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CHAPTER 14 Applications of recombinant DNA technology

Introduction Theme 1: Nucleic acid sequences as diagnostic tools is not new. The making of beer, wine, bread, yoghurt and cheese was practised by ancient Introduction to theme 1 civilizations, such as the Babylonians, the Romans and the Chinese. Much, much later came , Nucleic acid sequences can be used diagnostically in the production of basic chemicals (e.g. glycerol, two different ways. The first is to determine whether citric acid, lactic acid) and the development of anti- a particular, relatively long sequence is present in or biotics. In each of these examples, existing properties absent from a test sample. A good example of such of microorganisms were exploited. For example, an application is the diagnosis of infectious disease. Penicillium naturally make penicillin. What By choosing appropriate probes, one can ascertain the scientists have done is to increase the yield of in a single step which, if any, microorganisms are penicillin by repeated rounds of mutation and present in a sample. Alternatively, a search could be selection, coupled with optimization of the growth made for the presence of known antibiotic-resistance medium. Similarly, sexual crosses between related determinants so that an appropriate therapeutic plant species have created high-yielding and disease- regime can be instituted. In the second way in which resistant varieties of cereals. These improved cereals sequences are used diagnostically, the objective is to represent new combinations of genes and alleles determine the similarity of sequences from different already existing in wild strains. individuals. Good examples of this approach are With the development of gene manipulation tech- prenatal diagnosis of genetic disease and forensic niques in the 1970s, there was a major paradigm profiling (‘DNA fingerprinting’). shift. For the first time microorganisms could be made to synthesize compounds that they had never Detection of sequences at the gross level synthesized before, e.g. production in E. coli ( Johnson 1983). Soon all sorts of commercially or Imagine that a seriously ill individual has a disorder therapeutically useful proteins were being made in of the gastrointestinal tract. A likely cause is a bacteria, principally E. coli, and thus the modern microbial infection and there are a number of candid- biotechnology industry was born. As the techniques ate organisms (Table 14.1). The question is, which developed for manipulating genes in bacteria were organism is present and to which antibiotics is it extended to plants and animals there was a con- susceptible? The sooner one has an answer to these comitant expansion of the biotechnology industry questions, the sooner effective therapy can begin. to exploit the new opportunities being provided. Traditionally, in such a case, a stool specimen would Today there are many different facets to the com- be cultured on a variety of different media and would mercial exploitation of gene manipulation tech- be examined microscopically and tested with vari- niques as shown in Fig. 14.1. Rather than discuss ous immunological reagents. A simpler approach all these topics in detail, for that would take a book is to test the sample with a battery of probes and in itself, we have chosen to focus on six inter- determine which, if any, hybridize in a simple dot- disciplinary themes that reflect both the successes blot assay. With such a simple format it is possible to achieved to date and the likely successes in the next vary the stringency of the hybridization reaction to decade. accommodate any sequence differences that might POGC14 9/11/2001 11:11 AM Page 275

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Transgenic Nucleic animals acids

Therapeutic proteins Anti-sense

Improved farm /repair animals Diagnostic probes

Disease models Vaccines

Recombinant DNA technology ‘’

Vaccines Vaccines

Small molecules Small molecules

Therapeutic proteins Therapeutic proteins

Improved plants

Biopolymers

Transgenic Recombinant plants microbes

Fig. 14.1 The different ways that recombinant DNA technology has been exploited.

exist between the probe and the target. The down- Table 14.1 Pathogens causing infection of the side of this approach is that if one wishes to test the gastrointestinal tract. sample with 10 different probes, then 10 different dot blots are required; otherwise there is no way of Bacteria Salmonella spp. determining which probe has bound to the target. Shigella spp. Another disadvantage of this approach is that Yersinia enterocolitica sufficient target DNA must be present in the sample Enterotoxigenic E. coli to enable a signal to be detected on hybridization. Clostridium difficile Both of these problems can be overcome by using the Clostridium welchii Campylobacter spp. polymerase chain reaction (PCR). Aeromonas spp. In a traditional dot-blot assay, the sample DNA is Vibrio spp. immobilized on a membrane and hybridized with a selection of labelled probes. An alternative is a Rotaviruses ‘reverse dot blot’, where the probes are immobilized Enteroviruses on the membrane and hybridized with the sample. Enteric adenoviruses In this way, only one hybridization step is required, Protozoa because each probe occupies a unique position on Entamoeba histolytica the membrane (Fig. 14.2). For this approach to Giardia Iamblia work, the sample DNA needs to be labelled and this can be achieved using the PCR. This has the added POGC14 9/11/2001 11:11 AM Page 276

