- B-9.—Energy Products

Compound End use Product category End use Acetic acida ...... Miscellaneous acyclic Enzymes Acrylic acida...... Miscellaneous acyclic Ethanol dehydrogenase. Manufacturing processes Adipic acida ...... Misceilaneous acyclic Hydrogenate...... Manufacturing processes Bis (2-ethylhexyl) adipate. . . . . Plasticizer Biodegradation...... Petroleum byproducts removal Citronella...... Flavor/perfume material Citronellol ...... Flavor/perfume material Aliphatics Ethanol a...... Miscellaneous acylic Methane...... Fuel Ethanolamine...... Miscellaneous acyclic Ethanol ...... Fuel Ethylene glycola ...... Miscellaneous acyclic Inorganic Ethylene oxidea...... Miscellaneous acyclic Hydrogen...... Fuel Geraniol ...... Flavor/perfume material a Mineral leaching Glycerol ...... Miscellaneous acyclic Uranium...... Fuel Isobutylene ...... Miscellaneous acyciic, flavor/perfume material SOURCE: Genx Corp. Itaconic acid ...... Plastics/resin Linalool...... Flavor/perfume material Linalyl acetate...... Flavor/perfume material Methane...... Primary petroleum product Nerol...... Flavor/perfume material Pentaerythritol...... Miscellaneous acyclic Propylene giycola ...... Miscellaneous acyiic Table I- B- 10.—Total Market Values for the Sorbitol...... Miscellaneous acyclic Various Product Categories a-terpineol ...... Flavor/perfume material a-terpinylacetate ...... Flavor/perfume material Number of Current value Product category compounds ($ millions) alndicates compounds also identified by the Massachusetts Institute of Technology. The following additional chemicals were identified by MIT as Amino acids...... 9 $ 1,703.0 amenable to biotechnological production methods: acetaldehyde, acetoin, Vitamins...... 6 667.7 acetone, acetylene, acrylic acid, butadiene, butano~ butyl acetate, Enzymes...... 11 217.7 butyraldehyde, dihydroxyacetone, ethyl acetate, ethyl acrylate, ethylene, formaldehyde, isoprene, isopropanol, methanol, methyl ethyl ketone, methyl Steroid hormones ...... 6 376.8 acrylate, propylene, propylene oxide, styrene, vinyl acetate. Peptide hormones...... 9 263.7 SOURCE: Genex Corp. and the Massachusetts Institute of Technology. Viral antigens...... 9 N/A Short peptides...... 2 4.4 Nucleotides...... 2 72.0 Miscellaneous proteins ...... 2a 300.0 Table l-B-8.—Chemicals: Aromatics and 4 b Miscellaneous Antibiotics...... 4,240.0 Gene preparations...... 3 N/A b Pesticides ...... 2 100.0 Product category End use Aliphatics: Aromatics Methane...... 12,572.0 c Aniline ...... Cyclic intermediate Other...... 24 2,737.5 c Benzoic acid...... Cyclic intermediate Aromatics...... 10 1,250.9 Cresols...... Cyclic intermediate Inorganic ...... 2 2,681.0 Phenol ...... Cyclic intermediate Mineral leaching ...... 5 N/A Phthalic anhydride ...... Cyclic intermediate Biodegradation ...... N/A N/A Cinnamaldehyde...... Flavor/perfume material Totals ...... 107 $27,186.7d Diisodecyl phthalate...... Plasticizer Dioctyle phthalate...... Plasticizer %nly two of a number of compounds are considered here. %hese numbers refer to major classes of compounds; not actual numbers of Inorganic compounds. Ammonia ...... Manufacturing processes cThege numbers refer only to those compounds rePresenting the k9est market volume In classes specified in the text. Hydrogen ...... Manufacturing processes dcument value excluding methane = $14,614,7W,~. Enzymes SOURCE: Genex Corp. Pepsin...... Manufacturing processes Bacillus protease...... Manufacturing processes Mineral leaching Transition metals (cobalt, Inorganic intermediates; nickel, manganese, iron)...... catalysts Biodegradation ...... Removal of organic phosphates, aryl sulfonates, and haloaromatics

SOURCE: Genex Corp. 280 ● Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

Table I-B-11.-Amino Acids: Market Information

Current market data Market volume Bulk cost Market value Compound 1,000 lb $/lb ($ millions) Arginine ...... 900 12.73 11.46 Aspartate ...... 3,000 2.86 8.6 Cysteine...... 600 22.75 13.6 Glutamate...... 600,000 1.80 1,080.0 Lysine ...... 129,000 2.10 258.0 Methionine...... 210,000 1.40 294.0 Phertylalanine. . . . 300 38.18 11.46 Threonine...... 300 58.18 16.2 Tryptophan...... 225 43.18 9.71

SOURCE: Compiled by Genex Corp. from data in references 1,2, and 3.

Table l-B-12.—Amino Acids: Technical Information

Is precursor Time to implement renewable/non- Alternate commercial fermentation by Typical synthetic renewable precursor by genetically engineered Compound process Typical precursor limited fermentation strain Arginine ...... fermentation glucose renewable 5 yrs. +a and NH4 Aspartate ...... fermentation fumaric acid limited 5 yrs. and ammonia Cysteine ...... extraction protein renewable 5 yrs. hydrolysis Glutamate...... fermentation glucose renewable 5 yrs. + and NH4 Lysine...... fermentation glucose renewable 5 yrs. + and NH4 Methionine...... chemical B-methylmercapto nonrenewable glucose 10 yrs. + proplonaldehyde and NH4 Phenylalanine. . . . chemical a-acetamino- limited glucose 5 yrs. + cinnamic acid and NH4 5 yrs. Threonine...... fermentation glucose renewable 5 yrs. + and NH4 Tryptophan...... chemical a-ketoglutaric nonrenewable glucose 5 yrs. + phenylhydrazone and NH4 aArnmonlum ion. SOURCE: Compiled by Genex Corp. from data in references 2,3,4, and 5

Table l- B-13.-Vitamins: Market Information

Current market data Market volume Bulk cost Market value Compound 1,000 lb $/lb ($ millions) Nicotinic acid. . . . 1,400 1.82 2.5 Riboflavin (B2). . . . 22 15.40 0.34 Vitamin B12 ...... 6,991.60 153.8 Vitamin C ...... 90,0% 4.50 405.0 Vitamin D ...... 12 42.50 0.51 Vitamin E ...... 3,641 29.00 105.6

SOURCE: Compiled by Genex Corp. from data in references 1,6,7,8, and 9. Appendix l-B—A Timetable for the Commercial production of Compounds . 281

Table l- B-14.—Vitamins: Technical Information

Table I-B-1 5.—Enzymes: Market Information

Current market data Market volume Bulk cost Market value Compound 1,000 lb $/lb ($ millions) a-amylase. , . . . . . 600 19.33 11.6 Amyloglucosidase 600 Asparaginase. . . . (lnformation not available) Bacillus protease. 1,000 8.28 8.2 Ethanol dehydrogenase . (Information not available) Glucose isomerase 100 40000 400 Glucose oxidase . 0.80 Hydrogenate . . . . (Iformation not available) Papain...... 200 59.00 11.8 Pepsin...... 10 360.00 3.8 Rennin...... 24 696.00 40.0 Tyrosine hydroxylase . . . . (Information not available) Urokinase...... 60,000 IUa 89.5 alu = international units. SOURCE: Compiled by Genex Corp. from data in references 9,13,14,15, and 16. 282 ● Impacts of Applied Genetics— Micro-Organ/sins, Plants, and Animals

Table I- B-16.-Enzymes: Technical Information

Is precursor Time to implement renewable/non- Alternate commercial fermentation by Typical synthetic renewable precursor by genetically engineered Compound process Typical precursor limited fermentation strain a-amylase...... fermentation molasses renewable 5 yrs. Amyloglucosidase fermentation molasses renewable 5 yrs. Asparaginase. . . . extraction tissue culture renewable glucose 5 yrs. + and NH4 Bacillus protease. fermentation molasses renewable 5 yrs. Ethanol dehydrogenase (Information not available) glucose 10 yrs. + and NH4 Glucose isomerase. . . . . fermentation glucose renewable 5 yrs. +a and NH4 Glucose oxidase . fermentation molasses renewable 5 yrs. Hydrogenate . . . . (Information not available) glucose 10 yrs. + and NH4 Papain...... extraction papaya renewable glucose 5 yrs. + and NH4 Pepsin...... fermentation molasses renewable 5 yrs. Rennin...... fermentation molasses renewable 5 yrs. Tyrosine ...... extraction tissue culture renewable glucose 5 yrs. + and NH4 Urokinase...... extraction tissue culture renewable glucose 5 yrs. aArnrnonium ion. SOURCE: Compiled by Genex Corp. from data in references 4,5,13,14,16,17, and 18.

Table l=B=17.-Sterold Hormones: Market information

Current market data Market volume Bulk cost Market value Compound 1,000 lb $/lb ($ millions)

Corticoids. . . . ● . . 305.8 Cortisone ...... N/A 208.84 N/A Prednisone...... N/A 467.62 N/A Prenisolone . . . . . N/A 463.06 N/A Aldosterone . . . . . N/A N/A N/A Androgens 10,8 Testosterone . . . . (Information not available) Estrogens...... 60.2 Estradiol ...... (Information not available)

SOURCE: Compiled by Genex Corp. from data In references 1 and 4. Appendix l-B—A Time table for the Commercial Production of Compounds ● 283

Table I-B-18.—Steroid Hormones: Technical Information

Is precursor Time to implement renewable/non- Alternate commercial fermentation by Typical synthetic renewable precursor by genetically engineered Compound process Typical precursor limited fermentation strain Corticoids Cortisone Prednisone...... semisynthetic diosgenin or renewable glucose 10 yrs. stigmasterol Predisolone Aldosterone Androgens Testosterone . . . . semisynthetic chemical renewable glucose 10 yrs. modification of cholesterol Estrogens Estradiol ...... semisynthetic chemical renewable glucose 10 yrs. modification of cholesterol SOURCE: Compiled by Genex Corp. from data in references 4,19,20,21, and 22. .

Table l- B-19.—Peptide Hormones: Market Information

Current market data Market volume Bulk cost Market value Compound 1,000 lb $/lb ($ millions) ACTH a ...... N/A N/A 5.6 Bovine growth hormone...... 0.0 0.0 Endorphins...... (lnformation not available) Enkephalins . . . . . (Information not available) Glucagon ...... (Information not available) Human growth hormone...... N/A N/A 75.0 Insulin...... N/A N/A 163.1 Ovine growth hormone...... 0.0 0.0 0.0 Porcine growth hormone...... 0.0 0.0 Vasopressin. . . . . (lnformation not available)

aAdrenoco~icotropic hormone. SOURCE: Compiled by Genex Corp. from data in reference 4. 284 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Table I- B-20.-Peptide Hormones: Technical Information

Is precursor Time to implement renewable/non- Alternate commercial fermentation by Typical synthetic renewable precursor by genetically engineered Compound process Typical precursor limited fermentation strain ACTH a...... extraction adrenal cortex limited glucose 5 yrs. +b and NH4 Bovine growth hormone...... extraction anterior pituitary limited glucose 5 yrs. + and NH4 Endorphins...... extraction brain limited glucose 5 yrs. + and NH4 Enkephalins. . . . . extraction brain limited glucose 5 yrs. + and NH4 Glucagon ...... extraction pancreas limited glucose 5 yrs. + and NH4 Human growth hormone...... extraction anterior pituitary limited glucose 5 yrs. + and NH4 Insulin...... extraction pancreas limited glucose 5 yrs. + and NH4 Ovine growth hormone...... extraction anterior pituitary limited glucose 10 yrs. + and NH4 Porcine growth hormone...... extraction anterior pituitary limited glucose 10 yrs. + and NH4 Vasopressin. . . . . extraction posterior pituitary limited glucose, 5 yrs. + and NH4 aArjren~@iCOt@C hormone. bAmmonium ion. SOURCE: Compiled by Genex Corp. from data in references 4,23, and 24.

Table l- B-21.—Vjral Antigens: Market Information

Current market data Market volume Bulk cost Market value Compound 1,000 lb $/lb ($ millions) Avian leukemia virus...... (Information not available) Avian myeloblastosis virus...... (information not available) Epstein-Barr virus 0.0 0.0 0.0 Hepatitis virus . . . 0.0 0.0 0.0 Herpes virus. . . . . 0.0 0.0 0.0 Hoof and mouth disease virus . . 0.0 influenza virus . . . (lnformation not available) Reoviruses ...... 0.0 0.0 0.0 Rous sarcoma virus...... (Information not available) Rubella virus. . . . . (Information not available) Varicella virus . . . (information not available)

SOURCE: Compiled by Genex Corp. from data in reference 4. Appendix I-B—A Timetable for the Commercial Production of Compounds ● 285

Table l- B-22.—Viral Antigens: Technical Information

Is precursor Time to implement renewable/non- Alternate commercial fermentation by Typical synthetic renewable precursor by genetically engineered Compound process Typical precursor limited fermentation strain Avian leukemia. . . (Information not available) glucose 5 yrs. +a virus and NH4 Avian myeloblastosis. (Information not available) glucose 5 yrs. + virus and NH4 Epstein-Barr virus tissue culture Iymphoblasts renewable glucose 5 yrs. + and NH4 Hepatitis...... (Information not available) glucose 5 yrs. + viruses and NH4 Herpes ...... (Information not available) glucose 5 yrs. + viruses and NH4 Hoof and mouth. . (Information not available) glucose 5 yrs. + disease virus and NH4 Influenza...... (Information not available) glucose 10 yrs. + viruses and NH4 Reoviruses ...... (Information not available) glucose 15 yrs. + and NH4 Rous sarcoma . . . (Information not available) glucose 5 yrs. + virus and NH4 Rubella ...... tissue culture duck embryonic renewable glucose 5 yrs. + virus cells and NH4 Varicella ...... (Information not available) glucose 5 yrs. + virus NH4 aAmmonium ion. SOURCE: Compiled by Genex Corp. from data in references 4 and 25.

Table l-B-23.—Short Peptides, Nucleotides, and Miscellaneous Proteins: Market Information

Current market data Market volume Bulk cost Market value Product category 1,000 lb $/lb ($ millions) Short peptidesa Aspartame ...... 40 110.00 4.4 Glycine-histidine- Iysine...... (Information not available) Nucleotides b 5 1 -IMP c ...... 4,000 12.00 48.0 51-GMP d ...... 2,000 12.00 24.0 Miscellaneous proteinsa Interferon ...... N/A N/A 50.0 Human serum albumin...... 250 1,000.00 250.0 Monoclinal antibodies...... (Information not available)

aData from references 4 and 26. bData from references 4 and 27. c5’-inosinic acid. d5’-guanylic acid. ‘Data from reference 4. SOURCE: Compiled by Genex Corp. 286 ● Impacts of Applied Genet/cs—Micro-Organisms, Plants, and Animals

Table l- B-24.—Short Peptides, Nucleotides, and Miscellaneous Proteins: Technical information

Is precursor Time to implement renewable/non- Alternate commercial fermentation by Typical synthetic renewable precursor by genetically engineered Product category process Typical precursor limited fermentation strain Short peptidesa Aspartame ...... chemical phenylalanine & renewable glucose 5 yrs. + aspartic acid and NH4 Glycine-histidine- Iysine...... extraction human serum renewable glucose 5 yrs. + and NH4 Nucleotides c 51-lMP d ...... extraction yeast renewable glucose 10 yrs. + and NH4 51-GMP e...... extraction yeast renewable glucose 10 yrs. + and NH4 Mlscellaneous proteinsf Interferon ...... extraction or leukocytes, renewable glucose 5 yrs. + tissue culture Iymphoblasts, and NH4 or fibroblasts Human serum albumin ...... extraction human serum renewable glucose 5 yrs. + and NH4 Monoclinal antibodies . . . . . somatic cell various cells renewable glucose 10 yrs. + hybridization and NH4

%ata from reference 4 and 27. bAmmonium ion. cData from references 4 and ~. %’-inoalnic acid. ‘5’-guanyiic acid. ‘Data from reference 4. SOURCE: Compiied by Genex Corp.

Table I- B-25.-Antibiotics, Gene Preparations, and Pesticides: Market information

Current market data Market volume Bulk cost Market value Product category 1,000 lb $/Ib ($ millions) Antibiotic@ Penicillins...... 49,300 22.11 1,080.0 Tetracycline. . . . 29,300 34.13 1,000.O Cephalosporins. . 4,210 114.00 460.0 Erythromycins . . . (Information not available) Gene preperationsb Sickle cell anemia 0.0 0.0 0.0 Hemophilias. . . . . 0.0 Thallasemias . . . . 0.0 0.0 0.0 Pesticidesc Microbial...... N/A N/A 25.0 Aromatics ...... N/A N/A 75.0

aData from references 4,28, and 9. bData from references 4 and X. cData from references 4 and ~. SOURCE: Compiled by Genex Corp. Appendix I-B—A Timetable for the Commercial Production of Compounds ● 287

Table l- B-26.—Antibiotics, Gene Preparations, and Pesticides: Technical Information Is precursor Time to implement renewable/non- Alternate commercial fermentation by Typical synthetic renewable precursor by genetically engineered Product category process Typical precursor limited fermentation strain Antibiotics Penicillins. . . . . fermentation lactose & limited 10 yrs. semisynthetic nitrogenous oils Tetracycline . . . . fermentation lactose & limited — 10 yrs. nitrogenous oils Cephalosporins . . fermentation lactose & limited 10 yrs. nitrogenous oils Erythromycins . . . fermentation lactose & limited 10 yrs. nitrogenous oils Gene preprationsb Sickle cell anemia (No process exists glucose 15 yrs. +d currently) and NH4 Hemophilias. . . . . (No process exists glucose 20 yrs. + currently) and NH4 Thallasemias . . . . (No process exists glucose 20 yrs. + currently) and NH4 Pestlcidesc Microbial...... fermentation molasses & renewable 5 yrs. fishmeal Aromatics...... semi synthetic naphthalene nonrenewable — 10 yrs. aData from references 4,5,28, 3~t and 32. Coata from references 4 and ~. bData from reference 4. ‘Ammonium ion.

SOURCE: Compiled by Genex Corp.

Table l= B-27. -AIiphatics: Market Information Current market data Market volume Bulk cost Market value Compound 1,000 lb $/lb ($ millions) Acetic acid ...... 823,274 0.23 189.4 Acrylic acid...... 46,503 0.43 20.0 Adipic acid ...... 181,097 0.50 90.5 Bis (2-ethylehexyl) 43,015 0.49 21.1 adipate Citronella ...... 394 3.90 Citronellol...... 1,443 4.50 6.5 Ethanol ...... 1,048,000 0.24 251.5 Ethanolamine. . . . 320,236 0.46 147.3 Ethylene glycol . . 3,137,000 0.31 972.5 Ethylene oxide . . . 525,113 189.0 Geraniol ...... 2,307 3.25 7.5 Glycerol ...... 116,612 0.54 63.0 Isobutylene...... 597,712 0.95 567.2 Itaconic acid. . . . . 200 0.83 0.2 Linalool...... 3,341 2.60 8.7 Linalyl acetate . . . 1,535 3.50 5.4 Methane ...... 878,000,000 0.013 11,573.0 Nerol...... 462 4.20 1.9 Pentaerythritol. . . 117,085 0.62 72.6 Propionic acid . . . 62,848 0.21 13.2 Propylene glycol . 525,527 0.73 173.4 Sorbic acid ...... 20,000 2.15 43.0 Sorbitol...... 160,267 0.36 57.7 a-terpineol ...... 2,416 1.28 3.0 a-terpinyl acetate. 1,066 1.30 1.4 SOURCE: Compiled by Genex Corp. from data in references 1,4,9, and 33. 288 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Table l- B-28.—Aliphatics: Technical Information

Is precursor Time to implement renewable/non- Alternate commercial fermentation by Typical synthetic renewable precursor by genetically engineered Compound process Typical precursor limited fermentation strainb Acetic acid...... chemical methanol nonrenewable glucose 10 yrs. or ethanol Acrylic acid ...... chemical ethylene nonrenewable glucose 10 yrs. Adipic acid...... chemical phenol nonrenewable glucose 10 yrs. Bis (2-ethylhexyl). . . . chemical phenol nonrenewable glucose 20 yrs. adipate Citronella ...... chemical isobutylene nonrenewable glucose 20 yrs. Citronellol ...... chemical isobutylene nonrenewable glucose 20 yrs. Ethanol...... chemical ethylene nonrenewable glucose 5 yrs. Ethanolamine ...... chemical ethylene nonrenewable glucose 10 yrs. Ethylene glycol . . . . . chemical ethylene nonrenewable glucose 5 yrs. Ethylene oxide...... chemical ethylene nonrenewable glucose 5 yrs. Geraniol ...... chemical isobutylene nonrenewable glucose 20 yrs. Glycerol ...... chemical soap manuf. nonrenewable glucose 5 yrs. Isobutylene ...... chemical petroleum nonrenewable glucose 10 yrs. Itaconic acid ...... fermentation molasses renewable . . . 5 yrs. Linalool ...... chemical isobutylene nonrenewable glucose 20 yrs. Linalyl acetate ...... chemical isobutylene nonrenewable glucose 20 yrs. Methane...... chemical natural gas nonrenewable sewage 10 yrs. Nerol...... chemical isobutylene nonrenewable glucose 20 yrs. Pentaerythritol . . . . . chemical acetaldehyde & nonrenewable glucose 10 yrs. formaldehyde Propionic acid ...... chemical ethanol & limited glucose 10 yrs. carbon monoxide Propylene glycol . . . . chemical propylene nonrenewable glucose 10 yrs. Sorbic acid...... chemical crotonaldehyde & nonrenewable glucose 15 yrs. malonic acid Sorbitol...... chemical glucose renewable 10 yrs. a-terpineol ...... chemical isobutylene nonrenewable glucose 20 yrs. a-terpinyl acetate . . . chemical isobutylene nonrenewable glucose 20 yrs.