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Conventional dot blot

Membrane

Test DNA Test DNA Test DNA Test DNA Test DNA

Probe Probe Probe Probe Probe A B C D E

Reverse dot blot

Probe AProbe B Probe C Probe D Probe E

Labelled Fig. 14.2 Comparison of conventional test DNA dot blot assay with reverse dot blot method. Note that in the latter there is only a single hybridization reaction, regardless of the number of probes used, whereas in the former each probe has to be tested in a separate hybridization step.

benefit of amplifying the sample, thereby minimizing Despite the difficulties noted above, multiplex PCR the amount of target DNA required. The downside has been used successfully in the diagnosis of infec- of this approach is that the amplification step needs tious diseases. For example, Heredia et al. (1996) to be done with multiple pairs of primers (multiplex have used the method to simultaneously examine PCR), one for each target sequence. Although blood for the presence of HIV-1, HIV-2, human multiplex PCR is now an established method, con- T-cell lymphotropic (HTLV)-I and HTLV-II. siderable optimization is needed for each application Similarly, Grondahl et al. (1999) have used the (Elnifro et al. 2000). The major problems encoun- method to identify which of nine different organisms tered are poor sensitivity or specificity and/or pre- is responsible for respiratory infections. Once the ferential amplification of certain specific targets. clinical microbiologist knows the identity of a micro- The presence of more than one primer pair in the organism in a specimen, he/she can select those reaction increases the chance of obtaining spurious antibiotics that might be effective, provided that amplification products because of the formation of the organism in question does not carry multiple primer dimers. Such non-specific products may be drug-resistance determinants. The presence of these amplified more efficiently than the desired target. determinants can also be established using multi- Clearly, the design of each primer pair is crucial to plex PCR. Alternatively, if a virus causes the infec- avoiding this problem. Another important feature is tion, one can monitor the progress of the infection by that all primer pairs should enable similar amplifica- using quantitative PCR (see p. 23). This approach tion efficiencies for their respective targets. has been used to monitor cytomegalovirus infec- POGC14 9/11/2001 11:11 AM Page 277

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tions in kidney-transplant patients (Caballero et al. two 19-mer , one of which was 1997). It is worth noting that the development complementary to the amino-terminal region of the of multiplex PCR technology is being facilitated by normal β-globin (βA) gene and the other of which the rapid progress in the sequencing of microbial was complementary to the sickle-cell β-globin gene (for an up-to-date list, see http//igweb.integ- (βS). These oligonucleotides were radiolabelled and ratedgenomics.com/GOLD/), since the data gener- used to probe Southern blots. Under appropriate ated enable species-specific genes or sequences to be conditions, the probes could distinguish the normal identified. and mutant alleles. The DNA from normal homo- zygotes only hybridized with the βA probe and DNA from sickle-cell homozygotes only hybridized with Comparative sequence analysis: single- the βS probe. DNA of heterozygotes hybridized with polymorphisms (SNPs) both probes. These experiments, therefore, showed In prenatal diagnosis of genetic disorders, there is that hybridization probes can dis- a need to determine which alleles of a particular criminate between a fully complementary DNA and locus are being carried by the fetus, i.e. is the fetus one containing a single mismatched base. Similar homozygous for the normal or the deleterious allele results have been obtained (Fig. 14.3) with a point or is it heterozygous? In forensic DNA profiling the mutation in the α-antitrypsin gene, which is implic- requirement is to match DNA from the perpetrator ated in pulmonary emphysema (Cox et al. 1985). of a crime with that of a suspect. In each case, a The single-base changes that occur in the two definitive answer could be obtained by sequencing clinical examples just quoted are examples of single- the relevant samples of DNA. While this is possible, it nucleotide polymorphisms (SNPs, pronounced is not practicable for mass screening. An alternative ‘snips’). Many such polymorphisms occur through- could be to detect hybridization of specific probes. out an entire and in humans the frequency This has been done for the detection of sickle-cell is about once every 1000 bases. Their distribution is anaemia by Conner et al. (1983). They synthesized not entirely random. SNPs that alter amino acid