%Vherever glucose ia mentioned, other carbohydrates maybe aubatituted, including starch and cellulose. bln many Casea these times are bgsed on more readily developed fermentations using nonrenewable or limited hydrocarbons as Precursors. SOURCE: Compiied by Genex Corp. from data in references 4,33,34, and 35.

Table I= B-29.-Aromatics: Market Information

Current market data Market volume Bulk cost Market value Compound 1,000 lb $/lb ($ millions) Aniline ...... 187,767 0.42 78.9 Aspirin ...... 32,247 1.41 45.5 Benzoic acid ...... 36,822 0.47 17.3 Cinnamaldehyde. . . . 1,098 2.10 3.4 Cresols...... 94,932 0.54 51.2 Diisodecyl phthalate ...... 151,319 0.42 63.6 Dioctyl phthalate. . . . 391,131 0.42 164.3 p-acetaminophenol. . 20,000 2.65 53.0 Phenol ...... 1,431,000 0.36 515.2 Phthalic anhydride . . 646,289 0.40 258.5 SOURCE: Compiled by Genex Corp. from data in references 1 and 9. Appendix l-B—A Timetable for the Commercial Production of Compounds ● 289

Table l- B-30.-Aromatics: Technical Information

aAromatic refers t. benzene or benzene derivative. Eventually it is anticipated that Iignin, a renewable resource, would sewe as a Precursor.

SOURCE: Compiled by Genex Corp. from data in references 4 and 35.

Table I-B-31 .—lnorganics and Mineral Leaching: Market Information

Current market data Market volume Bulk cost Market value Product category 1,000 lb $/lb ($ millions) Inorganic Ammonia ...... 33,400,000 0.06 2,004.0 Hydrogen ...... 451,000 0.15 677.0 Mineral leaching Uranium ...... (Information not available) Transition metals. (Information not avaiiable) (cobalt, nickel, manganese, iron)

SOURCE: Compiled by Genex Corp. from data in reference 4.

Table l- B-32.-lnorganics and Mineral Leaching: Technical Information

SOURCE: Compiled by Genex Corp. from data in references 4 and 35. 290 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

Table l- B-33.-Projected Growth of Selected Markets min B Complex, “ in The Pharmacological Basis of Involving Applications of Genetic Engineering Therapeutics, 5th cd., L. S, Goodman and A. Gilman (eds.) (New York: MacMillan Publishing Projected Co., Inc., 1975), pp. 1,549-1,563. market Current market in 20 yrs. 12 Straw, J. A., “Fat-Soluble Vitamins: Vitamin D,” in Product category $ millions $ millions The Pharmacological Basis of Therapeutics, 5th Amino acidsa...... 300 cd., L. S. Goodman and A. Gilman (eds.) [New Miscellaneous proteins. 300 1,000 York: MacMillan Publishing Co., Inc., 1975), pp. Gene preparations . . . . . 0 1,579-1,590. Short peptides ...... 5 2,100 13 Aunstrup, K., “Industrial Approach to Enzyme Peptide hormones . . . . . Totals ...... 865 5,100 Product ion,” in Biotechnological Applications of Pro(eins and Enzymes, Z. Bohak and N. Sharon %nly four amino acids are considered here. (eds.) (New York: Academic Press, 1977), pp. SOURCE: Genex Corp. 39-50. 14. Aunstrup, K., Andresen, O., Falch, E. Z,, and Miel- References sen, T. K., “Production of Microbial Enzymes, ” in Microbial Technolo~: Microbial Processes, vol. 1, 1. Chemical Marketing Reporter, March and April H. J. Peppier and D. Perlman (eds.) (New York: 1980. Academic Press, 1979), pp. 282-311. 2. Hirose, Y., and Okada, H., “Microbial Production 15. Bernard Wolnak & Associates, in Food Prod., July of Amino Acids, ” in Microbial Technology: Micro- 1978, p. 41. bial Processes, vol. 1, H. J. Peppier and D. Perlman 16. Solomons, G. L., “The Microbial Production of En- (eds.) (New York: Academic Press, 1979), pp. zymes, ” in Biotechnological App/ica/ions of Pro- 211-240. teins and Enzymes, Z. Bohak and N. Sharon (eds.) 3. Hirose, Y., Sane, K., and Shibai, H., “Amino (New York: Academic Press, 1977), pp. 51-61. Acids, ” in Annual Reports on Fermentation Proc- 17 Chemical and Engineering News, Apr. 14, 1980, p. esses, vol. 2, (;. T. Tsao and D. Perlman (eds.) 6. (New York: Academic Press, 1978), pp. 155-190. 18 Levine, W. G., “Anticoagulants, Antithrombotic, 4. (;enex Corp. proprietary information. and Thrombolytic Drugs, ” in The Pharmacological 5. Weinstein, L., “Chemotherap~ of Microbial Dis- Basis of Therapeutics, 5th cd., L. S. Goodman and eases, ” in The Pharmacological Basis of Therapeu- A. Gilman (:eds.) (New York: MacMillan Publish- tics, 5th cd., L. S. Goodman and A. Gilman (eds.) ing Co., Inc., 1975), p. 1,365. (New York: MacMillan Publishing Co., Inc., 1975), 19 Gilman, A. G., and Muriad, F,, “Androgens and pp. 1,090-1,247. Anabolic Steroids, ” in The Pharmacological Basis 6. Cohn, V. H., “k’at-Soluble Vitamins: Vitamin K and of Therapeutics, 5th cd., L. S. Goodman and A. vitanlin E,” in The Pharmacological Basis of Ther- Gilman (eds.) (New York: MacMillan Publishing apeutics, 5th cd., L. S. Gmodrnan and A. Gilman Co., Inc., 1975), pp. 1,451-1,471. (eds.) (New York: MacMillan Publishing Co., Inc., 20. Gilman A. G., an-d- Muriad, F., “Estrogens and Pro- 1975), PP. 1,591-1,600. gestins, ” in The Pharmacological Basis of Ther- 7. Florent, J., and Nitiet, L., “Vitamin B,z,” in A4icro- apeutics, 5th cd., L. S. Goodman and A. C1ilman bial Technology Microbial Processes, vol. 1, H. J. (eds.) (New York: MacMillan Publishing Co., Inc., Peppier and D. Perlman (eds.) (New York: Aca- 1975), pp. 1,423-1,450. demic Press, 1979), pp. 497-520. 21. Haynes, R. C., and Lamer, J., “Adrenocortico- 8. Perlman, D., “Microbial Process for Riboflavin tropic Hormone: Adrenocorticol Steroids and Product ion,” in Microbial Technology: Microbial Their Synthetic Analogs: inhibitors of Adreno- Processes, vol. 1, H. J. Peppier (eds.) (New York: corticol Steroid Biosynthesis, ” in The Pharmaco- Academic Press, 1979), pp. 521-528. logical Basis of Therapeutics, 5th cd., L. S, Cmod- 9. Synthelic Organic Chemicals, March and April man and A. Gilman (eds. ) (New York: MacMillan 1980. Publishing Co., Inc., 1975), pp. 1,472-1,506. 10. Burns, J. J., “Water Soluble Vitamins, The Vita- 22. Sebek, O. K., and Perlman, D., “Microbial Trans- min B Complex, “ in The Pharmacological Basis of formation of Steroids and Sterols,” in Microbial Therapeutics, 5th cd., L. S. Goodman and A. Technology: Microbial Processes, vol. 1, H. J. Pep- Gilrnan (eds.) (New York: MacMillan Publishing pier and D. Perlman (eds.) (New York: Academic Co., Inc., 1975), pp. 1,549-1,563. Press, 1979), pp. 483-496. 11. Greengard, P., “Water Soluble Vitamins, The Vita- 23. Brazeau P., “Agents Affecting the Renal Conser- Appendix l-B—A Tjmetable for the Commercial Production of Compounds ● 291

l~ation of Water, “ in The Pharmacological Basis of 30. Ignoffo, C. M., and Anderson, R. F., “Bioinsec- Therapeutics, 5th cd., L. S. Goodman and A. ticides, ” in Microbial Technology: Microbial Proc- Gilman (eds.) (New York: MacMillan Publishing esses, vol. 1, H. J. Peppier and D. Perlman (eds.) CO., IIw., 1975), PP. 858-859. (New York: Academic Press, 1979), pp. 211-.z4o. 24 Haynes, R. C., and Lamer, J., “Insulin and Oral 31. German, M., and Huber, F. M., “@Lactam Anti- Hypogl.vcemic Drugs: Glucagon,” in The Pharma- biotics, ” in Annua[ Reports on Fermentation Proc- cological Basis of Therapeutics, 5th cd., L. S. Cmod- esses, vol. 2, G. T. Tsao and D. Perlman (eds. ) man and A. Gilman (eds. ) (New York: MacMi]]an (New York: Academic Press, 1978), pp. 203-222. Publishing Co., Inc., 1975), pp. 1,507-1)533. 32, Shibata, M., and [JLVeda, hf., “Microbial Transfor- 25 Jawetz, E., Melnick, J. L., and Adelberg, E. Z., Re- mation of Antibiotics, ” in Annuai Reports on views of Medical A4icrobioloqv, 1 Ith” cd., (Los Fermentation Processes, Yrol. 2, G. T. Tsao and D. Altos, Ca]if.: Lange Medical Publications, 1974). Perlman (eds.) (New York: Academic Press, 1978), 26. Thaler, M. M., Biochem. Bioph-vs. f?es. Comm. pp. 267-304. 54:562, 1973. 33. Lockwood, L. B., “Production of Organic Acids by 27. Nakiio, Y., “hficrobia] Production of Nucleosides Fermentation, ” in Microbial Technology: Microbial and Nuckwt ides, ” in Microbial Technology.. Micro- Processes, vol. 1, H. J. Peppier and D. Per]man bial Processes, twl. 1, H. J. Peppier and D. Perlman (eds.) (New York: Academic pl’ess, 1979), pp. (eds.) (Newf York: Academic Press, 1979), pp. 367-372. 312.3,55. 344 Roberts, J. D., and Caserio, M. C., Basic Principles 28, Perlman, D., “Microbial Production of Antibiot- oj’ Organic Chemistry (New York: Benjamin, Inc., ics, “ in Microbial Technology Microbial Processes, 1964), p. 1,096. \rol. 1, H. J. Peppier and D. Perlman (eds. ) (New 35. tVindholz, M. (cd.), The Merck Inde,x, 9th ed. (Rah- York: Academic Press, 1979), pp. 241-281. way, N. J.: Merck & Co., 1976). 29< Weatherall, D. J., and Ciegg, J. B., “Recent De\’el- opments in the Molecular (;enetics of Human Hemoglobin,” Cell 16:467-480, 1979. Appendix I-C Chemical and Biological Processes

A comparison was made of waste stream pollution Table I-C-1.— Waste Stream Pollution Parameters: for chemical and biological processes. Ideally, the Current Processes v. Biological Processes comparison should be between the two processes Compounds: Mixed chemicals, including ethylene used in the production of the same end product. oxide, propylene oxide, glycols, amines, and ethers Since such data do not currently exist at the industrial level, the comparison was made between the Current Biological Pollution parameters processes processes chemical production of a mixture of chemicals and Alkalinity (mg/l)...... 4,060 0 the biological production of alcohol and antibiotics. BOD5 a(mg/l)...... 1,950 4,000-12,000 one noteworthy parameter is the 5-day biochemical Chlorides (mg/l) ...... 430-800 0 oxygen demand (BOD5) —the oxygen required over a CODb(mg/l)...... 7,970-8540 5,000-13,000 5-day period by organisms that consume degradable Oils (mg/l) ...... 547 0 pH ...... organics in the waste stream. If the oxygen demand 9.4-9.8 4-7 Sulfates (mg/l) ...... 655 0 is too high, the discharge of such a stream into a Total nitrogen (mg/l)...... 1,160-1,253 50-200 body of water will deplete the dissolved oxygen to Phosphates (mg/l) ...... 0 50-200 the point that it threatens aquatic life. An important as.day biOk@Cal oxygen demand. variable that must be considered along with the BOD bchemical oxygen demand. is the COD (the chemical oxygen demand). Large dif- SOURCE: Office of Technology Assessment. ferences between the COD and BOD of a waste system can indicate the presence of nonbiodegradable substances. Although the conventional process stream shown in table 1-C-1 has less BOD5 than the as a fertilizer. These applications have been inten- biological process stream, its COD content probably sively investigated and have met with success. means that non biodegradables are present, and Because of the renewed interest generated by the specialized waste treatment is necessary. potentials of genetic engineering, some traditional BOD is one area where traditional fermentation fermentation systems are being redesigned. Immo- based processes have posed pollution problems. bilization allows the reuse of cells that would other- Batch fermentation processes typically generate wise be discarded. These systems can be used con- large quantities of dead cells and residual nutrients tinuously for several months as compared with the that cause a large BOD if they are dumped directly usual fermentation time in a batch process of about into a dynamic aquatic environment. (See table I-C-1.) one week or less. [remobilized operations create This difficulty can be circumvented by the use of waste cells much less often than batch systems, and spent cell material as an animal feed supplement or therefore generate less BOD.

292 Appendix I-D The Impact of Genetics on Ethanol— A Case Study

Objective plants to poor quality land, improved disease resistance, and so forth. However, the focus here is entire- This study examines how genetics can and will af- ly on the second area, the use of genetics to improve fect the utilization of biomass for liquid fuels produc- microbial-based conversion to produce ethanol. tion. There are two major areas where genetics are In order to assess the type and extent of im- applicable. One is in plant breeding to improve avail- provements in micro-organisms that might benefit ability (both quantity and quality) of biomass re- ethanol production, its process technology and sources (with existing and previously unused land); economics must first be examined. An overview of the second is in the application of both classical the biomass conversion technology is presented in mutation and selection procedures and the new ge- figure I-D-1; processes are defined mainly on the netic engineering techniques to develop more effi- basis of the primary raw material and the type of cient microbial strains for biomass conversion. Ex- pretreatment required to produce mono- or di- amples of goals in a plant breeding program would saccharides prior to fermentation. In addition, there include improvements in photosynthetic efficiencies, are several alternative fermentation routes to pro- increased carbohydrate content, decreased or modi- duce ethanol; these are characterized by the type of fied lignin content, adaptation of high productivity micro-organisms and will be examined with the in-

Figure l-D-l .—An Overview of Alternative Routes for Conversion of Biomass to Ethanol

SOURCE: Massachusetts Institute of Technology.

293 76-565 0 - 81 - 20 294 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

tent of quantifying the potential impact of genetic im- to break down the starch to about 15DE (dextrose provement on each one. It is interesting to note that equivalents). This process of liquefaction reduces the each type. of organism has its substrate restrictions, viscosity such that the solution can be easily mixed. and only the anaerobic bacteria such as Clostridium After further cooling to 30° C, glucoamylase is added thermosaccharolyticum and C. thermohydrosulfori- along with a starting culture of yeast so that saccha- cum can utilize all of the available substrate. rification and fermentation proceed simultaneously. The resulting fermentation, to produce typically 8 to Substrate pretreatment 10 percent ethanol (volume per volume), requires 42 to 48 hr for completion. This compares with a 16- to Pretreatment refers to the processing that is re- 20-hr fermentation if sugar as molasses or cane juice quired to convert a raw material such as sugarcane, is used as the substrate. Thus, the use of starch re- starch, or cellulosic biomass to a product that is quires the addition of enzymes prior to and during fermentable to ethanol. In most cases, the pretreat- fermentation, as well as large fermenter capacity as a ment is either extraction of a sugar or hydrolysis of a consequence of the slower fermentation time com- polysaccharide to yield a mono-or disaccharide. pared with sugar substrates. Improvement in the economy of ethanol fermenta- EXTRACTION OF SUGAR tion based on starch is possible by developing a Sugar crops such as sugarcane, sugar beets, or micro-organism that can produce wamylase and sweet sorghum are highly desirable raw materials glucoamylase and thus eliminate the need to add for producing ethanol. These crops contain high these enzymes. Since the rate of fermentation de- amounts of sugars as sucrose. In addition, the yield pends on the rate of starch hydrolysis, increased lev- of fermentable material per acre is high; sugarcane els of glucoamylase may enhance the rate of starch and sugar beets yield 7.5 and 4.1 dry tons of biomass hydrolysis and thus increase the rate of ethanol proper acre, respectively. ’ duction, This would lower the capital requirements Sugar is extracted from cane or beets with hot as well as the cost of enzyme addition. water and then recrystallized. The resulting sugars are utilized directly by organisms having invertase CELLULOSIC BIOMASS activity (to split sucrose to glucose plus fructose). Processes for the utilization of cellulosic biomass Molasses, a sugary byproduct of the crystallization of to produce liquid fuels all have three features in com- sucrose, may also contain sucrose although in most mon: cases it is inverted with acid. 1. They employ some means of pretreatment to at The primary use for sugar crops is food sugar. least effect some initial size reduction and, more Sugar sells for over 20 cents/lb. Molasses, which cur- often, cause a disassociation of lignin and cellu- rently sells for about $100/ton (about 10 cents/lb lose; sugar) is used extensively as an animal feed. Substan- 2. they involve either acid or enzymatic hydrolysis tial amounts of both sugar and molasses are im- of the cellulose and hemicellulose to produce ported into the United States for food uses and are mono- and disaccharides; and therefore unavailable for ethanol production, There 3. they employ fermentation to produce ethanol or are proposals to increase sugar production for use as some other chemical. an energy crop; however, this will require the A wide variety of process schemes have been pro- development of new land for sugar production. posed for the conversion of cellulosic biomass to STARCH liquid fuels; a summary of the major steps in two The primary raw material for ethanol fermenta- acid hydrolysis and three enzymatic hydrolysis tion in the United States is cornstarch. Corn proc- schemes in shown in figures I-D-2 and I-D-3. The in- essed by wet milling, yields about 36 lb of starch itial size reduction is required to increase the from each 56 lb bu; this amount of starch will pro- amount of biomass surface area that can be con- duce 2.5 gal of absolute ethanol. Corn yields are tacted with acid, solvent, steam, enzymes, or typically 80 to 120 bu/acre so that 200 to 300 gal of chemicals that might be used to disassociate the ethanol can be derived per acre of corn per year. cellulose and hemicellulose from the lignin. Pretreatment of starch is initiated by a gelatiniza- Pretreatments that have been investigated to tion step whereby a starch slurry is heated for 5 min facilitate the process are summarized in table at 105° C. After cooling to 98° C, œ-amylase is added I-D-1. The problems with pretreatment are that they require energy, equipment, and often chemicals; they result in an irretrievable loss of sugar, and in ‘Paul B. Weisz and John F. Marshall, Science 206:24, 1979. undesirable side-reactions and byproduct forma- Appendix l-D—The Impact of Genetics on Ethanol—A Case Study ● 295

Figure l-D-2.– Alternative Schemes for Acid Hydrolysis of Cellulosic Biomass for Ethanol Production

SOURCE: Massachusetts Institute of Technology.

tion. Furthermore, if acids, alkali, or organic chem- tures is used to cause cellulose hydrolysis (scheme icals are used, they must be recycled to minimize A). A major problem with this approach is the irre- cost or disposed of in order to prevent pollution. versible loss of sugars to undesirable side-product In starch processing, prior to ethanol fermenta- formation. After separation of residual solids (mostly tion, mechanical grinding, steam, and enzymes are lignin), which can be burned to provide energy for employed. The energy requirements are small and distillation, the sugar solution is fermented by yeast contribute relatively little to the final ethanol cost. to ethanol. The pentose sugars also can be fer- The objective in the development of cellulose-based mented, but by organisms other than the ethanol processes should be to minimize both energy and producing yeast, to other chemicals, some of which chemical requirements. The development and scale- could be used as fuels (e.g., ethanol, acetic acid, up of effective pretreatment technology are under acetone, butanol, 2,3-butanediol, etc.). active investigation and require continued financial An alternative (scheme B, figure I-D-2) to the above support to better develop several alternative routes. is to use a solvent, after pentose sugar removal, to The most promising routes are: steam treatment, sol- dissolve the cellulose, allowing its separation from vent delignification, dilute acid, cellulose dissolution, lignin. This cellulose solution is easily and efficiently and direct fermentation. hydrolyzed to sugars. The advantage of this ap- Several different acid hydrolysis schemes have proach over the direct acid hydrolysis is that the been proposed. However, most appear as in flow yield of sugar is much higher. In the harsh acid hy- scheme A or B in figure I-D-2. Dilute acid is used to drolysis, considerable sugar is destroyed. However, hydrolyze the hemicellulose to pentose sugars pri- the major disadvantage of both these schemes is that marily and then stronger acid at higher tempera- they require recycling or disposal of acids and ‘Proceedings of 3rd Annual Biomass Energv System Conference, National solvents. A second problem is that almost nothing is Technical Information Set-vice, SfXl~rP-33-285, 1979. known about how to scale-up some of the newly de- 296 . Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

Figure I-D-3.—Alternative Schemes for Enzymatic Hydrolysis of Cellulosic Biomass for Ethanol Production

SOURCE: Massachusetts Institute of Technology.