Marker Marker DNAMM MZ ZZ DNAMM MZ ZZ

Fig. 14.3 Schematic representation of the use of oligonucleotide probes α to detect the normal 1-antitrypsin gene (M) and its Z variant. Human DNA obtained from normal (MM), heterozygous (MZ) and homozygous variant (ZZ) subjects is digested with a restriction endonuclease, electrophoresed and fragments Southern blotted on to a nitrocellulose membrane. The patterns shown were obtained on autoradiography of the filter following hybridization with either the normal (M-specific) or M-specific probe Z-specific probe variant (Z-specific) probe. (C TTT CTC GTC GAT GGT CAG) (C TTT CTT GTC GAT GGT CAG) POGC14 9/11/2001 11:11 AM Page 278

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(a) (b)

MstII MstII MstII AA Amn AS SS *

β-globin gene

* ProGlu Glu Base sequence in CCTGAGGAG normal (βA) gene 576 Base sequence in βS CCTGTGGAG mutant (sickle, βS) gene βA Pro Val Glu

Fig. 14.4 Antenatal detection of sickle-cell genes. Normal individuals are homozygous for the βA allele, while sufferers from sickle-cell anaemia are homozygous for the βS allele. Heterozygous individuals have the genotype βAβS. In sickle-cell anaemia, the 6th amino acid of β-globin is changed from glutamate to valine. (a) Location of recognition sequences for restriction endonuclease MstII in and around the β-globin gene. The change of A → T in codon 6 of the β-globin gene destroys the recognition site (CCTGAGG) for MstII as indicated by the asterisk. (b) Electrophoretic separation of MstII-generated fragments of human control (AA, AS, SS) and DNA from amniocytes (Amn). After Southern blotting and probing with a cloned β-globin gene, the normal gene and the sickle gene can be clearly distinguished. Examination of the pattern for the amniocyte DNA indicates that the fetus has the genotype βAβS, i.e. it is heterozygous.

sequences occur much less frequently than silent between a polymorphic restriction site and the locus substitutions and SNPs in non-coding regions (Cargill of interest and this can be used to trace the inher- et al. 1999). However, they are stably inherited. itance of the gene. When this approach was first In some instances, these polymorphisms result in developed, a major limitation was the availability the creation or elimination of a restriction- of suitable polymorphic markers. Following the site and this can be used diagnostically. A classical sequencing of the entire human genome, an ency- example is sickle-cell anaemia (Fig. 14.4), where clopedia of SNPs is being created (currently 1.4 the mutation from GAG to GTG eliminates restric- million) and this will greatly facilitate association tion sites for the enzymes DdeI (CTNAG) and MstII studies. Indeed, this approach is now being used to (CCTNAGG) (Chang & Kan 1981, Orkin et al. 1982). match patients with appropriate drugs (see p. 292). The mutation has been detected by digesting mutant Again, when this approach was developed, RFLPs and normal DNA with the and were detected by genomic Southern blotting. This performing a Southern-blot hybridization with a laborious step can be bypassed by use of the PCR. cloned β-globin DNA probe. Enough DNA can be synthesized in the PCR reaction Many of the SNPs that cause genetic diseases so that after digestion with the restriction enzyme do not lie within a restriction-enzyme site, as is the and electrophoresis, the DNA bands are directly case in sickle-cell anaemia. However, the restriction visible following staining with ethidium bromide. fragment length poymorphisms (RFLPs) caused by Another advantage of PCR is that the gel step can be other SNPs can be used diagnostically, as shown omitted altogether and the SNPs detected directly, in Fig. 14.5. In this case there is a close linkage using microarrays (‘DNA chips’) (p. 116). POGC14 9/11/2001 11:11 AM Page 279