Table I- D-1 .-Alternative Pretreatment Methods for most promising directions that need development Lignocellulose Materials are: ● the scale-up of high rates and high yield labora- Chemical methods Physical methods tory hydrolysis systems, and Sodium hydroxide (alkali) Steam ● the development of methods for acid and chem- Ammonia Grinding and milling ical recycle schemes. Chemical pulping Irradiation Ammonium bisulfite Freezing There are three types of approaches that have Sulfite been employed for enzymatic hydrolysis of cellulosic Sodium chlorite biomass. These are summarized in figure I-D-3. They Organic solvents all involve some initial size reduction to increase the Acids surface area available for enzymatic attack. In SOURCE: Office of Technology Assessment. schemes A and B, the incoming cellulosic biomass is split into two streams; one is used to grow organisms that produce cellulolytic enzymes called cellulases, veloped technology, such as that developed by and the other is used to produce sugar. groups working at Purdue University, New York Uni- In scheme A, the cellulases are recovered and then versity, and Dartmouth College. There are several added to a separate enzyme hydrolysis reaction. engineering problems involving both heat and mass They hydrolyze both the cellulose and hemicellulose, transfer and acid/solvent recycle that need to be eval- and the resulting sugar solution is then passed to an uated at larger scale. At least some of this work will ethanol fermentation stage where hexoses are con- be done at the process development unit now being verted by a yeast fermentation to ethanol. Utilization built at the Georgia Institute of Technology. The of the pentose requires a separate fermentation. Re- Appendix l-D—The Impact of Genetics on Ethanol—A Case Study ● 297

sidual lignin, which is removed before (by solvents Table I-D-2.—A Comparison of the Distribution of extraction) or after hydrolysis, is used to provide Manufacturing Costs for Several Ethanol energy for ethanol recovery, Extensive work on this Production Processes approach has been done at the University of Califor- nia, Berkeley, and the U.S. Army Natick Laboratories. Grain Substrate Molasses Corn Sorghum In scheme B, the cellulase is not recovered but Cost component (%) rather, the whole fermentation broth from cellulase Capital...... 9 12 10 production is added to the cellulosic biomass along Operating ...... 20 26 30 with ethanol-producing yeast. The result is a simul- Feedstock...... 71 62 60 taneous cellulose hydrolysis (saccharification) and Total ...... 100 100 100 fermentation. (In the production of ethanol from Cost on energy basis starch, the starch hydrolyzing enzymes are added at ($MMBtu) ...... 12.5 14.9 12.7 Cost/gal ethanol ($/gal) . . . . 1.05 1.25 1.07 the same time as the yeast for simultaneous sacchari- Capital investment fication and fermentation.) This technology has been ($/annual gal) ...... 1.02 1.05 1.75 demonstrated by the Gulf Oil Co. After fermentation, SOURCE: “Comparative Economic Assessment of Ethanol From Biomass,” the ethanol is recovered and the residual lignin can Mitre Corp., report HCP/ET-2854). again be used for energy for distillation. The problem of unused pentose sugar still remains and will require a separate fermentation step. A third alternative (scheme C, figure I-D-3) shows a cell hr), by increasing the content and/or activi- simpler approach, namely a direct fermentation on ty of those enzymes in the pathway to ethanol; cellulose. This approach has been developed at the increase the utilization of other materials in the Massachusetts Institute of Technology. It utilizes substrate, e.g., the use of oligosaccharides, espe- bacteria that will produce cellulase to hydrolyze the cially branched, in starch, and the use of con- cellulose and hemicellulose and ferment both the taminating sugars such as galactose or mannose hexose and pentose sugars to ethanol in a single- for hemicellulose; and stage reactor. The advantage of this approach is a develop a route for the utilization of pentose su- minimal requirement for pretreatment, a combined gars, especially xylose, present in hemicellulose. enzyme production, cellulose hydrolysis and ethanol The potential effect of oligosaccharides or con- fermentation, and simultaneous conversion of both taminating sugar utilization is relatively small, since pentose and hexose sugars to ethanol. This concept they represent typically 1 to 3 percent of the total is new and work still needs to be done to increase the sugar content. However, if cellulosic biomass con- ethanol concentration, minimize side product forma- taining 15 to 25 percent hemicellulose is used, then tion, and increase the rate of ethanol production. the impact of pentose conversion to ethanol is great. Again, residual lignin will be used to provide the Cellulosic biomass is made up primarily of cellu- energy for ethanol distillation. lose, hemicellulose (mostly xylan) and lignin. Other components such as protein, ash, fats, etc., typically FERMENTATION OF ETHANOL comprise about 10 percent. The composition of bio- An examination of the economics for ethanol pro- mass can be expressed in terms of the following duction shows that the dominant cost is the process equation: raw material. As seen in table 1-D-2 the feedstock rep- F,. + F,, + F,, = 10 [1] resents 60 to 70 percent of the manufacturing cost. 1 – 1 \ Thus, it is clear that any improvement in substrate where F , F , F~) and F are the weight fractions of utilization efficiency is of substantial benefit. The C H A theoretical yields of ethanol from glucose, sucrose, cellulose, hemicellulose, lignin, and ash, respectively. Assuming that the ash is 10 percent (F = 0.1) and and starch or cellulose are 0.51, 0.54 and 0.57 gram A that F and F only fermentable components (g) ethanol/g material, respectively; the differences C H are the result from the addition of a molecule of water on in the biomass, then: F = F = 0.9 – F (2) hydrolysis. There are several approaches to improve C H L the yield above the typical value of 90 to 95 percent The maximum amount of ethanol from one unit of currentl y achieved. These are: biomass (Y~~,~) is; . increase the ratio of ethanol produced per unit YWCFC = YE/HFH = Y~jB (3) weight of cells, e.g., through cell recycle, Where Yi,,C and Yk;lH are the yield of ethanol for cel- vacuum fermentation, immobilized cells, or im- lulose and hemicellulose, respectively. Equation 2 provement in specific productivity (g ethanol/g can be rearranged to relate the fractions of cellulose: 298 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

0.9 – F c = F L - FH (4) of hemicellulose present. From the value in figure Substituting this into equation 3 gives: I-D-4 and the observation that 70 percent of the

YE/B + YE/c(0.9 – FL – FH) + YE/HFH (5) manufacturing cost is the raw material cost, it is From equation 5, the effect can be calculated of possible to estimate the economic benefit of pen- hemicellulose content and conversion yield on the tose utilization. Equation 7 relates the overall overall conversion of biomass to ethanol. Assuming a ethanol yield to the manufacturing cost: C E = CB x 6.6 lignin content of 15 percent (FL = 0.15) and using YE/c (7) Y ().7 = 0.57 g/g the following equation is obtained: E/B

= 0.43 + F H(YE/H - 0.57) (6) Y E/B where CE is the manufacturing cost per gallon of

The theoretical yield value on hemicellulose, ethanol, CB is biomass cost (cents/lb), 6.6 is the con-

YE/H, is not well-defined because so little is known version from pound to gallon of ethanol, and 0.7 is about the biochemistry of anaerobic pentose the 70-percent factor for relative biomass cost to metabolism. If one mole of ethanol is produced per ethanol cost. For a biomass costing 2 cents/lb and mole of xylose, the yield is 0.3 g ethanol/g xylose. It containing 20 percent hemicellulose, the manufac- two moles of ethanol could be obained, YE/H would turing cost is reduced from 59 to 43 cents/gal, when be 0.61; however, neither the mechanism nor the the yield on pentose goes from zero to 0.6. thermodynamics of the conversion is sufficiently At the present time, there are few organisms that well-defined to allow one to expect this value. The produce more than one mole of ethanol per mole of maximum observed values are about 0.41 g pentose and none of the usual alcohol producing ethanol/g xylose.3 The sensitivity of the overall yeasts will ferment pentoses to ethanol. Addition or yield to this value is shown in figure I-D-4. The im- improvement of the ability to use pentose will have a pact of pentose utilization depends on the amount major impact on the economics of ethanol production. %. D. Wang and [:. Cooney, Massachusetts Institute of Technolo~v, un- The second major cost in ethanol production re- published results. lates to the cost of operation. Typically, 20 to 30 percent of the final manufacturing cost is accounted for by the sum of labor, plant overhead, administration,

Figure l= D-4.-Effect of Pentose Yieid (YE/H) on chemical supplies, and fuel costs. The chemical sup- Overall Yield of Ethanol from Cellulosic Biomass plies represent less than 1 cent/gal ethanol and may be neglected. The labor, overhead, and marketing (YE/B) with Varying Fractions of Hemicellulose (FH). costs vary with plant size, but represent 11 to 7 0.5 cents/gal for a 20 to 100 million gal/yr plant, respectively. Any improvement in the reduction of plant size or complexity will reduce this cost; however, the economic impact is small. The major component of the operating cost is the fuel charge for plant operation and for distillation. Plant operations, e.g., mixing, pumping, sterilization, starch gelatinization, biomass grinding, etc., represent about 20 to 30 percent of the energy cost. The remainder is for ethanol distillation and residual solids drying. Considerable effort has been focused on methods to improve the energy efficiency, of distillation to reduce it from the 160)000 Btu/gal required for beverage alcohol. While considerable differences in opinion exist as to the minimum, a reasonable expectation is about 40,000 Btu/gal although current technology requires 69,000 Btu/gal. 4 Forty thousand Btu is about half of the energy content of ethanol per gallon. A discussion of process improvements relating to 0 0.2 0.4 0.6 ethanol recovery has two components. The first is Yield on pentose (g ethanol/g pentose) 4Report of (he Gasohol Stud-y C,roup of the Energy Research Advisory Soard, Department of Energv, Washington, D. C., 1980. ‘M. Gibbs, and R, D. DeMoss, “Ethanol Formation in Pseudomonas /indneri, ” Arc/I. Biochem SOURCE: Massachusetts Institute of Technology. Biophys. 34:478-479, 1951. Appendix l-D—The Impact of Genetics on Ethanol—A Case Study . 299

related to operating costs and the second is related to The third major cost for ethanol manufacturing is energy efficiency. If coal is used to provide energy the capital investment, which represents about 4 to for distillation, and it is valued at $30/ton, with 12 percent of the manufacturing cost. The capital in- 10,500 Btu/lb or $1.50/million Btu, then the energy vestment is determined by the complexity of the cost for distillation (optimistically assuming 40,000 processes and the volumetric productivity of ethanol Btu/gal) is $6/gal. If lignin from cellulosic biomass is production. Thus, the development of a micro-orga- used as a fuel, the cost is reduced further. On the nism that will require a minimum amount of feed- other hand, if oil at $40/bbl (130,000 Btu/gal and 42 stock pretreatment and will produce ethanol at a gal/bbl) or $7/million Btu is used, then the energy higher rate will reduce the net capital investment.

cost is 28 cents/gal of ethanol. The volumetric productivity (QE) for ethanol pro- From a common sense, economic, and political duction is given by:

point of view, it does not seem reasonable to utilize QE = qpX liquid fuel to produce liquid fuel from biomass. where q is the specific productivity expressed in g Therefore, it will be assumed that petroleum will not P ethanol per g cell hr, and X is the culture density. be used for distillation and that either coal or bio- Therefore, there are two approaches to obtain high mass will be employed. productivity: first, to choose or create an organism In order to assess the impact of process improve- with a high specific rate of ethanol production and ments on the energy demand, it is necessary to look second, to design a process with high cell density. at an overall material balance. This is summarized in The application of genetics can be used to enhance figure 1-D-5. Only a portion of the entering biomass the intracellular enzyme activity of the enzymes feedstock is fermented to ethanol and there are two used for ethanol production. The resulting increase product streams, one containing ethanol and the in q , will result in reduced capital investment re- other solids, both must be separated from water. It is p quirements. important to note that as the ethanol concentration is There are four types of ethanol processes based increased, the energy requirement for both ethanol on different organisms; they are: recovery from the water and for drying will de- 1. Saccharomyces cerevisiae and related yeast, crease. Therefore, the impact of developing ethanol 2. Saccharomyces cerevisiae/Trichoderma reesei, tolerant micro-organisms is seen as a reduction in 3. Zymomonas mobilis, and energy cost. 4. Clostridium thermocellum/thermosaccharolyticum, or thermohydrosulfuricum. Figure l-D-5. - Process Schematic for Material and The first is the traditional yeast based process using Energy Balance S. cerevisiae to ferment soluble hexose sugar to eth- Biomass anol. In the second, the substrate range is extended to cellulose by the use of cellulase produced by T. reesei. The third approach utilizes Z. mobilis; this organism is a particularly fast and high ethanol yielding one. Its range of fermentable substrates, however, is limited to soluble hexose sugars. In many tropical areas of the Americas, Africa, and Asia, alcoholic beverages prepared from a mixed fermentation of plant steeps are popular. Bacteria Process heat from the genus Zymomonas are commonly employed. In the early 1950’s, the genus Zymononas acquired a certain fame among biochemists by the discovery that the anaerobic catabolism of glucose follows the Enter-Doudoroff mechanism.’ This was Ethanol very surprising, since Zymomonas was the first example of an anaerobic organism using a pathway mainly in strictly aerobic bateria.6 In spite of its extensive use in many parts of the world, its great social implications as an ethanol pro-

Solids ‘M. Gibbs and R. D, de Moss, “Ethanol Formation, in Psuedornonas Iindneri,” Arch. Biochem. Biophys., 34;478-$79, 1951. 6J. Swings and J. Del.ey, “-rhe Biologv of Zymomonas, ” Bacteriological Re- SOURCE: Massachusetts Institute of Technology. views 41: 1-46, 1977. 300 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals ducer, and its unique biochemical position, Zymo- 2. Manipulations require further knowledge in a monas has not been studied extensively. T specific area or the development of an entirely The organism most often studied is Zymomonas new genetic system in ethanol producing mi- mobilis, which can produce up to 1.9 moles of crobes—e.g., there is no genetic system for the ethanol per mole of glucose. Recent studies reported thermophilic anaerobic bacteria. Knowledge on from Australia, have established the Z. mobilis can how to genetically alter ethanol tolerance of ferment high concentrations of glucose rapidly to both bacteria and yeast is lacking. ethanol in both batch and continuous culture with The economics of the fermentation of a substrate higher specific glucose uptakes rates for glucose and into alcohol is primarily controlled by three factors: ethanol production rates than for yeasts currently 1. Ethanol yield.—The amount of product pro- used in alcohol fermentations in Australian.89 duced per unit of substrate determines the ma- For example, several kinetic parameters for a Z. jor raw materials cost of the fermentation. mobilis fermentation were compared with Saccha- 2. Final ethanol concentration. —The cost of separat- romyces carlsbergensis10 specially selected for its ing the ethanol from the fermentation broth is a sugar and alcohol tolerance.ll Both specific ethanol function of the ethanol concentration in that productivity and specific glucose uptake rate are sev- broth. eral times greater for Z. mobilis. This result is mainly 3. Productivity. —The amount of ethanol produced due to lower levels of biomass formation and glucose per liter of fermenter volume per hour deter- consumption. The lower biomass produced would mines the capital cost of the fermentation step, seem to be a consequence of the lower energy avail- once the type of fermenter and the annual out- able for growth with Zymomonas than with yeasts— put have been chosen. Productivity is not inde- the Enter-Doudoroff pathway producing only 1 mole pendent of the final ethanol concentration, and of adenosine triphosphate (ATP) per mole of glucose, so an optimum compromise between these vari- compared to glycolysis with 2 moles ATP per mole ables must be chosen. glucose. In none of the first three examples can ethanol be produced from pentose sugar. The impact of genetics on ethanol yield The fourth approach utilizes a mixed culture of Most microbes that are chosen for making ethanol Clostridia, which will utilize cellulose and hemicellu- already produce nearly the theoretical maximum lose, hexoses, and pentoses for ethanol production. yield. In these cases little improvement can be made. The yield may be lower when the microbe has The application of genetics for been chosen for its other technical advantages such improving microbial strains as ability to degrade cellulose. Lower yield of a microbial end product, like ethanol, can result from In the previous sections, the process steps have the diversion of substrate to cell mass or to an alter- been identified that are particularly sensitive to the native product. Both of these faults can be readily at- quality of the microbial strains. The following are im- tacked. A number of cell changes (e.g., leaky mem- provements of microbial characteristics that are branes) can cause the microbe to waste energy, re- either now possible or might be so in the future and quiring it to metabolize more substrate into alcohol that will have an impact on the overall economics of to make the same cell mass. Where the thermo- the process. The effect of new genetic techniques re- dynamics and redox balance of the fermentation quiring future research is similar for all micro-orga- allow, unwanted waste products can be eliminated nisms in two ways. by mutation of the relevant pathways. Only limited 1. Manipulations could be attempted today with work has been done on this type of research with in- less effort and greater chance of success if tools dustrially significant bacteria. like cell fusion and recombinant DNA (rDNA) techniques were available for all of the mi- The impact of genetics on final ethanol crobes of interest. concentration —— T;ibbs, et al., op. cit. ‘K. J. Lee, D. E. Tribe, and P. L. Rogers, “Ethanol Production by Zymo- This is amenable to genetic manipulation, both em- monas mobilis in Continuous Culture at High Glucose Concentrations,” Bio- pirical and planned. An improvement in ethanol tol- technology Lett. 421-426, 1979. erance decreased both separation costs and ferment- 9P. L. Rogers, K. J. ke, and D. E. Tribe, Biotechnoi. Lett. 1:165-170, 1979. IOlbid. er capital cost (through increased productivity). I ID. Rose, Proc. Bichem. 11 [2), 1976, pp. IO-12. When traditional distillation is used, the effect on Appendix I-D—The Impact of Genetics on Ethanol—A Case Study ● 301

the separation cost of increased ethanol tolerance is energy level of the cell, expressed through smaller once ethanol concentrations have reached adenosine monophosphate (AMP), adenosine di- approximately 6 percent. However, the importance phosphate (ADP), and adenosine triphosphate of increased ethanol concentration to fermenter pro- (ATP) levels, partially controls the rate of gly- ductivity remains. colysis. A defective cell membrane would pro- It is likely that the most important inhibitory ac- vide an energy sink, to keep glycolysis at its tion of ethanol takes place at the cell membrane. maximum rate. Strategies such as this could be Strategies for manipulating the cell membrane com- attempted now. position and properties, and understanding in this 2. Increase the amount of each transport and cata- area, are increasing rapidly. bolic enzyme in the fermentation pathway. This requires the ability to isolate the genes of in- Genetics and ethanol tolerance terest and to amplify them with in vivo or in vitro recombinant techniques in the microbe of The study of ethanol tolerance by micro-orga- interest. This is not an immediate prospect. nisms has been approached using strains with 3. Accomplish complete deregulation of the fer- altered genetic makeup. Several kinds of Escherichia mentation pathway in the microbe of interest. coli mutants have been isolated having different Essential catabolic enzymes are difficult to tolerances to ethyl alcohol. 12 Solvent resistant strains manipulate, and this is also not an immediate either had larger amounts of total phospholipid (type prospect. III) or had an altered phospholipid and membrane- Genetic manipulation of the microbe can influence bound protein composition (type 11). On the other fermentation processes in other ways as well. These hand, mutants with a lesion mapping close to pss are less important than improvements in yield, final gene (which codes for phosphotidylserine syn- ethanol concentration, and productivity, but they thetase) were either solvent sensitive or resistant .13 also affect the cost. Examples are: The physiology of an E. coli ethanol resistant mu- type of fermenter used; tant has been characterized similarly .14 This strain nonsubstrate nutrients; had pleiotropic growth defects including abnormal strain stability; cell division and morphology. It also had an altered cell separations for byproducts, recycle, or eth- lac permease that was not due to a mutation in the Y anol recovery (i.e., increased size for recovery); gene. It was concluded that altered membrane com- operating conditions, i.e., higher growth tem- position was responsible for this abnormal behavior. peratures for yeast and mesophilic bacteria; and More recently, ethanol tolerant mutants have been range and efficiency of substrate utilization (i.e., isolated from C. thermocellum. 15 Indirect evidence complete utilization of all sugars). lead to the conclusion that strain S-4 was defective in More detailed examples are: hydrogenate, since this strain produced lower ● Type of fermenter. —If the organism, whether it amounts of acetic acid.l6 A different ethanol resistant be a yeast or a bacterium, can be made to grow isolate of the same bacterium, strain C9, proved to under conditions of pH, ethanol concentration, tem- have a lower activation energy for growth than the perature, etc., that preclude contamination, inexpen- wild type, a property that has been related to mem- sive lined basins can be used instead of tanks, since brane composition. steam sterilization of the fermenter is not required. There are three categories of changes that could In this case, some operating and capital costs asso- influence the fermentation process: ciated with sterilization are avoided as well. 1. Manipulate the existing controls on metabolism. A type of continuous beer fermenter requires Consider an example. In many organisms the growth in the form of fast-settling pellets. In other fermenters, fast-settling particles (such as mycelia)

IZI), p. (;l~rk and J, P, Beard, “Aitered phospholipid Composition in Mutants present problems that are best avoided by agglom- of’ Escherichia Co/i %msitive or Resistant to organic Solvents, ” J. Gen. eration of the cell mass. This type of control over the Mirrobio/. 1 13:267-274, 1979. IJA. ohta and I. Shibuva, “Membrane Phospho]ipid Synthesis and Pheno- growth form of micro-organisms is amenable to t,vpic (correlation of an ~. Co/ipss Mutant,” J. Bacferio/. 132:434443, 1977. genetic manipulations. 14L, ,,~, ~,l.ied ~lld A, N~\,i(:k, “organic Soh,ents as Probes for the structure ● Nonsubstrate medium costs. —In addition to the iind k’unrtion of the Bacterial Membrane: Effects of Ethanol on the Wild Type and as F,thanol Resistant hfutant of’ Fscherichia Co/i, ” J. Bacteriol. carbon-energy substrate and water, growing cells 114:239-248, 1973 must be supplied with other nutrients. Some orga- ‘%. D. Wang, ‘production of P;thanol From (;ellulose by [,loslridiurn Ther. nisms can make all of their biochemical from quite mocellum, ” M.S. Thesis, Department of Nutrition and Food Science, ,Massa. chusetts Institute of Technology, ]979. simple sources of nitrogen, phosphorus, sulfur, Idbid. magnesium and trace metals. Others require more 302 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals complex molecules, ready-made, such as amino acids systems, and by adding protective systems. This is and vitamins. not at all a near-term possibility. The more cheaply these nutrient needs can be ● Range and efficiency of substrate utilization.-A provided, the better. Whenever an organism can be single-step conversion of a substrate to ethanol is given genes from another source by applied biotech- highly desirable. This often requires that the ethanol nology techniques, there is a possibility that complex fermenting organism possess a degradation capabili- nutrient requirements can be obviated. However, ty it does not have. this requires that all the genes in a given pathway be As an example, consider ligno-cellulose. It consists located in the source and be made to function in the of hexosans, pentosans, and lignin, All of these com- new microbes. The feasibility of this is uncertain, but ponents should be used. Assume that one cellulase- solutions would decrease the cost of producing etha- producing candidate does not use pentoses, while a nol with yeast as well as clostridia. related noncellulase producing organism does, this is ● Stain stability. —Many of the suggested ethanol exactly the situation with clostridia. If the second processes propose to employ continuous culture. organism can be given the cellulase genes of the first, Although this offers several advantages over batch a microbe better-suited to direct conversion could be culture, it is somewhat vulnerable to deleterious mu- created. The pace at which such a manipulation tations of the microbe used, particularly if the mi- could be developed cannot be predicted with con- crobe has been extensively altered in ways that make fidence, although this is not necessarily a long-term it less competitive. prospect. These deleterious genetic changes are almost en- Another obvious area that merits attention is the tirely catalysed by biological systems in the microbe. enhancement of cellulase activity. Classical genetic Alteration of these systems, so that the frequency of manipulations, employing mutation and selection or unwanted genetic changes is decreased, could great- screening, should result in micro-organisms better ly extend the period of operation that is possible equipped to degrade cellulose. E.g, it should be possi- before having to shut down and restart the fermen- ble to isolate strains that are deregulated in cellulase tation. So far, this is a possibility only in microbes production (hyperproducers) as well as those in that have a highly developed genetics. It may be that which the cellulase is not subject to product inhibi- strain stabilization of this sort would not be possible tion. In addition, it is tempting to think about the in other microbes until their genetics are highly de- possibilities of amplifying cellulase genes by means veloped. of DNA technology and cloning. However, this latter It is also possible to design strategies using current approach must await further understanding of the strain development techniques that might lead to biochemistry and genetics of the cellulase system as genetically stable strains, but these are unproven. well as the development of the appropriate genetic ● Cell separations. —Many fermentation schemes systems in cellulolytic micro-organisms, incorporate cell recycle to boost productivity. This requires that cells be separated from effluent broth. Utilization offermentation byproducts Others need to separate cells from other residue as a byproduct. In addition, some of the low-energy alter- Presently for each gallon of ethanol produced, ap- natives to distillation, such as adsorption, could re- proximately 14 liters of stillage is formed. 17 If ethanol quire separation of the cells from the broth prior to is mixed with gasoline to make gasohol (10 percent ethanol recovery. ethanol), the total stillage produced annually in the In these cases, microbes that can be made to floc- United States would be in the billions of liters. Surely culate and redisperse, or that can be made to rever- a problem of this magnitude deserves serious atten- sibly change their morphology would allow cheap tion. The utilization of stillage or fermentation by- gravity separations (settling or flotation). products could be greatly improved by genetic ● Operating conditions.—An increase in the means in several ways, In actuality, only a rational temperature an organism will tolerate is advanta- long-range genetic approach can increase the value geous for heat removal and in situ ethanol removal of such a fermentation byproduct. Value can be in- schemes. The feasibility of accomplishing this is creased in two main ways. The first is to increase the uncertain. nutritive value of the fermentation byproduct fol- The extreme of productivity improvement via cell lowed by developing economical processing technol- recycle is an immobilized cell reactor, It is conceivable that cells could be made less prone to i~w. E. Tyner, “The Potentjal of Obtaining Energy From Agriculture, ~W- degradation under the conditions of immobilization, posium on Biotechnology The Energy Production and Conservation, Gatlin- by modifying sensitive components and degradation berg, Term., 1979. Appendix l-D—The Impact of Genetics on Ethanol—A Case Study ● 303

ogies that stabilize and preserve nutritive value. The quality. Most of these types of studies remain to be second approach is to increase the functionality of done. However, the potential for innovative applica- the byproducts so that more useful products can be tions is great, but such applications may not result developed. because of the current lack of any Government agen- For this one can envisage clever and novel ways to cy that has a sound program for funding biotech- utilize mutants to increase the value in a manner nology research. similar to those described. 18 l9 20 Ethanol production is not compatible with producing a valuable byprod- Recommendations and areas in which uct. E.g., a filamentous yeast may be useful for direct applied genetics should have an impact texturization or fortification of an animal food but production of ethanol may not be suitable with such There has been little published research done in an organism. A possible solution to this type of con- the United States on the genetic improvement of flict involves the development and engineering of ethanol production processes with bacteria such as two-stage fermentation processes. In the first stage, Zymomonas and clostridia, and only limited studies ethanol producing organisms are propagated under with yeast. In light of previous discussion, the follow- optimal economic conditions for ethanol production. ing points have been identified as being the most im- After the production phase is over, the organisms portant and relevant in the application of genetics are then transferred to a second-stage reactor, for improving ethanol-producing processes: where desirable phenotypic properties are then ex- ● improvements on ethanol yield; pressed. Signals for expression of phenotypic prop- ● increased ethanol tolerance to achieve higher erties can be extrinsic environmental parameters, final ethanol concentrations in the fermentation such as temperature, or levels of oxygen or carbon broth; dioxide, or intrinsic parameters, such as specific ● increased rates of ethanol production; nutrient requirements. ● elimination of unwanted products of anaerobic Thus the large-scale utilization of fermentation catabolism, that is, direction of catabolism byproducts as feed or other materials will then towards ethanol; become more valuable when genetic engineering can ● enhanced cellulolytic and/or saccharolytic capa- decrease processing costs and increase product bilities to improve rates of conversion of cellulose and/or starch to fermentable sugars; ● laA. J, Sinskey, J. Boudrant, C. Lee, J. DeAngelo, Y. Miyasaka, (;. Rha, and S. incorporation of pentose catabolic capabilities R. Tannenbaum, “Applications of Temperature-Sensitive Mutants for Single- into ethanol producers; Cell Protein Production,” in Proceedings of U.S./U.S.S.R. Conference on Mech- ● anisms and Kinetics of Uptake and Utilization of substrates in Processes for development of strains capable of hydrolyzing the Production of Substances by Microbiological Means, Moscow-Pushchino, cellulose and starch as well as of producing p. 362, June 4-11, 1977. PB. 283-330-T. ethanol from pentoses and hexoses; 19J, Boudrant, J. DeAngelo, A. J. Sinskey, and S. R. Tannenbaum, “process ● Characteristics of Cell Lysis Mutants of Saccharomyces cerviciae,” Biotech. improved temperature stability of micro-orga- Bioeng. 21:659, 1979. nisms and/or their enzymes; and ZOY, Mivasaka, A. J. Sinskey, J. DeAngelo, and C. Rha, “Characterization of ● improved harvesting properties of cellular bio- a Morphological Mutant of saccharomyces cervisiae for Single-Cell Protein Production,” ./. Food Science 45:558 ;563, 1980. mass produced during fermentation. Appendix II-A A Case Study of Wheat

Wheat is a major food staple in the diet of a large as cold, wheat, wind dessication, and excessive mois- percentage of the world’s population. Wheat grain in ture, and inherent yield capacity in the absence of the United States is used almost exclusively for significant production limitations. human consumption, although temporary localized The quality of wheat’s end products has been oversupply may result in some wheat feeding to live- improved significantly through breeding. Varieties stock. have been tailored to meet the demands of various Attempts to improve wheat plant populations by industries. The bread bakeries needed a higher pro- selection began several thousand years ago. The de- tein and more gluten strength to make a lighter, sirable attributes selected included the ability to larger loaf, while the cookie producer needed a low- withstand severe environmental stresses such as protein flour with desirable dough-spreading prop- heat, cold, and drought and the stability of the seed erties. head (which tends to disarticulate in wild forms). Wheat seeds moved from country to country Wheat productivity and management along with explorers and colonists. New varieties played major roles in the establishment of many The pattern of wheat productivity (yield per acre) productive wheat cultures—e.g., the Mennonite set- in developed countries is remarkably similar. When tlers introduced hard red winter (Turkey Red) wheat yields are plotted over the centuries, there is a long into the Kansas area from Russia in the late 19th period of barely perceptible increases in yield, from century. And two private breeders—E. G. Clark of the time of first records of production to the end of Sedgenick, Kans., and Danne of Elreno, Okla.–de- the first third of this century (the period of 1925-35). veloped varieties that set new levels of productivity Since around 1935, yield has increased sharply. Re- and straw strength in hard winter wheats which cent data suggest that yield increases may be leveling were sought by millers for their excellent flour off. Why increases have been so substantial after recovery. generations of little success, is a complex question in- Breeding programs expanded during the first half volving genetic resources, economic development, of the 20th century. At first, the U.S. Department of social interaction, and adoption of mechanical and Agriculture (USDA) played a lead role; hut the biological innovations. emergence of the Land Grant System and the estab- Until recently, the U.S. commercial seed compa- lishment of the State experiment station concept nies, with one or two exceptions, have not been in- prompted individual States to launch breeding pro- terested in wheat breeding programs as a profitmak- grams designed to address the particular production ing venture. Since wheat has a perfect flower and problems faced by farmers within their respective can fertilize itself, the farmer can purchase seed of a boundaries. new variety and reproduce it from generation to As the State experiment stations began to assume generation. However, the discovery of cytoplasmic more responsibility, USDA programs and personnel male sterility and nuclear restorer genes has stim- began to concentrate in central locations to assemble ulated industry interest in the possibility of devel- the optimal number of personnel for the greatest in- oping hybrid wheat. The farmer would purchase the teraction and productive output. If the present trend hybrid seed each year; the inbred lines used to make continues, there will be virtually no USDA scientists the hybrid would be the exclusive property of the engaged in actual wheat breeding. Instead they will originating company. Although progress has been have assumed the roles of basic researchers and re- good, problems exist with the sterility and restorer gional coordinators supplying information to the systems, the ability to produce adequate amounts of public and private breeders. hybrid seed, and the identification of economic levels Disease and insect resistance have been the pri- of hybrid vigor. The next 5 years should reveal the mary breeding goals of many programs. The dramat- potential for success in hybrid wheat. ic losses associated with severe pest problems have Several milestones of progress have been set in focused the attention of producers, researchers, and wheat. Yield has risen dramatically. Genetic protec- legislators on these areas of need. Other traditional tion against pests and other hazards has been a ma- breeding objectives have included improved use jor contributor to increased yields. In addition, re- properties, tolerance to environmental stresses such cent advances using semidwarf genes have been as-

304 Appendix II-A—A Case Study of Wheat . 305

sociated with significant yield improvement. The opment are producing a new array of complex prob- shorter, stiffer stems of the semidwarf plants allow lems. maximization of resources without yield reductions. Improvement in the inherent yield components of Genetic vulnerability in wheat stems per unit area, kernels per stem, and kernel weight has also contributed extensively to yield im- Genetic vulnerability is defined as a high degree of provement. genetic uniformity in a crop grown over a wide acre- The use of applied genetics in wheat improvement age. Wheat, which is produced on about 62 million occurs in close harmony with total wheat manage- acres annually in the United States, has a relatively ment systems. The farmer must integrate a huge as- high level of uniformity and genetic vulnuerability. sortment of alternatives in each decision—e.g., an in- In 1974,102 hard red winter wheat varieties were dividual producer may be deciding on a nitrogen grown on 36.6 million acres, with four varieties oc- program. If the farm is irrigated, the producer cupying 40 percent of the acreage. Hard red spring selects nitrogen amounts and application timing wheat varieties totaled 80 percent on 14.7 million based on soil tests, intended crop and variety, the acres, with three varieties occupying 52 percent of end use of that crop, and watering schedules. [f the the acreage. Similar situations occurred with other farm is rainfed, the producer takes into account soil classes of wheat. Plant pests, including diseases and tests, crop considerations, and rainfall probabilities. insects, have periodically caused moderate to servere In both cases product prices at the time of sale wheat crop losses in years favorable to the develop- must be predicted since they govern potential gross ment of strains capable of attacking current forms of return, which in turn affects the costs of maintaining resistance. a profit margin. Genetic interaction in this system is Incorporating genetic resistance to pests has tradi- intricate. The farmer must first select the variety tionally been the responsibility of public breeders. most likely to produce at the maximum economic Wheat is a self-fertilized plant that can be faithfully level. For irrigated land, it may be a short high- reproduced from generation to generation. Private yielding semidwarf either for the cookie trade or the industry has been reluctant to invest R&D money in export market. The farmer knows that part of the improvements since the farmer, following the initial value of his product is dependent on low protein. seed purchase, can reproduce the crop without re- However, inappropriately high levels of nitrogen, turning to the seed company. Thus, public breeders which greatly improve yield, will also raise the pro- have been the main source of new varieties and have tein of the crop beyond acceptable levels. If the ex- had the responsibility of delivering genetic import market is strong and the total U.S. supply re- provements to the producer. Wheat breeding produced, the higher protein may be of little economic grams are generally designed to respond to State pro- consequence. duction needs. Goals and objectives are established In the case of the dryland farmer, the variety by technical advisory groups that include breeders selected may be taller with lower yield potential but and scientists, growers, use industry representa- with much better levels of adaptation and tolerance tives, and extension workers. to adverse environments. It may be designed for the Genetic variability is available to the breeder from bread industry or the export market. Part of the naturally occurring sources and artificially induced value is related to high-protein content. Since mois- mutations. Naturally occurring variability has been ture conservation and use is critical, nitrogen ap- collected from native plant populations throughout plications and amounts must be selected so that the the world and is maintained in the World Wheat C ol- plants do not waste their moisture reserve. However, lection by the Science and Education Administration nitrogen applied too late may not receive enough of USDA located in Beltsville, Md. Currently, about rain to penetrate the soil and become available to the 37,000 accessions are contained in the collection. plants. If the plants “burn up” because of unwise Breeders use the collection as a reservoir from which water use early in the season, the seeds will be high to draw exotic genes needed to improve the value of in protein but low in yield. If inadequate nitrogen is their breeding programs. In addition to variability available, the crop will generally be low in protein. within wheat varieties, the breeders can use special The abbreviated protein story is but one of many genetic techniques to draw valuable genes from re- examples of farm management interaction with ap- lated species such as rye and various forage grasses. plied genetics in wheat production. Recent changes This approach, while time-consuming and costly, has in energy price and availability, environmental re- been used in a number of variety development pro- straints, marketing structures, and technology devel- grams. Mutations induced by artificial means have 306 ● lmpacts of Applied Genetics-Micro-Organisms, Plants, and Animals not been used extensively by the breeders, since ly evaluated, characterized, and documented, forc- desired mutations without detrimental effects are ing breeders to spend time and resources carrying very difficult to obtain. Enough natural genetic out their own evaluation work. The committee has variability seems to exist to satisfy needs in the proposed a standard set of descriptors for all acces- foreseeable future. sions in the collection, as well as an information The National Wheat Improvement Committee has management system to efficiently bring the informa- stated that the World Wheat Collection is inadequate- tion to the breeders. Appendix II-B Genetics and the Forest Products Industry Case Study