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unique sequences and repetitive sequences. Many of 10 kb the repetitive DNA sequence elements are arranged 10 kb in tandem and are known as satellite DNA. Three M N types of satellite DNA can be distinguished on the basis of the level of repetition and the repeat-unit P 7 kb M A length (Table 14.2). Not only is satellite DNA dis- persed throughout the genome, it is highly variable 7 kb Gene map of and provides a valuable tool for genetic individual- Chromosomes of parents parents ization. An example of this is shown schematically in Fig. 14.6. In this case the RFLPs detected are due to variations in the number of repeat units (VNTR – – Mother + Father + polymorphisms) between restriction sites, rather 10 kb than changes in the location of the restriction sites, as discussed in the previous section. VNTR polymorphisms are of importance to 7 kb + + Brother + Fetus + clinical geneticists because a number of important hereditary diseases are associated with alterations Gene map of in the degree of repetition of microsatellites (for Family studied by gene mapping brother and fetus reviews see Bowater & Wells 2000, Gutekunst et al. 2000, Usdin & Grabczyck 2000). Probably the best Fig. 14.5 An example of prenatal diagnosis using length polymorphism (RFLP) linkage analysis. The example of such a disease is Huntington’s chorea, parents are both carriers for a deleterious gene (A): one of which is caused by an expansion of a CAG trinu- their chromosomes carries this determinant, the other its cleotide repeat in exon 1 of the gene coding for a normal allele (N). One of the parental chromosomes carries protein of unknown function, which has been a polymorphic restriction enzyme site P, which is close named huntingtin. Expansion beyond 40 repeat enough to A or N for them not to be separated in successive generations. On the chromosome which does not contain this units correlates with the onset and progression of site (–), a particular restriction enzyme cuts out a piece of DNA the disease (for review see Reddy et al. 1999). 10 kb long which contains another locus (M), for which we have a radioactive probe. On the chromosome containing the polymorphism (+), a single base change produces a new site Forensic applications of VNTRs and hence the DNA fragment containing locus M is now only 7 kb. On gene mapping of the parents’ DNA using probe M, The existence of VNTR polymorphisms is of great we see two bands representing either the + or − chromosomes. utility in paternity testing and criminal investigations, A previously born child had received the deleterious gene since they allow ready comparison of DNA samples A from both parents and on mapping we find that it has the in the absence of detailed genetic information by the ++ chromosome arrangement, i.e. only a single 7 kb band. generation of a DNA profile or fingerprint. In prin- Hence the mutation must be on the + chromosome in both parents. To identify the disease in a fetus in subsequent ciple, a multilocus DNA fingerprint can be generated pregnancies, we shall be looking for an identical pattern, either by the simultaneous application of several i.e. the 7 kb band only. (Reproduced courtesy of Professor probes, each one specific for a particular locus, or by D. Weatherall and Oxford University Press.) applying a single DNA probe that simultaneously detects several loci. When DNA profiling was first developed (Jeffreys et al. 1985a), multilocus probes were used and these were derived from a tandemly Variable number tardem repeat repeated sequence within an intron of the myo- (VNTR) polymorphisms globin gene (Fig. 14.7). These probes can hybridize In higher eukaryotes, genes and their associated to other autosomal loci – hence their utility. The introns occupy only a small proportion of the first criminal court case to use DNA fingerprinting genome. The intergenic DNA, which makes up the was in Bristol, UK, in 1987, when a link was shown majority of the genome, is composed of a mixture of between a burglary and a rape. In the following POGC14 9/11/2001 11:11 AM Page 280

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Table 14.2 Classification of satellite DNA. Degree of repetition Repeat-unit Type of repeat per locus Number of loci length (bp)

Satellite 103–107 One to two 1000–3000 per chromosome Minisatellite 10–103 Thousands per 9–100 genome Microsatellite 10–102 Up to 105 1–6 per genome

H1 H1 A Core sequence G G A G G T G G G C A G G A G G Individual A Probe 33.6 [(A G G G C T G G A G G )3]18

Probe 33.15 (A G A G G T G G G C A G G T G G)29 C Probe 33.5 (G G G A G T G G G C A G G A G G)14 Individual B Fig. 14.7 Probes used for DNA fingerprinting.