The Weyerhaeuser Co. yields and shorter harvest cycles, as well as rapid production of tree stands for seed production. The Weyerhaeuser CO., which has its main head- The immediate results of 10 years of research are quarters in Centralia, Wash., is the largest forest not overly impressive at first glance. To date, 3,000 products company in the United States. In 1970, tissue-cultured Douglas firs have been planted for Weyerhaeuser initiated a program to research the comparison analysis and research of handling tech- mass propagation of Douglas fir trees by tissue niques, transfer procedures, etc. culture. Douglas firs are the main species in many of The cost effectiveness of a tissue culture program the Nation’s forests, over $3.1 billion (or about 8.5 is determined by several factors, of which labor in- billion board feet) worth were harvested in 1979. tensity varies the most. The more streamlined the While they are normally propagated by seed in the system can be made, the fewer labor-requiring steps field, the classical development of improved seed that are needed—the less direct costs will be in- does not adequately satisfy the criteria of the rapid curred. Ideally, cells would be cultured in sterile con- availability of trees of superior quality. ditions and then planted for the direct embryogene- Specially selected clones have the potential to dou- sis of plantlets that are ready for the field. Steps that ble the productivity of forestlands; each year that involve cutting shoots and rooting them on another unimproved trees are planted is another year of media or repeated subculturing procedures are cost- “suboptimum” harvests 40 years from now. With the ly and cumbersome. The major problem affecting steadily increasing demand for forest products, cost so far is the difficulty of achieving high volume planting substantially improved trees as soon as pos- plant regeneration from the tissue cultures. Efficient sible is of great economic importance. systems with more successful regeneration will re- Weyerhaeuser’s tissue culture research began in duce the labor and materials involved in culturing 1974 with a project at the Institute of Paper Chem- and result ultimately in a lower cost per plant. istry to produce Douglas firs. The project was ex- In addition to problems of cost, Weyerhaeuser has panded with a contract for additional research at the run into the classic difficulty with woody species— Oregon Graduate Center. Although the intention was the inability to obtain required results from plants to propagate select strains of mature trees, the main more than 1 year old. In addition, the risk of induced focus of the program, in 1974 to 1978, was to devel- genetic variability increases with every subculture of op a basic, consistent system for propagation. From the tissues. The triggering techniques for effective 1978 to the present, Weyerhaeuser has been con- manipulation of mature versus embryonic and imma- ducting most of its applied research into Douglas firs ture tree tissues are not well understood, and un- at its own research facilities in Centralia, Wash. Basic locking the Douglas fir system may well provide in- research is still being funded at the Institute of Paper sight into some basic physiological questions. Chemistry, which services the entire forest industry. Some commercial companies do not want to get While specific figures for the tissue culture systems deeply involved in basic research because it is ex- research have not been made available, the annual tremely expensive and time-consuming. However, it research and development budget at Weyerhaeuser has been up to the major forestry companies, such as specifically for biological work with forest species is Weyerhaeuser, to independently fund essentially on the order of $7 million to $8 million. ’ basic research into the biological triggers for organo- The project in mass propagation of Douglas fir by genesis and embryogenesis of Douglas fir. tissue culture was initiated to establish a reliable, By comparison, no other plant has been as intense- economic means for mass production of superior ly researched for mass propagation purposes and trees. The cloning of these trees could bring higher proved so unyielding. Among other things, this indicates that questions of basic plant cell physiology will have to be addressed before major break- 1 llRX M(’(kdlough, The \V(>}ffll-llat?lls(>l’ (k)., persomd (sonln~lllli(’ali(]il [Ma.v throughs can be expected. The goals of the Weyer- 1 !)80) with the I%inl Resourc[ls Institute in the working report, Commercia/

[ Isf.s Of planf I’issup (:1~/fure and Rjfenfial [mparf of Genetic Engineering on haeuser program are exacting and demand the re- l.’orf-.sl~y, prfy)iirfd lilldf?l’ (’olltl’il(’t 1[) () ”l’,\, 1980 finement of present techniques into a precise in-

307 308 ● Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

dustrial science. While it may seem that the invest- Elite trees are selected from wild stands for ment has been disproportional to the returns at this straightness of trunks, height, specific gravity of point, it must be remembered that they are the fore- wood, and proper branch drop (branches that drop runners of a new technology, both in terms of work- without tearing the stem). There are no major pests ing with mature tree tissues of an especially intricate in redwoods, so pest and disease resistance have not species and in terms of imposing stringent industrial been a concern. Two methods of selection are used. standards on a mass biological production system. Clones of special trees are produced by rooting the uppermost branches of the tree, a process that takes Simpson Timber Co. up to 1 year. Although the rooting percentage may be as low as 10 percent, this method has the advan- The Simpson Timber Co., whose central headquar- tage of producing mature cloned plants that can con- ters are in Seattle, Wash., is a large producer of red- tinue to produce flowers and seed. Simpson is using wood and other forest products, and has been in- roughly 200 elite trees for these clones. volved over the past 5 years in a program to develop Mite trees can also provide clones through tissue a mass production system for the coast redwoods cultures of their needles, a process that is less time- through tissue culture. Approximately $250,000 has consuming but which produces seed very slowly been invested in research performed at the Universi- because of the time involved in maturation. Simpson ty of” California, Irvine, by Dr. Ernest Ball, a recog- Timber Co. has planted out 2,500 tissue cultured red- nized authority in the field of tissue-cultured redwoods for field comparisons with seedling material. woods. z The results so far have been encouraging, but it may Coastal redwoods are normally a field-seeded crop take another 10 to 15 years before definite conclu- and have a production cycle of around 50 years. The sions can be drawn. The major factors being ana- major reason for consideration of tissue culture over lyzed are field growth rates and outplanting survival seed is the greater speed with which superior trees percentages. Clones of elite varieties will also have to might be developed through tissue culture as com- be compared to the parent trees for the traits origi- pared to using seed stock. Simpson Timber Co., nally selected for, such as wood quality. Since the which has been involved in a controlled breeding operation] cost of tissue-cultured plantlets is about program along conventional lines as well, and is ap- twice that of seedlings, the quality of tissue cultured proaching the creation of homozygous strains. Since plants must be markedly superior if the program is a sequoia seedling does not reach sexual maturity to be cost effective, before it is 15 to 20 years old, and since about 10 Dr. Ball is confident that the tissue culture system generations are normally required to produce a true which has been developed for the rapid multiplica- homozygous strain,3 the classical process is time-con. tion of elite redwood trees is ready for: implementa- suming and contains no guarantees that the end tion at a commercial production facility.4 Simpson products will be better than the clones selected Timber (:0. is planning the construction of a tissue through tissue culture. culture lab at their California headquarters within the next 2 years, The pilot plant is expected to cost $250,000 and produce upwards of 200,000 plantlets in its first year of production. As mass production 21,, ]$1)/’.s1 Ihll, I It)iwr.silcv 01” ( ‘illil’ol’l)id, Iri’int’, J)(~l’Sollill (’ot)]tl)l]t)i(’ illioll” techniques are perfected, the company plans to ex- [kl;I}r I !)80) u ill] tht> I’lilllt RW()(II”WS Illslilulr in lhr w(wking I’op(wl, (;f)r?l. IIllvv’l;ll 1 !$t’.v ()/” /%1/)/ /$ss11(’ ( ‘1///1/17’ ;t!lfl Pf)lw)li;d /11)/); )(’1 ()/’ (;!’l)/’/; (” \,;!l,qmfv’J’- pand the facility to a production capacity of over 1 Ilt,< ()/1 /4’fJf’f’.Sff’l’o 1)l’(>j)ill’(*(l 1111( 1(’1’ (,0111 I’il{.l l{) ( )“1’ \ , I !IMII million plantlets per years

!. I< II III’S l{,l(l(4i(li, Sinll)s(nl 1 11111 )1’r ( “II , 1)l>l’~llllill (’l)lllllltlllll’ illi(lll (\ld\ I! K+()) ,,1111 III(, 1}1,1111 1{(.wNII.(.I.h IIISIIIIII(. ill Illt, 11 (M’hIll~ I’t’l)llt’l. ( ‘f Jlllf IIf’l’f”iil/ 1 ‘.s1’s 11/” /41!1( I’isslw ( “Idt::rc ;111[1 /’01( ”111(;11 Ifllll.w’l (If” (;twrtic’ I,;tl,qittf,f’f .itt,< (m Il}illl, 01) (,11 1<”~)l’(’,$lt’t’, 1)1’l,l)ill’l’(l 1111[11,1’ (1)1111 ’il(.l 10 ( )’I \, I !)80 “’l{ml(,lill~, of) (,il Appendix II-C Animal Fertilization Technologies

Sperm storage estrus detection, quality of semen, timing of insemination, and semen handling. DEFINITION DISADVANTAGES The freezing of semen to – 196° C, storage for an indefinite time, followed by thawing and successful 1. With increased herd size, estrus detection has insemination. become a major problem. 2. Inexperienced dairymen are buying semen and in- STATE OF THE ART seminating their own cows, resulting in lowered fertility and no feedback on semen fertility. Conception rates at first insemination with frozen sperm average between 30 to 65 percent for most ADVANTAGES species. This technology is not a key to the success of 1. Widespread use of genetically superior sires. artificial insemination (AI), but because of the con- 2. Services of proven sires at a lower cost. venience it is now an essential ingredient. Current 3. Elimination of cost and danger of keeping bulls on operational procedures are adequate for the dairy in- the farm. dustry. 4. Control of certain diseases. S. Use of other breeding techniques including cross- ADVANTAGES breeding. 1. Greater use of selected bulls as AI studs. 6. Continued use of valuable sire after his death. 2. Elimination of the need to maintain expensive and dangerous bulls on dairy farms. FUTURE 3. Sperm can be tested for disease and treated for Greater use of AI in beef cattle will depend on the venereally transmitted diseases. availability of successful and inexpensive estrus syn- 4. Ease of transport and therefore of increasing po- chronization technology, on relaxed restrictions of tential offspring. the various breed associations, and on accurate progeny records, FUTURE Little change is anticipated in semen processing. Estrus synchronization Freeze-dried semen is unlikely to be successful enough to use. Sperm banking can be expected to in- DEFINITION crease, especially on AI studs. Banking provides Estrus (“heat”), is the period during which the cheap storage while bulls (slaughtered) are being female will allow the male to mate her. This sexual progeny tested, and insurance against loss of bulls behavior is subtle and varies widely among individ- through natural causes. For preservation of semen uals. Thus the synchronization of estrus in a herd, from bulls of less populous breeds, banking can be using various drug treatments, greatly enhances AI - completed in about a year, after which the-bull can and other reproduction programs. be slaughtered. STATE OF THE ART Effective methods for synchronization of estrus Artificial insemination periods for large numbers of animals have been available for more than two decades, and several ap- DEFINITION proaches are now available which result in normal Manual placing of sperm into the uterus. fertility. Several schemes involve use of prosta-

glandin F2 (PGF2) for the cow and ewe. However, FDA STATE OF THE ART approves usage only for controlled breeding in beef Highly developed for most species. Representative cows and heifers, nonlactating dairy heifers, and in use rates in the United States are: dairy cattle, 60 per- mares. cent; beef cattle, 5 percent; turkeys, 100 percent. The major limitation to the use of AI is the low na- ADVANTAGES tional average conception rate at first service, 1. Time a heifer’s entry into a milking stream. around 50 percent. The success or failure of AI is 2. Increase productivity by breeding heifers earlier determined by a multiplicity of factors including in life. 309

76-565 0 - 81 - 21 310 . Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

3. Ability to breed large numbers of cattle over a bandry, and much current effort is directed towards shorter calving interval. developing and testing a commercial procedure. 4. Increase use of AI, especially in beef cattle, sheep, and swine. Embryo recovery FUTURE DEFINITION Estrus cycle regulation should allow selected sires to be more widely used to improve important traits The collection of the fertilized ova from the in beef cattle. It should also gain widespread and oviducts or uteri. Collection of embryos is a rapid acceptance among dairymen as well. - necessary step for embryo transfer or storage, and for many experiments in reproductive biology. Both surgical and nonsurgical methods are used. Superovulation STATE OF THE ART DEFINITION Surgical. -Methods are available for recovering Superovulation is the hormonal stimulation of 40 to 80 percent of ovulations from cattle, sheep, multiple ovarian follicles resulting in release from goats, swine, and horses. The development of adhe- the ovary of a larger number of oocytes (ova) than sions and scar tissue following surgery limits these normal. techniques. Surgical recovery is the only method for sheep, goats, and pigs. It is presently practiced STATE OF THE ART almost exclusively when a suspected pathology of the Superovulation with implantation into surrogate oviducts renders an individual subfertile, or when mothers increases the number of offspring, usually embryos must be recovered before the individual from highly selected dams. Adequate procedures are reaches puberty. presently available for superovulation of laboratory Nonsurgical.- Non-surgical embryo recovery and domestic animal species, except the horse. The techniques are preferred for the cow and horse. drugs used to induce superovulation are the go- Fifty to eighty percent of cow ovulations can be nadotropins, pregnant mare’s serum gonadotropin recovered, and 40 to 90 percent of the operations on (PMSG) and follicle stimulating hormone (FSH), in horses to recover the single ovulation are successful. some instances followed by other treatments to stim- ADVANTAGES ulate ovum maturation and ovulation. Superovulated ova result in normal offspring with the same success 1. Nonsurgical embryo transfer can be performed an rates as achieved with normally ovulated ova. unlimited number of times. 2. Requirements for equipment, personnel, and time DISADVANTAGES are low in nonsurgical recovery. This is especially 1. Greatest drawback is that degree of success can- important in milk cattle: since the nonsurgical pro- not be predicted for an individual animal. cedure is performed on the farm, milk production 2. Batches of hormones for ovulation treatment vary is not interrupted. widely in quality. 3. A single embryo can be obtained between super- 3. PMSG is scarce, and has been declared a drug by ovulation treatments. the Food and Drug Administration (FDA). Thus, 4. Embryos can be obtained from a young heifer most use of PMSG is now illegal. before it reaches puberty. 4. There is insufficient data to judge the effect of 5. The technology is especially important for re- repeated superovulation. search, e.g., in efforts to produce identical twins, embryo biopsies for sex determination, etc. FUTURE FUTURE Methods for superovulation will improve consistency of results. Additional understanding of basic Methods of collecting embryos have not changed physiological mechanisms will facilitate such efforts. appreciably since about 1976, nor are significant ad- New work in superovulatory technology involves ac- vances predicted for the future. tive immunization against adrostenedione (a hormone involved in regulation of follicular develop- Embryo transfer ment). This treatment prevents atresia and reliably increases the frequency of multiple ovulations. The DEFINITION technology has definite commercial potential for cat- Implantation of an embryo into the oviduct or tle husbandry and limited potential for sheep hus- uterus. Appendix II-C—Anim81 Fertilization Technologies ● 311

STATE OF THE ART in progeny testing of females, obtaining twins in beef Surgical.—Pregnancy rates of 50 to 75 percent cows, obtaining progeny from prepubertal females, are achievable in cows, sheep, goats, pigs, and and in combination with in vitro fertilization and a horses. Surgical transfer is the only practical method variety of manipulative treatments (e.g., production in sheep, goats, and pigs, and is the predominant of identical twins, selfing, genetic engineering, etc.) method for cows and horses. A number of factors determine the success of surgical transfer: age and Embryo storage quality of embryos, site of transfer, degree of syn- chrony between estrous cycles of the donor and re- DEFINITION cipients, number of embryos transferred, in vitro Maintenance of embryos for several hours or days culture conditions, skill of personnel, and manage- (short-term) or for an indefinite length of time (freez- ment techniques. The 50- to 60-percent success rate ing). in cattle compares with AI success rates at first service. (Pregnancy rates should not be confused with STATE OF THE ART survival rates, which may be much lower.) Short-term.-The requirement for embryos Nonsurgical. —This method is an adaptation of from farm animal species has not been defined, AI. Reported success rates are much lower than although adequate culture systems for the short in- those with surgical transfer. Nonsurgical transfer is terval between recovery and transfer have been not used in sheep, goats, or pigs. developed by trial and error. Whereas the important ADVANTAGES parameters of culture systems have been identified 1. Obtaining offspring from females unable to sup- (e.g., temperature, pH, etc.), optimal conditions have port pregnancy, not been determined. Cow embryos may be stored 2. Obtaining more offspring from valuable females. for three days in the ligated oviduct of the rabbit. 3. With a homozygous donor, undesirable recessive Long-term (freezing).-No completely adequate traits among animals used for AI can be rapidly protocol exists for freezing embryos of farm species. detected. One-third to two-thirds of embryos are killed using 4. Introducing new genes into specific pathogen-free present methods. Pregnancy rates of 32 to 50 per- swine herds. cent for cattle, sheep, and goats have been reported 5. Coupled with short- or long-term embryo storage, after freezing. No successful freezing of swine or transportation of animals as embryos. horse embryos followed by development to term has 6. Increasing the population base of rare or endan- been reported. Despite disadvantages (one-half of gered breeds of animals by use of closely related embryos are often killed) advantages are such that in breeds for recipients. some’ situations embryo freezing, and embryo sell- 7. Separation of embryonic and maternal influences ing, are already profitable. in research. ADVANTAGES DISADVANTAGES 1. Amplification of advantages of embryo transfer. 1. Personnel requirements in surgical transfer ac- 2. Elimination of requirements for large recipient count for a large share of high costs and thus limit herds when embryo transfer is being used. applicability in animal agriculture. 3. Reduction of costs in animal transport. 2. Provision of suitable recipients is the greatest 4. Control of genetic drift in animals over prolonged single cost in embryo transfer. time intervals. FUTURE FUTURE Surgical transfers will remain the method of Anticipated development of embryo culture tech- choice for sheep, goats, and pigs in the foreseeable nology would be of significance in efforts toward in future. For cows and horses, however, nonsurgical vitro maturation of gametes, in vitro fertilization, sex methods will be increasingly used rather than sur- determination, cloning, and genetic engineering, all gical techniques (and this will be apparent) within of which involve prolonged manipulation of gametes the next year or two. It is likely that half of the com- and embryos outside of the reproductive tract. mercial transfer pregnancies in cattle in North Amer- As freezing rates improve, nearly all embryos re- ica in 1980 will be done nonsurgically, even if suc- covered from cattle in North America will be frozen. cess rates are only 60 to 80 percent of those obtain- Probably as many as half of the embryos will be able with surgical transfer. Among future appli- deep-frozen for 2 to 3 years. It is unlikely that suc- cations, a role for embryo transfer can be predicted cess rates will ever approach 90 percent of those 312 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals without freezing. However, 70- to 80-percent success DISADVANTAGE rates may be attainable within several years. It ap- The major disadvantage of twinning is intensive pears that embryos can be stored indefinitely with management necessary for periparturient complica- little deterioration. tions, unpredictable gestation periods, depressed lactation, etc. Sex selection FUTURE DEFINITION Technical feasibility for twinning, in conjunction Tests to determine the sex of the unborn or deter- with embryo transfer, management adjustments, mination of sex at fertilization by separating x- bear- and selection for good recipients, can be predicted. A ing from y-bearing sperm. reliable procedure for twinning in sheep can also be STATE OF THE ART expected. The technology would most likely be first used in Europe and Japan, where there are shortages Sexing of embryos.—Through karyotyping of calves to fatten for beef. nearly two-thirds of embryos can be sexed. Tech- niques using identification of the condensed X chromosomes are unreliable. A third method, identifica- In vitro fertilization tion of sex-specific gene products, is under development. DEFINITION Sexing of sperm.—A 100-percent method has The union of egg and sperm outside the reproduc- not been achieved in any mammalian species; and no tive tract. For some species, the technology includes standard protocol for farm species exists. successful development of the embryo to gestation FUTURE and birth. Before this technology can be applied commercially, it must be simple, fast, inexpensive, reliable, and STATE OF THE ART nonharmful for embryos. Such techniques could un- In vitro fertilization has been accomplished in doubtedly be developed. There would be numerous several laboratory animal species, including the rab- medical and experimental applications. bit, mouse, rat, hamster, and guinea pig and nine There is much interest in research in this area other mammalian nonlaboratory species, including because of its use in understanding male fertility man, cat, dog, pig, sheep, and cow. However, normal with AI in humans, and in enhancing sperm survival development following in vitro fertilization and em- after frozen storage. bryo transfer has only been accomplished in the rabbit, mouse, rat, and human. Consistent and repeat- Twinning able success with in vitro fertilization in farm species has not yet been accomplished. DEFINITION None of the cases of reported success of in vitro Artificial production of twins, either using embryo fertilization, embryo transfer, and normal develop- transfer or hormone treatments, ment in man is well documented. Most of the in vitro fertilization work to date has STATE OF THE ART concentrated on the development of a research tool Currently, embryo transfer is the most effective so that the physiological and biochemical events in method for inducing twin pregnancies in cattle, fertilization and early development could be better resulting in pregnancy rates of between 67 to 91 per- understood. More practical application of in vitro cent, of which 27 to 75 percent deliver twins. Other fertilization techniques would include: methods include transferring one embryo into a cow 1. a means for assessing the fertility of ovum which has been artificially inseminated, and hor- and/or sperm; monal induction of twinning, which is a modification 2. a means to overcome female infertility with em- of superovulation. This latter method is not reliable. bryo transfer into a recipient animal; and 3. when coupled with ovum and/or embryo stor- ADVANTAGE age and transfer, a means to facilitate combina- The advantage of twinning in nonlitter-bearing tion of selected ova with selected sperm for pro- species is the improved feed conversion ratio of production of individuals with predicted character- ducing the extra offspring. istics at an appropriate time. Appendix /l-C—Animal Fertilization Technologies ● 313

FUTURE Cloning: nuclear transplantation Rapid progress in research is anticipated and many of the potential applications of in vitro fertiliza- DEFINITION tion to animal breeding should become practical The production of genetically identical mammals within the next 10 to 20 years. With further develop- by inserting the nucleus of one cell into another, ment of in vitro fertilization methodology, along with before or after destroying the original genetic com- storage of unfertilized oocytes (gamete banking), fer- plement. These occur by separation of embryos or tilization of desired crosses should become possible. parts of embryos early in development but well after In the more distant future, genetic engineering and fertilization has occurred. sperm sexing along with in vitro fertilization may STATE OF THE ART become possible. Experimentalists have found in certain amphibia Parthenogenesis that transplantation of a nucleus from a body cell of an embryonic (tadpole) stage into a zygote following DEFINITION destruction or removal of the normal nucleus can lead to development of a sexually mature frog. The initiation of development in the absense of sperm. FUTURE STATE OF THE ART The ideal technique for making genetic copies of any given outstanding adult mammal would involve Parthenogenesis has not been satisfactorily dem- inserting somatic (body) cell nuclei into ova, which onstrated or described for mammalian species. The may take years of work to perfect if indeed it is possi- best available information leads to the conclusion ble, There is some evidence that adult body cells are that maintenance of parthenogenetic development to irreversibly differentiated. produce normal offspring in mammals approximates How identical will clones be? They can be ex- impossibility. pected to be fairly similar in appearance. They would be less similar than identical twins, however, which Cloning: production of identical twins share ooplasm and uterine and neonatal environments. Furthermore, certain components are inher- DEFINITION ited exclusively from the mother, e.g., the mitochon- The production, using a variety of methods, of drial genome and perhaps the genome of centrioles. genetically identical individuals. The random inactivation of one or the other of the X STATE OF THE ART chromosomes may also limit similarities. Other differences among clones would result from the pre- There are several ways to obtain genetically iden- natal environment: in litter-bearing species even tical livestock. The natural way is identical twins, uterine position can affect offspring. In single-bear- although these are rare in species other than cattle ing species the maternal effect may be pronounced. and primates. Both natural and laboratory methods Environmental differences in later life may greatly depend on the fact that the blastomeres of early em- affect certain traits, even if those traits have a strong bryos are totipotent (i.e., each cell can develop into a genetic component. complete individual if separated from the others.) Serious technical barriers must be overcome For practical purposes, highly inbred lines of some before realistic speculation of possible advantages in mammals are already considered genetically iden- animal production can be foreseen. tical; F1 crosses of these lines are also considered genetically identical and do not suffer from the depressive effect of inbreeding. cell fusion ADVANTAGE DEFINITION An advantage of identical twins is the experimen- The fusion of two mature sex cells or the fertiliza- tal control provided by one animal through which tion of one ovum with another. An analogous scheme two sets of environmental conditions can be com- for the male would be accomplished by microsurgi- pared for effects on certain end points, e.g., native v. cal removal of the female pronucleus and substitu- surrogate uterine environments for gestational de- tion of nuclei from two sperm. Combining sex cells velopment, nutrition on milk production, etc. from the same animal is called “selfing.” 314 . Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

STATE OF THE ART FUTURE Combination of ova has led to early development Practical applications of chimera technology to to the blastocyst stage in the mouse but no further livestock are not obvious at this stage of develop- development following transfer has been reported. ment. The main objective of this research is to pro- Initial success in experimentation with manipulation vide a genetic tool for better understanding of devel- of pronuclei has been reported. opment, and maternal-fetal interactions. FUTURE Recombinant DNA Cell fusion technology may someday prove useful for getting genetic material from a somatic cell into a DEFINITION fertilized l-cell embryo for the purpose of cloning. In The introduction of foreign DNA into the germ- conjunction with tissue culture technology the tech- plasm. nology would have a role in gene mapping of chromosomes for the cow and perhaps other species. STATE OF THE ART Combining ova of the same animal, selfing, The mechanics of changing the DNA molecules of would rapidly result in pure genetic (inbred) lines for farm animals directly have not yet been worked out. use as breeding stocks. The technique would also The plasmid methods used in bacteria may not be ap- lead to rapid identification of undesirable recessive plicable, traits which could be eliminated from the species. FUTURE Chimeras None of these techniques, no matter how great the potential, will be of any use in animal breeding until DEFINITION knowledge of genetics is greatly advanced. Before A chimera is an animal comprised of cell lines one can alter genes, they must be identified, from a variety of sources. They can be formed by Prior to exploitation of recombinant DNA technol- fusing two or more early embryos or by adding extra ogy in animal breeding, it is necessary to identify cells to blastocysts. gene loci on chromosomes, i.e., genetic mapping. Work toward this goal has only recently been initi- STATE OF THE ART ated and rapid progress cannot be anticipated. Multi- Live chimeras between two species of mouse have variate genetic determinants of characteristics of been produced. Such young have four parents in- economic importance are anticipated to be the rule. stead of two; hexaparental chimeras have also been produced. Appendix III-A History of the Recombinant DNA Debate

The history of the debate over the risks from stitute of Medicine> requesting the appointment of rDNA techniques and the Government’s response committees to study potential hazards to laboratory 4 may be divided into four phases. * Phase I covered workers and the public; and by a narrow majority the period from the first awareness of risks to to arrange for the letter to be published in the widely human health from experiments involving recombi- read journal, Science, to alert the broader scientific nant DNA (rDNA) in the summer of 1971 to the end community.5 of the Conference at the Asilomar Center in Feb- NAS appointed a committee of prominent scien- ruary 1975, which resulted in prototype guidelines tists involved in rDNA research. In July 1974, the covering the research, Phase 11 covered the period panel asked for a temporary worldwide moratorium from Asilomar through the development by the Na- on certain types of experiments, and called for an in- tional Institutes of Health (NIH) of the Guidelines of ternational conference on potential biohazards of June 1976. In this period, the public first became the research through a letter published in Science significantly involved in the debate and most, if not and its British counterpart, Nature.6 This letter also all, of the policy issues were clearly framed. Phase requested the Director of NIH to consider estab- 111, from mid-1976 through mid-1978, involved con- lishing an advisory committee to develop an experi- gressional consideration of the issues in an atmos- mental program to evaluate potential hazards and phere that went from almost imminent passage of establish guidelines for experimenters. legislation to the cessation of such efforts. Phase IV In response, the Director of NIH, after authoriza- covers the postlegislative period, when NIH and its tion by the Secretary of HEW, established the Recom- organizational parent, the Department of Health, binant DNA Molecule Program Advisory Committee Education, and Welfare (HEW) (now the Department (later renamed the Recombinant DNA Advisory Com- of Health and Human Services) undertook to develop mittee, RAC) on October 7, 1974, along the lines sug- satisfactory voluntary standards in areas over which gested by the NAS Committee. The Committee’s they had no legal authority and to accommodate charter described its purpose as:7 growing pressure for public involvement, while The goal of the Committee is to investigate the cur- avoiding a full regulatory role. rent state of knowledge and technology regarding Phase I began in the summer of 1971, when sev- DNA recombinant, their survival in nature, and eral scientists became concerned about the safety of transferability to other organisms; to recommend a proposed experiment to insert DNA from SV40 programs of research to assess the possibility of virus, a monkey tumor virus that also transforms hu- spread of specific DNA recombinant and the possible hazards to public health and to the environment; and man cells into tumor-like cells, into a type of bacteria to recommend guidelines on the basis of the research naturally found in the human intestine. After results. This Committee is a technical committee, estab- months of discussion, the scientist who had pro- lishedto look at a specific problem. (Emphasis added.) posed the experiment decided to defer it. Meanwhile, The international conference called for by the as rDNA techniques became more refined, debates NAS Committee letter was held at the Asilomar Con- about safety increased; at the June 1973 Gordon ference Center, Pacific Grove, Calif., in February Research Conference, safety issues were discussed. 1975. The organizing committee made it clear that its The participants voted: to send a letter to the Na- purpose was to focus on scientific issues rather than tional Academy of Sciences (NAS) and the National In- to become involved in considering ethical and moral questions. However, in one session the few lawyers “For a detailed history through 1977, see footnote 1. For a history and a discussion of the broader issues, see footnotes 2 and 3. ‘J. Swazey, J. Sorenson, and C. Wong, ‘(Risks and Benefits, 4Swazey, et al., op. cit., p. 1,023. Rights and Responsibilities: A Historv of the Recombinant DNA Re- ‘Letter from Maxine Singer and Dieter Soil to the National search Controversy,” Southern California Law Review SI:1019, Academy of Sciences (NAS) and the National Institute of Medicine, September 1978. reprinted in Science, vol. 181, 1973, p. 1114. ‘C. Grobstein, A Double Image of the Double Helig (San Fran- %etter from Paul Berg, et al. to the editor, reprinted in Science, cisco: W. H. Freeman Co., 1979). VO]. 185, 1974, p. 303. 3D. Jackson, and S. Stich (eds. ), The Recombinant DNA Debate The charter of the Recombinant DNA Molecule Program Ad- (Englewood Cliffs, N. J.: Prentice-Hall, Inc., 1979). visory Committee, Oct. 7, 1974.

315 316 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals invited confronted the scientists with some of these advised the Director of NIH, an agency whose mis- questions.’ The conference report concluded that al- sion was to foster biomedical research, not to stop or though a moratorium should continue on some ex- otherwise regulate it. These issues were brought out periments, most work involving rDNA could con- in a petition to NIH signed by 48 biologists in August tinue with appropriate safeguards in the form of 1975. Criticizing a proposed draft of the guidelines as physical and biological containment, setting substantially lower safety standards than In Phase II, the debate widened to encompass those accepted at Asilomar, the petition argued for broader social and ethical issues, such as the re- broader representation on RAC from other fields of lationship between scientific freedom of inquiry and scientific expertise and from the public-at-large. RAC the protection of society’s interests, in whatever itself had been sensitive to these limitations; in the manner those were defined. Such issues led natural- summer of 1975, an attempt was made to recruit ly to questions about who makes the decisions and nonscientists.l0 One nonscientist was added in the role of the public in that process. Finally, deci- January 1976, and another was added in August sionmaking mechanisms were developed. Issues 1976. raised and actions taken during this phase in many In December 1975, RAC submitted revised draft respects controlled the subsequent development of guidelines to the Director of NIH, Dr. Donald the Federal response to the debate, and created Fredrickson, Although they were stricter than those problems that continue to the present. At this stage, drafted at Asilomar, some criticized them as being participation in the debate went beyond the scien- “tailored to fit particular experiments that are al- tific community. ready on the drawing boards.”11 The consensus of Questions of ethics and public’ policy had been RAC, on the other hand, was that the guidelines were raised earlier, but they now received much wider at- excessively strict, but that it was necessary to be tention. On April 22, 1975, Sen. Edward M. Kennedy, overly cautious because of its limited expertise in Chairman of the Subcommittee on Health of the public health.l2 In any event, Dr. Fredrickson ar- Senate Committee on Labor and Public Welfare, held ranged for public hearings on the proposed guide- a half-day hearing on science policy issues arising lines at a 2-day meeting in February 1976 of the Ad- from rDNA research. In May 1975, a 2-day con- visory Committee to the Director, a diverse group of ference on “Ethical and Scientific Issues Posed by scientists, physicians, lawyers, philosophers) and Human Uses of Molecular Genetics” was held under others. A similarly diverse group of scientists and the joint sponsorship of the New York Academy of public interest advocates were invited to attend. Sciences and the Institute of Society, Ethics, and the Some modifications to the Guidelines proposed by Life Sciences. In addition to molecular biologists, par- Dr. Fredrickson as a result of that meeting were ticipants included lawyers, sociologists, psychiatrists, adopted and others were rejected by RAC in April and philosophers. 1976.13 The issue of public participation arose as decision- The final major issue arising during this period making mechanisms were developed. RAC was orig- concerned NIH’s lack of authority to set conditions inally composed of 12 members from “the fields of on research funded by other Federal agencies or by molecular biology, virology, genetics and microbiol- the private sector. In a June 2, 1976, meeting be- Ogy.”9 Critics first noted the need for more expertise tween Dr. Fredrickson and some 30 representatives in the fields of epidemiology and infectious diseases, of industry, including pharmaceutical and chemical since most molecular biologists were trained as companies, it became clear that some rDNA research chemists. * RAC’s membership was increased to 16 was being done; however, the representatives ap- and the range of expertise was widened to include peared hesitant to commit themselves to voluntary the fields of epidemiology, infectious diseases, and compliance with the proposed guidelines.l4 The pri- the biology of enteric organisms, by amendment to the charter on April 25, 1975. Since some members were conducting the re- IODr+ E]izabeth Kutter, a former RAC member, persona] cot’n- search in question, critics claimed that a conflict of munication, Sept. 11, 1980. interests existed. They also noted that the Committee 1 IN, wade, ~,Re~ombinant DNA: NIH sets strict Rules to ~unch New Technology,” Science, vol. 190, 1975, pp. 1175, 1179. %3wazey, et al., op. cit., p. 1,034. ‘zKutter, op. cit. Whe charter of the Recombinant DNA Molecule Program Ad- ‘sIbid. visory Committee, Oct. 7, 1974, op. cit. ltsubcommittee on science, Research and Technology of the ● One of the members of the original RAC (Stanley Falkow) did House Committee on Science and Technology, Genetic Engineering have substantial expertise with enteric organisms and E. coli in Human Genetics, and Cell Biology: DNA Recombinant Molecule Re- particular. search (Supp. Report 11) 94th Cong,, Zd sess,, 1976, p, 51. Appendix III-A—History of the Recombinant DNA Debate ● 317

mary reason was their concern over protection of although two nonscientists were already members. proprietary information.l5 The number of nonscientists remained the same Phase 11 culminated with the promulgation on until December 1978.20 Also, RAC’s responsibilities June 23, 1976, of the Guidelines for Research Involv- were defined in greater detail, including the respon- ing Recombinant DNA Molecules (“1976 Guidelines”) sibility for reviewing large-scale experiments. Never- covering institutions and individuals receiving NIH theless, RAC continued formally at least to be “a tech- funds for this research. nical committee, established to look at a specific Phase III was characterized by attempts to remedy problem.” the limited applicability of the Guidelines. Soon after The second development was a growing belief their publication, Senators Kennedy and Javits sent a among scientists that the risks of the research were letter to President Ford, calling his attention to the less than originally feared. This was based on the fol- Guidelines. They noted that any risk was not limited lowing: 1) a letter from Roy Curtiss at the University to federally funded research, and urged him to of Alabama to the Director of NIH, explaining risk take necessary steps to implement the Guidelines assessment experiments using Escherichia coli, from throughout the research community. In October which he concluded that the use of E. coli K-12 host- 1976, the Secretary of HEW, with the approval of the vectors posed no danger to humans; 2) the conclu- President, formed the Federal Interagency Advisory sions of a committee of experts in infectious diseases Committee under the chairmanship of the Director assembled by NIH in June 1977 in Falmouth, Mass., of NIH to determine the extent to which the Guide- that the alleged hazards of the research were un- lines could be applied to all research and to rec- substantiated; and 3) a prepublication report on ex- ommend necessary executive or legislative actions to periments showing that genetic recombination oc- ensure compliance.l6 In March 1977, the Committee curs naturally between lower and higher life forms, concluded that existing Federal law would not per- and suggesting that the rDNA technique was not as mit the regulation of all rDNA research in the United novel as presumed. States to the extent deemed necessary;17 it further The third development affecting the legislation recommended new legislation, specifying the ele- was a concerted lobbying effort by scientists against ments of that legislation, is what they considered to be some of the overly During 1977, several bills to deal with this and restrictive provisions of the bills, especially S. other problems were introduced in Congress. They 1217.21 22 23 The efforts included wide circulation of addressed in different ways the issues of the extent reports (including some in draft form) as soon as of regulatory coverage, the mechanisms for regula- available, which supported the conclusion that tion, and Federal preemption of State and local regu- the research was less hazardous than originally lation. The major bills were those of Rep. Paul supposed. Rogers, H.R. 7897 (and its substitute, H.R. 11192) and By the end of 1977, the legislation was in limbo. of Sen. Edward Kennedy, S. 1217. * This situation continued in early 1978, although While hearings were being held, three devel- some hearings were held. On June 1, 1978, Senators opments occurred which, by the end of 1977, had Kennedy, Javits, Nelson, Stevenson, Williams, and dissipated much of the impetus for legislation. The Schweiker addressed a letter to HEW Secretary first was the expanded role of RAC. On September Joseph Califano, which acknowledged the likelihood 24, 1976, its charter had been amended once more to that legislation would not pass and urged that defi- provide for additional expertise in the areas of ciencies in the regulatory system be addressed botany, plant pathology, and tissue culture. More- through executive action based on existing authority, over, its membership was increased from 16 to 20 so if that were to be the case. that four members would be “from other disciplines During Phase IV, NIH and its parent organization, or representatives of the general public. ” This was HEW (now DHHS), have attempted to operate in the the first official provision for public representation regulatory vacuum left by the lack of legislation. In response to the consensus that developed in 1977 on ‘sIbid., pp. 52. Iafnterim Repofl of the Federal Interagency Committee On flec0l7?- binant DNA Research: Suggested Elements for Legislation, Mar. 15, ~William Gartland, Director of the Office of Recombinant DNA 1977, pp. 3.4. Activities, NIH, personal communication, June 19, 1980. “[bid., pp. 9-10. ZIB. Cu]]iton, “Recombinant DNA Bills Derailed: COngre55 Still ‘“Ibid., pp. 11-15. Trying to Pass Law,” Science, vol. 199, Jan. 20, 1978. pp. 274-277. ● For a more complete discussion of the legislation, see footnote “D. Dickson, “Friends of DNA Fight Back,” Nature, vol. 272, 19. April 1978, pp. 664-665. ‘g.’’ Recombinant DNA Molecule Research, ” Congressional Re- ZSR. ~win, ,, Recombinant DNA as a political pa~n,~’ ~e~ ~ien. search Service, issue brief No. IB 77024, update of Jan, 2, 1979. fist, VO]. 79, Sept. 7, 1978, pp. 672-674.

76-565 0 - 81 - 22 318 ● Impacts of Applied Genetics-Micro-Organisms, Plants, and Animals

the question of risk, RAC proposed revisions to the ● Major actions, such as decisions to except other- Guidelines, which placed most experiments at a wise prohibited experiments on a case-by-case lower containment level. They were published for basis or to change the Guidelines, could be made public comment in September 1977. * As with the only on the advice of RAC and after public and original Guidelines, public hearings were held in the Federal agency comment. course of a 2-day meeting of the Advisory Committee The increased public responsiveness of the IBC’s to the Director in December 1977, in which a diverse was crucial, since the revised Guidelines placed ma- group of individuals and organizations were permit- jor responsibility for compliance on them. This had ted to comment. However, at this point, HEW took a been proposed in the July version and had not been much more active role in a situation that had been changed by the hearings. * Califano also announced handled almost entirely by NIH.24 he would appoint 14 new members to the RAC, in- When RAC’s charter was renewed on June 30, cluding people knowledgeable in fields such as law, 1978, Secretary Califano reserved the power to ap- public policy, ethics, the environment, and public point its members instead of delegating it to the health. Z’* ● All of these changes were envisioned to Director of NIH as in the past.** And the new pro- “provide the opportunity for those concerned to posed Guidelines, published in the Federal Register raise any ethical issues posed by recombinant DNA on July 28, 1978, were accompanied by an introduc- research” and to change the role of the RAC to “serve tory statement by Secretary Califano announcing a as the principal advisory body to the Director of NIH 60 day public comment period to be followed by a and the Secretary of HEW on recombinant DNA public hearing before a departmental panel chaired Policy .’’28* * * by HEW General Counsel Peter Libassi. * * * The In addition to broadening public participation, Secretary was particularly interested in comments Califano attempted to deal with a major limitation of on: new mechanisms to provide for future discre- the Federal response—the Guidelines did not cover tionary revision of the Guidelines; and the composi- private research, He directed the Food and Drug Ad- tion of the various advisory bodies, especially the ministration (FDA) to take steps to require that any RAC and the local Institutional Biosafety Committees firm seeking approval of a product requiring the use 25 (IBCS). of rDNA techniques in its development or manu- The public hearing called for by Secretary Cali- facture, demonstrate compliance with the Guidelines fano and held on September 15, 1978, was a sig- for the work done on that product; an FDA notice of nificant event in the history of Federal actions on the its intention to propose such regulations accom- rDNA issue. Testimony was heard from represent- panied the revised Guidelines in the Federal Register. atives of industry, labor, the research community, In addition, he requested the Environmental Protec- and public interest groups; more than 170 letters of tion Agency (EPA) to review its regulatory authority comment were received and subsequently reviewed. in that area. He believed if both agencies could As a result, the revised final Guidelines of December regulate research on products within their jurisdic- 22, 1978, were significantly rewritten to increase tion, “virtually all recombinant DNA research in this public participation in the decisionmaking process:26 country would be brought under the requirements 29 ● Twenty percent of the members of the IBCs had of the revised guidelines. ’’ * In the meantime, the to represent the general public and could have no connection with the institution. ● As part of the revision process, HEW held a meeting in October ● Most of the records of the IBCs had to be public- 1978 for IBC chairpersons in order to exchange information and ly available. experiences gained under the 1976 Guidelines. Z71bid.

● Shortly thereafter, in October 1977, the Final Environmental ● ● This was implemented by an amendment to the RAC Charter Impact Statement for the 1976 Guidelines was published. on Dec. 28, 1978, which increased the membership to 25 and Z4D. Fredrickson, “A History of the Recombinant DNA Guide- changed the composition to the following categories: 1) at least lines in the United States,” Hecornbinmt DNA Technical Bulletin, eight specialists in molecular biology or rDNA research; 2) at least vol. 2, Ju]y 1979. pp. 87, 90. six specialists in other scientific fields; and 3) at least six persons ● ● The statement providing for delegation of authority that ac- knowledgeable in law, public policy, the environment, and public companied the updated Charter was not signed by Califano. See or occupational health. In addition, the Charter was amended to also, footnote 24. grant nonvoting representation to representatives of various Fed- * ● ● The other members of the HEW panel were Dr. eral agencies. Fredrickson, Julius Richmond, who was the Assistant Secretary ZsIbid. for Health, and Henry Aaron, who was the Assistant Secretary for ● * ● The Charter was never amended to change or delete the Planning and Evaluation. final sentence of the “Purpose” section, which states, “This Corn. =43 F.R. 33042, July 28, 1978. mittee is a technical committee, established to look at a specific ~estatement of Secretav Califano accompanying the revised problem.” Guidelines, 43 F.R. 60080, Dec. 22, 1978. ‘“Ibid., p. 60081. Appendix I//-A—History of the Recombinant DNA Debate ● 319 revised Guidelines provided, for the first time, for April 11, 1980, NIH published Physical Containment voluntary registration of projects with NIH, in which Recommendations for Large Scale Uses of Organisms the registrant would agree to abide only by the con- Containing Recombinant DNA Molecules in the form tainment standards of the Guidelines.31 of Draft Part VII to the Guidelines.33 Besides setting Other major changes were embodied in the new large scale containment levels, this document recom- Guidelines. Because of the consensus that the ex- mends that the institution: appoint a biological safety periments posed lower risks than originally thought, officer with specified duties; and establish a worker some types of experiments were exempted, while health surveillance program for work requiring a containment levels were lowered for almost all high (P3) containment level. Finally, a more ad hoc re- others. In order to provide greater flexibility, these quirement has been used since October 1979 for ap- Guidelines permitted exceptions on a case-by-case provals of industrial requests for cultures up to 750 basis, and included procedures for their change on a liters (1); the approvals were conditioned on NIH piecemeal basis without going through the whole in- designated observers being permitted by the com- ternal process at HEW. For major changes, the pro- panies to inspect their facilities.34 At least one inspec- cedure was: 1) publication of the proposed changes tion has taken place. in the Federal Register at least 30 days prior to a RAC On November 21, 1980, NIH adopted the third ma- meeting; 2) RAC consideration of the proposed jor revision to the Guidelines.35 It contained these changes; and 3) publication in the Federal Register of significant changes: institutions sponsoring the the final decision of the Director, NIH. The standard research are no longer required to register their for all actions of the Director under the Guidelines projects with NIH pursuant to an informational docu- was “no significant risk to health or to the environment called a Memorandum of Understanding (MUA) merit. ”32 Lastly, the new Guidelines delegated project whenever the containment levels are specified in the approval to the IBCs. Guidelines; and NIH will no longer review IBC deci- The problems posed by voluntary compliance and sions on experiments for which containment levels commercialization have continued to be addressed are specified in the Guidelines. by NIH. In a second major revision to the Guidelines On November 21, 1980, NIH also promulgated on January 29, 1980, a section (Part VI ) was added to revised application procedures for large-scale pro- specify procedures for voluntary compliance. * ● On posals. The application must include the following information: 1) the registration document submitted to (cominuedfmm p. 318) the local IBC; 2) the reason for wanting to exceed the ● Subsequently, Califano sent similar letters to the Secretaries of 10-1 limit; 3) evidence that the rDNA to be used was Agriculture [February 1979) and Labor (July 1979) requesting rigorously characterized and free of harmful se- them to consider how their agencies’ authorities could be used to require private sector rDNA research to comply with the quences; and 4) specification of the large-scale con- Guidelines. qo tainment level proposed to be used as defined in the aoMinutes of the Interagency Committee on Recombinant DNA NIH Physical Containment Recommendations of Research, p. 3, July 17, 1979, preprinted in Recombinant DNA Re- April 11, 1980. search, vol. 5, p. 132, et. seq. alsec. IV.F.3, 1978 Guidelines. 3zSec. IV-E-l-b. ● “Several responses to the FDA notice had questioned the agen. cy’s legal authority to regulate private rDNA research. Conse. In addition to adding part VI to the Guidelines, the most signifi- quently, Dr. Fredrickson and Dr. Donald Kennedy, then Commis- cant change in the January 1980 Guidelines was the addition of sioner of Food and Drugs, developed a draft supplement to the sec. 111-0, which permitted most experiments using E. coli K-12 Guidelines, specifying procedures for voluntary compliance by in. host-vector systems to be done at the lowest containment levels. dustry. It was published for comment on Aug. 3, 1979 (44 F.R. s345 F.R. 24%8, Apr. 11, 1980. 45868) and incorporated as part of the proposed revised Guide. 3444 F.R. 69251, NOV. 30, 1979. 33 lines of November 30, 1979. (44 F.R. 69210, 69247). 45 F.R. 77372, Nov. 21, 1980. Appendix III-B Constitutional Constraints on Regulation

Under the checks and balances of our system of For many years, the Supreme Court has conceptual- government, the Constitution, as ultimately inter- ized the right of free expression in terms of a market- preted by the Supreme Court, requires certain pro- place of ideas—through the open and full discussion cedural and substantive standards to be met by stat- of all ideas and related information, the valuable, utory or other regulation imposed upon an activity. valid, or useful ones will be accepted by society, These requirements depend on the nature of the ac- while the ridiculous or even dangerous ones will be tivity involved. In the present case, it will be useful to so demonstrated and discarded. This is a consensual consider first the regulation of basic research and process; no person, group, or institution has suffi- then the regulation of technological applications, cient wisdom to prejudge ideas and deny them such as the production of pharmaceuticals by using admittance to that intellectual marketplace, even if genetic engineering methods. they threaten fundamental cultural values, for such values, if worthwhile, will survive. Under this con- Research cept, scientists would certainly have virtually unre- strained freedom to think, speak, and write. With respect to research, the fundamental ques- Difficulties arise with actions, such as experimen- tion is what limitations, if any, may be placed on the tation, which may be essential to the implementation search for scientific knowledge. The primary appli- of freedom of expression. Recent Supreme Court cable constitutional provision is the first amendment, cases have recognized a limited protected interest of which has been broadly interpreted by the Supreme the media to gather information as an essential ad- Court to severely limit intrusion by the Government junct to freedom of publication. By analogy, it may on all forms of expression.123 Another constitutional be argued that scientists would also be protected in safeguard, known as equal protection, is secondarily their research, as a necessary adjunct to freedom of involved. expression. On the other hand, the information If the Supreme Court were to recognize a right of gathering cases usually involve access to Govern- scientific inquiry, its boundaries would not exceed ment facilities, such as courtrooms or prisons. They those for freedom of expression.4 There is disagree- are based on the principle that actions by the Gov- ment among commentators on this issue concerning ernment should be open to public scrutiny—a con- the boundaries of the first amendments and certain- cept not directly applicable to the present issue. ly disagreement on the application of generally ac- More importantly, the Court has long recognized cepted principles to particular cases. Moreover, that actions related to expression can be regulated there have been no judicial decisions dealing with the and that regulation may increase with the degree of precise issue at hand. However, it is possible to out- the action’s impact on people or the environment. line general principles derived from judicial deci- The Court would probably apply what has been sions interpreting the first amendment, and indicate called a structured balancing test;6 i.e., regulation how they might be applied by the courts to attempts would be deemed valid only when the Government to regulate genetic research. sustains the burden of proving: 1) that there are There are very few limitations on the written or “compelling reasons” for the regulation; and 2) that spoken word. The prohibitions against obscenity or the objective cannot be achieved by “less drastic “fighting words”* clearly would be inapplicable here. means, ” i.e., by more narrowly drafted regulations having less impact on first amendment rights. ‘Harold P. Green, “The Soundaries of Scientific Freedom” Regulation of Scientt~ic Inquiry: ticietal Concerns With Research, Keith M. Wulff (cd.) The second part of the test is fairly straightfor- (Washington, D. C.: AAAS, 1979), pp. 139-143. ward. Governmental restrictions must be kept to a aThomas 1. Emerson, “The Constitution and Regulation of Research,” fteg- minimum. E.g., where possible, they should be reg- u/ation o~scientt~ic inquiry: Societal Concerns With Reseamh, Keith M. Wulff (cd.) (Washington, D. C.: AAAS, 1979), pp. 129-137. ulatory rather than prohibitory, temporary rather ‘John A. Robertson, “The Scientists’ Right to Research: A Constitutional than permanent, involve the least burden, and so on. Analysis,” Soufhern Ca/~j’ornia Law Review 5 1:1203, September 1978. 4 The difficult part of this test lies in determining Green, op. cit., p. 140. ‘Emerson, op. cit., pp. 131-134. “’Fighting words” are those provoking violent reaction or imminent disorder. ‘Ibid., p. 134.

320 Apppendix III-B—Constitutional Constraints on Regulation ● 321

what is a compelling reason. The protection of health plied in a discriminatory way without compelling or the environment is the most clearly acceptable reasons. reason for regulation. In addition, the protection of Congress could therefore, mandate by law that individual rights and personal dignity is generally certain kinds of research not be funded or be con- considered an acceptable reason. E.g., the National ducted in certain ways. An example is the National Research Act7 requires that all biomedical and be- Research Act, discussed previously. However, this havioral research involving human subjects sup- approach may have some serious practical limita- ported under the Public Health Service Act be re- tions because of the difficulty of determining which viewed by an Institutional Review Board in order to molecular biological research might lead to the pro- protect the rights and welfare of the subjects. scribed knowledge. Much discretion would have to The above discussion relates to protection from be left to the funding agency, which is likely to be un- physical risks due to the process of research. Could sympathetic or even hostile to such an approach, if it the Government regulate or forbid experimentation views its primary mission as fostering research. solely because the product (knowledge) threatens cultural values or other intangibles such as the genet- Applications and products ic inheritance of mankind? Religious or philosophical Although fears have been expressed that current objections to research, based solely on the rationale genetic technologies may lead to applications that that there are some things mankind should not would be detrimental, no one can reasonably con- know, conflict with the basic principles of freedom clude, at the present time, that this will actually oc- of expression and would not be sufficient reason on cur. For this reason, the most constitutionally per- constitutional grounds to justify regulation. Even if missible approach in all probability will be to regu- the rationale underlying this objection were expand- late the applications of the science. In such situa- ed to include situations where knowledge threatens tions, whatever harms occur tend to be more tangi- fundamental cultural values about the nature of ble and the governmental interests, therefore, more man, control of research for such a reason probably clearly defined. Moreover, since fundamental con- would not be constitutionally permissible. The ra- stitutional rights are generally not involved, statutes tionale would again conflict with the marketplace of and regulations are subjected to a lower level of ideas concept that is central to freedom of expres- scrutiny by the Federal courts. sion. However, what if the knowledge were to pro- The constitutional authority for Federal regulation vide the means to alter the human species in such a of the applications of technologies such as genetic en- way that the physical, psychological, and emotional gineering lies in the commerce clause, article I, sec- essence of what it is to be human could be changed? tion 8 of the Constitution, which grants Congress the No precedent exists to provide guidance in determin- power “To Regulate Commerce with foreign Nations, ing an answer. Were the situation to arise, the and among the Several States. ” In contrast to sit- Supreme Court might fashion another limitation on uations involving fundamental rights, the Supreme the concept of free expression in the same way it Court has interpreted this clause as giving Congress developed the obscenity or “fighting words” doc- extremely broad authority to regulate any activity in trines. any way connected with commerce. It has been vir- The discussion thus far has had as its premise a tually impossible for Congress not to find some con- direct regulatory approach to research. There is a nection acceptable to the courts between commerce more indirect approach, which would be constitu- and the goals of a particular piece of legislation. * The tionally permissible and could accomplish much of standard of review of such legislation by the Federal what direct regulation might attempt, including pre- courts is to determine if it bears a rational re- vention of the acquisition of some forms of knowl- lationship to a valid legislative purpose. If so, the edge. This is the use of the funding power. The Court will uphold the legislation and will not second lifeblood of modern science in the United States is guess the legislators. This standard of review rec- the Federal grant system. Yet it is generally agreed ognizes that a statute results from the balancing of that Government has no constitutional duty to fund competing interests and policies by the branch of scientific researches This is a benefit voluntarily pro- Government created to function in that manner. vided to which many kinds of conditions may be attached. The only constitutional limitation on such an *See Wickard v. Filburn, 317 U.S. Ill 0942) in which the Supreme Court up- approach would be the concept of equal protection— held civil penalties for violation of acreage allotments established by the Agricultural Adjustment Act of 1938, covering the amount of wheat that in- any restrictions must apply to all or must not be ap- dividual farmers could plant, even if the wheat was intended for selfcon- sumption. The rationale was that even though the individual farmer’s wheat had no measurable impact on interstate commerce, Congress could properly determine that all wheat of this category, if exempted from regulation, TPublic Law 93-348 (1974), 42 U.S. C. S289 1-3 could undercut the purpose of the Act, which was to increase the price ‘Green, op. cit., p. 141. farmers received for their various crops. Appendix III-C Information on International Guidelines for Recombinant DNA

The following information is based largely on in- Germany United States ternational surveys undertaken by The Committee France U.S.S.R. on Genetic Experimentation of the International Italy Council of Scientific Unions reported as of July IV. Nations that had modified the guidelines of other, 1979.’ indicated, countries: I. Nations that had established guidelines for conduct of rDNA research or were using the guidelines of Australia (UK, U. S.) Mexico (U. S.) other nations: Belgium (UK, U. S.) Netherlands (U. S.) Brazil (U. S.) New Zealand Australia Italy Bulgaria (U. S. S. R., U. S.) Norway (U. S.) Belgium Japan Czechoslovakia (U. S. S. R., Poland (U. S.) Brazil Mexico U. S., Fed. Rep. Ger.) South Africa (U. S.) Bulgaria Netherlands Denmark (UK) Sweden (U.S) Canada New Zealand East German Democratic Switzerland (U. S.) Czechoslovakia Norway Republic (UK, U. S., Taiwan (U. S., UK) Denmark Poland Netherlands) Yugoslavia German Democratic South Africa Finland (U.S. mainly) (European Science Republic Sweden Hungary (U. S.) Foundation) Federal Republic of Switzerland V. Nations in which entirely voluntary guidelines have Germany Taiwan been adopted: Finland United Kingdom France United States Finland Hungary U.S.S.R. Israel Yugoslavia VI. Nations with guidelines that are enforceable through control of research funding: II. Nations that had not established guidelines or had not responded with updated information: Australia” Japan Canada Netherlands Country Yes No Czechoslovakia Norway Austria x Denmark South Africa Ghana x Federal Republic of Sweden India x Germany’ Switzerland e Iran x France Taiwan f Jamaica x German Democratic United Kingdom Korea x Republic United States Nigeria x a’isubmissions may be made directly to the Academy of Science or Singapore x through a granting agency. In the latter case, it is a requirement for the ap- Sri Lanka x plicant to observe the recommendations of the Academy’s Standing Com- Sudan x mittee if the agency makes a grant for the work. Otherwise, the guidelines are voluntary with the worker required to make an annual report on prog- Turkey x ress, or more frequently if conditions of the experiment (such as volumes) are changed appreciably.” 111. Nations that had drafted their own guidelines: b’~ntrol through Academy of Sciences and Ministry of Health.” c~veral research organ~tions require MCeiVers of grants to apply ‘he Canada Japan NIH guidelines until their own national guidelines are completed. Federal Republic of United Kingdom dThe Netherlands Organization for the Advancement of Pure Research will only subsidize projects which have been given the committee’s conaent.

e!(waiting for re5pon5e from National Advisory CCJmmittee. ” f“Notification of proposals to GMAG became compulsory August 1, 1978. ‘Report to COGENE ftmm the working gruup on Recombinant DNA Guide- In addition, funding bodies require, as a condition of funding, GMAG’s ad- lines, May 1980. vice to be sought and followed. ”

322 Appendix I//-C—Information on International Guidelines for Recombinant DNA ● 323

VII. Nations in which guidelines are legally en- the Control on Genetic Manipulation, Amsterdam, forceable: March 1977, p. 54.) Bulgaria, Switzerland: None of the above. Hungary Taiwan: No response. U.S.S.R. Finland...... “At present, the guidelines are entirely voluntary, but in the near them in the context of information about the ‘centre’ in which the work is to future, the intention is to include go on.” them in the law of infectious dis- X. Nations in which special provisions for agriculture eases when they will become legally and/or industrial research and applications have enforceable. ” been made: South Africa. . “At present the guidelines are not Czechoslovakia. “10 liter maximum volume of the legally enforceable. They will only culture containing recombinant become so if regulations under the DNA” existing Health Act of 1977 and the German Animal Diseases and Parasites Act of Democratic 1956 are promulgated; and none are Republic. . . . . “The GDR Guidelines will be com- intended at present. ” pulsory for industrial and agricul- United Kingdom “The regulation to notify GMAG tural applications. 10-liter maximum does not strictly mean that the deviations may be allowed by the Williams Guidelines themselves are Minister of Health if suggested by legally enforceable. But, under the the Committee. ” Health and Safety at Work Act Federal Republic (within which the Regulations were of Germany . . “Specification of containment of introduced), it is expected that ac- plants” count will be taken of the relevant France ...... “Industry, maximum volume of cell Codes of Practice and the advice culture is set at 10 liters” given by GMAG.” Norway ...... “The Guidelines cover both agri- VIII. Nations in which observance of the guidelines is culture and industry. Application of monitored by a nationally-directed mechanism: recombinant DNA research outside an approved laboratory is prohib- Australia Norway ited. Otherwise the Committee fol- Czechoslovakia South Africa lows the NIH Guidelines.” German Democratic Sweden United Kingdom “Agriculture, industry; see Williams Republic United Kingdom Report, paragraphs 1.3, 2.7, 5.13 France United States and appendix II, section 34. ” Hungary U.S.S.R. United States . . “Agriculture. NIH Guidelines pro- Japan Yugoslavia vide containment levels for cloning total plant DNA, plant virus DNA IX. Nations in which a license or other authorization for recombinant DNA activity is granted: and plant organelle DNA in E. coli K-12, and provide general guidance —to an institution: U.S.S.R. for the use of plant host-vector sys- —to an indivdual laboratory: Hungary, Czechoslo- tems. 10 liter maximum. A proposed vakia Supplement to the Guidelines for —to an individual scientist: Australia, Canada, Ger- voluntary compliance by the private man Democratic Republic, Federal Republic of sector is under consideration by Germany, Finland, France, Japan, Norway, South RAC. Development of a monograph Africa, Sweden, United Kingdom”, United States for large-scale applications has been and U.S.S.R. proposed.” Netherlands: “There are gentlemen’s agreements, U.S.S.R...... “Guidelines are compulsory for in- signed by the individual scientist, the institution dustrial and agricultural applica- and the Committee.” The reports of the Committee tions. 10 liter maximum. Deviation also recommend legislation that will require regis- is allowed by the Recombinant DNA tration of research projects in this field and make Commission. ” binding the guidelines and supervision of their Other observance. (Report of the Committee in Charge of respondents, . No 324 ● impacts of Applied Genetics—Micro-Organisms, Plants, and Animals

XI. Number of laboratories currently engaged in re- XII.Countries in which specific training for workers combinant DNA activities: and safety officers in recombinant DNA activities is Country Any labs? How many? required by the guidelines: Australia...... yes 16 Country Yes No Other a Austria ...... no a a Australia . . . . . Belgium...... yes 6 Bulgaria...... x Brazil...... yes 5 x a Canada ...... Bulgaria...... , yes no response Czechoslovakia. X b Canada ...... yes 10-15 German Czechoslovakia ., . . . . . yes 3 Democratic a c Denmark...... yes several Republic . . . . . x German Democratic Federal Republic Republic ...... yes 5 of Germany . . X d Federal Republic Finland. .,.... x of Germany...... yes 10-20 France ...... x Finland ., ...... yes 3(3-4 planned) Hungary...... x a France. ., ...... yes 12 Japan. ..., . . . x a Ghana ...... no Netherlands. . . X e a Hungary ...... yes 1-2 Norway ...... X f a India ...... no South Africa. . . x Iran...... noa x a Sweden ...... Israel...... yes 1 Switzerland . . . “recommended” a Jamaica...... no Taiwan...... “recommended” Japan ...... yes 35 United Kingdom Xg a Korea ...... no United States . . X h Netherlands ...... yes 7 U.S.S.R...... x New Zealand...... yes 2 Yugoslavia . . . . no response Nigeria ...... no Norway ...... yes not stated Other respondents: no or no response to question. Philippines ...... noa Poland ...... yesa 3 aAustra]ia: URequireexpefii~ through Biosafety COmmiWW.” Singapore ...... noa bczechoslovakia: ~spific training is recommended.” a c~rman Democratic Republic: “Training courses are organized by the South Africa ...... yes 3 Committees in cooperation with Akademie fur Arztliche Fortbildungder a Sri Lanka...... no DDR.” a dFederal Republic oftkrmany: “Experience as required by law on the Sudan ...... no a control ofcommunicable diseases.” Sweden...... yes 2 eNether]ands: “Thescientistss hould be trained in microbiology.” Switzerland. ., ...... yesa 18 fNorway: The Committee certifies training and expertise of personnel are adequate. ” Taiwan ...... yes 2 gunited I(ingdorn: “Detai]S of training are required; the employer is legally a Turkey ...... no obliged to provide suitable training. ” United Kingdom ...... yes 45 hunited States: ‘“SFific training not required. However, local biohazards a committees are required to certify to the NIH that the training and expertise United States...... yes 50 of the personnel are adequate. ” U.S.S.R ...... yesa 6 Yugoslavia...... yesb 4

a~wd on replies from previous Questionnaires. bln preparation. Appendix III-C—lnformation on international Guidelines for Recombinant DNA ● 325

X111. Countries in which the guidelines are applicable and compliance at laboratories; only to biological agents containing recombinant ZKBS (Zentrale Commission fur DNA, or also cover the recombinant DNA mole- die biologische Sicherheit) has cules themselves: overall responsibility. France ...... “Local safety committees” Only to Hungary. ... , . . . “National Institutes of Public biological Also recombinant Health” agents DNA molecules Country Japan ... , ...... “Principal Investigator and Safety Australia...... x Officer” Bulgaria ...... Netherlands . . . . . “Site Inspection Commission” Canada...... (a) (a) New Zealand. . . . . “Local controlling Committees Czechoslovakia . . x are charged with monitoring German observance of Guidelines. Democratic Biological Safety Officers are ap- Republic...... x pointed to take immediate Federal Republic of responsibility.” Germany...... x Norway ...... “Physical containment: Norwe- Finland...... x gian National Institute of Public France ...... x Health. Biological containment: Japan ...... x Committee. ” Netherlands . . . . . x South Africa. . . . . “Above P3, Biosafety Committee New Zealand. . . . . x of Institute involved and Norway ...... x SAGENE. Below P3, SAGENE South Africa. . . . . x only.” Sweden...... x Sweden...... Not applicable. Switzerland . . . . . x Switzerland . . . . . “At the responsibility of either Taiwan...... x the individual investigator or a United Kingdom . . x local biohazards committee.” b United States . . . . X Taiwan...... No response U. S. S. R...... x United Kingdom . . The Health and Safety Executive United States . . . . “Observance of containment is to a(;uidelines appty to a]i, but containment is not required for naked DNA. b’The [guidelines-apply to recombinant DNA experiments that are not ex- be monitored by biohazards com- empt under Section I-E of the Guidelines. Recombinant DNA molecules that mittees located in institutions in are not in organisms or viruses are exempt from the Guidelines (I-E-l ).” which the research is conducted. XIV. Groups/Committees responsible for carrying out Effectiveness of containment monitoring of containment procedures: procedures is to be monitored by the principal investigator who is Country Group to report problems to the NIH.” Australia...... Institutional Biosafety Commit- U. S. S. R...... “Local biosafety commission, tees. State Sanitary Inspection control Bulgaria ...... National Committee group of Recombinant DNA Com- Canada ...... “University and Medical Re- mission. search Council Biohazards Com- mittees” Czechoslovakia. . . “Under consideration of the Na- tional Institutes of Public Health.” German Democratic Republic ...... “Monitoring is carried out by local Biosafety Officers, who are representatives of the Committee in their institutions. ” Federal Republic of Germany...... Officers for Biological Safety monitor the health of employees 326 ● Impacts of Applied Genetics—Micro-Organisms, Plants, and Animals xv. Countries in which the guidelines apply to all gene New Zealand. . . . . “Yes, with the approval of the combinations instructed by cell-free methods, or National Committee.” only to molecules containing combinations of United Kingdom . . “The question has not arisen.” genes from different species: Other respondents No

Molecules aAr@ there an circumstances under which such dissemination can be car- v All gene com- containing ried out? binations con- combinations of structed by cell- genes from XVII. Countries in which the guidelines are restricted Country free methods different species to recombinant DNA activities or also cover other areas of genetic experimentation: Australia...... x Canada...... x Recombinant Other areas of Czechoslovakia. . . x DNA genetic German Country activities experimentation Democratic Australia. ... , ., . x a Republic...... x Bulgaria ...... x Federal Republic of Canada ...... X b X a Germany...... Czechoslovakia. . . x Finland...... x German France . . . , . . . . . x Democratic Japan ...... x Republic ...... x b Netherlands . . . . . X Federal Republic of New Zealand. . . . . x Germany...... x Norway ...... x Finland...... x South Africa . . . . . x France ...... x Sweden...... x Hungary...... x Switzerland . . . . . x Japan ...... x Taiwan. . . . , . . . . Netherlands . . . . . x United Kingdom . . New Zealand. . . . . X C United States . . . . Norway ...... x U. S. S. R...... x South Africa. . . . . X d aFederal Republic of Germany Selfwloning experiments involving non- Sweden...... x pathogenic donors and hosts shall be reported to ZKBS. Switzerland ... , . x bNetherlands “The definition of recombinant DNA has recedy been United Kingdom . . x modified and includes the insertion of chemically synthesized DNA molecules into a vector. ” United States . . . . x cunited Kingdom “The G~u@s provision] interpretation Of their Own re- U. S. S. R...... x mit is that they are concerned with work involving genetic manipulation, defined for these purposes as: the formation of new combinations of her- a“At present, the terms of reference of the Academy Committee refer itable materials by the insertion of nucleic acid molecules, produced by only to in vitru experiments (i.e., the use of restriction enzymes and ligases). whatever means outside the cell, into any virus, bacterial plasmid, or other An ad hoc Academy Committee is about to investigate in vivo experimenta- vector sbystem so as to allow their incorporation into a host organism in tion, with the following terms of reference: which they do not naturally occur but in which they are capable of con- 1. Examine whether, other than by using the technique of in vitru re- tinued propagation.” combinant DNA construction, new hybrid nucleic acid molecules can be produced that are potentially dangerous to humans, animals, or plants. XVI. Countries in which the guidelines restrict the in- In so doing, the committee should give particular attention to the following possibilities: tentional dissemination into the environment of —The use of mixed infections involving human or animal viruses, or the biological agents containing recombinant DNA: use of bacteria or fungi. –The introduction of foreign DNA into plants and the production of a All respondents . . Yes new plant pathogens. Australia...... Not explicity so 2. Consider whether there are certain classes of viral pathogens (e.g., polio) on which experimentation should not be carried out unless a spe- German cial need is demonstrated.” ‘ Democratic b“work with animal viruses and cells” C“i.e., cell fusion with approval of National Committee” Republic ...... “Exceptions have to be discussed d“other c]osely related areas are also covered.” by the Committee and require special permission by the Minis- ter of Health.” Appendix //l-C—Information on International Guidelines for Recombinant DNA . 327

XVIII. Countries in which the recombinant DNA ad- research, possess specific knowl- visory committee includes public representa- edge in the implementation of tives as well as scientists: safety measures in biological research work, particularly how- Country Yes No ever in microbiology, cytobiolo- Australia...... x gy, or hygiene and, in addition, 4 Bulgaria ...... x outstanding individuals, for ex- Canada ...... x ample from the trade unions, in- Czechoslovakia. . . x dustry, and the research-promot- Denmark ...... x ing organizations. German Finland...... 27 members: 6 molecular biol- Democratic ogy, 3 genetics, 3 microbiology, 1 Republic ...... x virology, 1 plant physiology, 3 in- Federal Republic of fectious diseases, 3 epidemiology, Germany. ., . . . . x 2 enteric bacteria, 1 cell cultures, Finland...... x 3 public health, 1 occupational France . . . ., . . . . x health. Hungary...... x France ...... 13 members, 4 observers, 1 sec- Italy ...... , x retary Japan ...... x Hungary. , ...... Scientists Netherlands . .,. . x Italy ...... 8 molecular biologists, 4 micro- New Zealand. . . . . x biologists, 1 civil servant (Health Norway ...... x Ministry). South Africa . . . . . x Japan ...... (Combines both Steering Com- Sweden...... x mittee and Advisory Group): 7 re- Switzerland . . . . . x combinant DNA scientists, 7 sci- Taiwan...... x entists in other fields, 6 special- United Kingdom . . x ists in medicine and biohazards, 2 United States . . . . x lawyers, 2 specialists in physical U. S. S. R...... x containment, 3 public represent- Composition of DNA advisory committees is as fol- atives. lows: Netherlands . . . . . 14 scientists representing genet- Australia...... 8 scientists ics, molecular biology, bacteriolo- Canada ...... 5 laymen (1 lawyer, 1 business- gy, virology, botany, medicine, man, 3 generalists); 6 scientists (2 ethics and social aspects of health M.D.s, 3 virologists/cancer spe- and health-care. To be added: a cialists, 1 recombinant DNA spe- committee composed of scientists cialist) and representatives of industry Czechoslovakia. . . 6 members representing molec- and trade unions. ular biology, genetics, microbiol- New Zealand. . . . . 1 molecular biologist, 1 microbial ogy, medicine geneticist, 1 virologist, 1 botanist Denmark ...... 9 scientists and administrative (molecular biologist), 1 human representatives. geneticist (medically qualified). German Norway ...... 3 biochemists, 2 medicine, 1 vet- Democratic erinary medicine, 1 lawyer, 1 Republic ...... 3 geneticists, 1 biochemist, 2 bac- artist. teriologists, 2 virologist, 1 jurist, South Africa . . . . . One each from: Council for Sci- 1 representative of trade union entific and Industrial Research, of GDR. Medical Research Council, De- Federal Republic of partment of Health, Department Germany...... 4 experts working in the field of of Agricultural Technical Serv- recombinant DNA research; 4 ex- ices. Three from universities, perts who, though not working public and legal professions. in the field of recombinant DNA Sweden...... No response 328 ● Impacts of Applied Genetics— Micro-Organisms, Plants, and Animals

Switzerland ... , . 12 members representing medi- ogy: 2, Plant Genetics: 2, Law: 2, cine, microbiology, molecular Environmental Concerns, Lab- biology, antibiotics, industry, oratory Technician, Infectious university management, and 7 Diseases, Occupational Health, governmental departmental Education: 1 each. assessors. U. S. S. R...... 8 scientists United States . . . . Molecular biology: 6, Molecular Yugoslavia ...... 3 geneticists Genetics: 5, Ethics: 3, Microbiol- Appendix IV Planning Workshop Participants, Other Contractors and Contributors, and Acknowledgments

Planning workshop participants Odelia Funke George E. Garrison Philip Bereano F. Eberstadt & Co. University of Washington Reinaldo F. Gomez Ralph Hardy Massachusetts Institute of Technology E. L Du Pent de Nemours & Co., Inc. John Hamilton Patricia King Neal F. Jensen Georgetown University Law Center Scott A. King Charles Lewis F. Eberstadt & Co. U.S. Department of Agriculture Harvey Lodish Herman Lewis Massachusetts Institute of Technology National Science Foundation L. D. Nyhart Pamela Lippe Massachusetts Institute of Technology Friends of the Earth ChoKyun Rha Victor McKusick Massachusetts Institute of Technology Johns Hopkins University Medical School William Scanlon Elena O. Nightingale Andrew Schmitz Institute of Medicine University of California, Berkeley Gilbert Omenn Michael J. Shodell Office of Science and Technology Policy The Sterling-Hobe Corp. Donna Parratt David Tse Congressional Research Service Massachusetts Institute of Technology Walter Shropshire James Welsh Smithsonian Radiation Biology Laboratory Montana State University Leroy Walters George Whiteside The Kennedy Institute Massachusetts Institute of Technology Bernard Wolnak and Associates Other contractors and contributors Nancy Woods OTA intern Betsy Amin-Arsala Richard J. Auchus Acknowledgments Massachusetts Institute of Technology Fred Bergmann A large number of individuals provided valuable National Institutes of Health advice and assistance to OTA during this assessment. Charles L. Cooney In particular, we would like to thank the following Massachusetts Institute of Technology people: Richard Curtin John Adams Robert A. Cuzick Pharmaceutical Manufacturers Association Massachusetts Institute of Technology Rupert Amann Roslyn Dauber Colorado State University Arnold L. Demain William Amen, Jr. Massachusetts Institute of Technology Cetus Corp. Richard B. Emmitt Daniel Azarnoff F. Eberstadt & Co. Searle Laboratories Emanuel Epstein A. L. Barr University of California, Davis West Virginia University Robert F. Fleischaker K. J. Betteridge Massachusetts Institute of Technology Animal Diseases Research Institute 329 330 ● Impacts of Applied Genetics —Micro-Organisms, Plants, and Animals

Jerome Birnbaum Michael Goldberg Merck, Sharp & Dohme Research Laboratories Food and Drug Administration Gerald Bjorge Maxwell Gordon U.S. Patent and Trademark Office Bristol Laboratories Hugh Bollinger Lorance L. Greenlee Plant Resources Institute Keil and Witherspoon G. Eric Bradford Ralph Hardy University of California E. I. Du Pent de Nemours and Co,, Inc. Robert Brackett W. C. D. Hare Parke, Davis & Co. Animal Diseases Research Institute Robert Byrnes Paul Harvey Genentech, Inc. U.S. Department of Agriculture Daniel Callahan Harold W. Hawk The Hastings Center Agricultural Research Center Alexander M. Capron Richard L. Hinman President’s Commission for the Study of Ethical Pfizer, Inc. Problems in Medicine and Biomedical and Peter Barton Hutt Behavioral Research Covington & Burling Robert Church E. Keith Inskeep University of Calgary West Virginia University H. Wallis Clark Irving Johnson University of California tiny Research Laboratories Gail Cooper Charles Kiddy Environmental Protection Agency Agricultural Research Center Joseph P. Dailey Thomas D. Kiley Revlon Health Care Group Genentech, Inc. Frank Dickinson Carole Kitti Agricultural Research Center National Science Foundation Donald R. Dunner Duane C. Kraemer Finnegan, Henderson, Farabow, Garrett & Texas A&M University Dunner Sheldon Krimsky Roger B. Dworkin Tufts University Indiana University School of Law W. W. Lampeter Richard P. Elander Lehn und Versuchtsgut Bristol-Myers Co. Earl Lasley Peter Elsden Monsanto Farmers Hybrid Colorado State University F. Douglas Lawrason Haim Erder Schering Corp. University of Pennsylvania Bernard Leese James F. Evans Plant Variety Protection Office Pennsylvania Embryo Transfer Service Stanley Leibo Kenneth Evans Oak Ridge National Laboratory Plant Variety Protection Office Morris Levin Richard Faust Environmental Protection Agency Hoffman-La Roche, Inc. Herman Lewis Herman Finke National Science Foundation Sterling Systems Peter Libassi Robert H. Foote Verner, Lipfert, Bernhard & McPherson Cornell University Paul J. Luckern Orrie M. Friedman U.S. Department of Justice Collaborative Research, Inc. Clement Markert William J. Gartland, Jr. Yale University National Institutes of Health Ralph R. Maurer Kenneth Goertzen U.S. Meat Animal Research Center Seed Research, Inc. Appendix IV—Planning Workshop Participants, Other Contractors and Contributors, and Acknowledgments ● 331

Robert McKinnell Thomas J. Sexton University of Minnesota U.S. Department of Agriculture Edward Mearns, Jr. Brian F. Shea Case Western Reserve University School Ralph Silber of Medicine Stanford University Alan S. Michaels Elizabeth L. Singh Stanford University Charles G. Smith Elizabeth Milewski Revlon Health Group National Institutes of Health Animal Diseases Research Institute Henry I, Miller Davor Solter Food and Drug Administration Wistar Institute of Anatomy and Biology Paul Miller Mark Sorrells W. R. Grace Cornell University A. V. Nalbandov G. F. Sprague University of Illinois University of Illinois Claude H. Nash Richard Staples Smith Kline & French Laboratories Cornell University Dorothy Nelkin Gerald G. Still Resources for the Future U.S. Department of Agriculture Gordon Niswender Charles W. Stuber Colorado State University North Carolina State University Elena Ottolenghi-Nightingale Bernard Talbot National Academy of Medicine National Institutes of Health David Padwa Rene Tegtmeyer Agrigenetics, Inc. U.S. Patent and Trademark Office Seth Pauker Clair E. Terrill National Institute of Occupational Safety U.S. Department of Agriculture & Health Robin Tervit J. B. Peters Colorado State University West Virginia University Stephen Turner Nancy Pfund Bethesda Research Laboratories, Inc. Stanford University School of Medicine L. D. Van Vleck James Punch Cornell University The Upjohn Co. Robert Walton Neils Reimers W. R. Grace Stanford University Charles Weiner Ira Ringler Massachusetts Institute of Technology Abbott Laboratories Ray W. Wright, Jr. Roman Saliwanchik Washington State University The Upjohn Co. Susan Wright Robert B. Samuels University of Michigan Beckman Instruments Oskar R. Zaborsky George E. Seidel, Jr. National Science Foundation Colorado State University M. S. Zuber Sarah M. Seidel University of Missouri Colorado State University o