Multilocus probes can also be used to prove or Individual C disprove paternity and a unique example, which was part of an immigration test case, is shown in Box 14.1. The technique also has application in many other areas, such as pedigree analysis in cats and dogs ( Jeffreys & Morton 1987), confirming cell-line authenticity in animal cell cultures (Devor et al. 1988, Stacey et al. 1992) and monitoring the behaviour and breeding success of bird populations (Burke & Bruford 1987). In criminal cases, a major disadvantage of multi-

Individual Individual Individual locus probes is the complexity of the DNA fingerprint A B C provided. Showing innocence is easy, but proving identity is fraught with problems. The issue boils Fig. 14.6 Restriction fragment length polymorphisms down to calculations of the probability that two caused by a variable number of tandem repeats between profiles match by chance as opposed to having come the two HinfI restriction sites. The upper part of the diagram shows the DNA structure for three different individuals. from the same person (Lewontin & Hartl 1991). For The lower part of the diagram shows the pattern obtained on this reason forensic scientists have moved to the use electrophoresis of HinfI cut DNA from the three individuals of single-locus probes and an example is shown in after hybridization with a probe complementary to the Fig. 14.8. The latest variation of the technique, sequence shown in pink. introduced in the UK in 1999, targets 10 distinct loci, and the likelihood of two people sharing the year, DNA-fingerprinting evidence was used in the same profile is less than one in a billion (thousand USA. It is worth noting that DNA evidence has been million). Chance matches are even less likely in the used to prove innocence as well as guilt (Gill & USA, where the FBI routinely examines 13 VNTR Werrett 1987). loci. Another advantage of single-locus probes is POGC14 9/11/2001 11:11 AM Page 281

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Box 14.1 Use of DNA fingerprinting in an immigration test case

In 1984, a Ghanaian boy was refused entry into were present in the mother and/or at least one of the Britain because the immigration authorities were siblings. Analysis of the data showed the following. not satisfied that the woman claiming him as her son • The probability that either the mother or the was in fact his mother. Analysis of serum proteins and father by chance possess all 61 of the alleged son’s erythrocyte antigens and enzymes showed that the bands is 7 × 10−22. Clearly the alleged son is part alleged mother and son were related but could not of the family. determine whether the woman was the boy’s mother • There were 25 maternal-specific fragments in or aunt. To complicate matters, the father was not the 61 identified in the alleged son and the chance available for analysis nor was the mother certain probability of this is 2 × 10−5. Thus the mother and of the boy’s paternity. DNA fingerprints from blood alleged son are related. samples taken from the mother and three children • If the alleged mother of the boy in question is in who were undisputedly hers, as well as the alleged fact a maternal aunt, the chance of her sharing the son, were prepared by Southern blot hybridization to 25 maternal-specific fragments with her sister is two of the mini-satellite probes shown in Fig. B14.1. 6 × 10−6. Although the father was absent, most of his DNA When presented with the above data (Jeffreys et al. fingerprint could be reconstructed from paternal- 1985b), as well as results from conventional marker specific DNA fragments present in at least one of analysis, the immigration authorities allowed the boy the three undisputed siblings but absent from the residence in Britain. In a similar kind of investigation, mother. The DNA fingerprint of the alleged son a man originally charged with murder was shown to contained 61 scorable fragments, all of which be innocent (Gill & Werrett 1987).

U M X B S1 S2 MXBS1S2 kb 20

10

8

6 Fig. B14.1 DNA fingerprints of a Ghanaian family involved in an immigration dispute. Fingerprints of blood DNA are shown for the mother (M), the boy in dispute (X), his brother (B), sisters (S1, S2) and an unrelated 4 individual (U). Fragments present in the mother’s (M) DNA are indicated by a short horizontal line (to the right of each fingerprint); paternal fragments absent from M but present in at least one of the undisputed siblings (B, S1, S2) are marked with a long line. Maternal and paternal fragments transmitted to X are shown with 2 a dot. (Photo courtesy of Dr A. Jeffreys and the editor of Nature.) POGC14 9/11/2001 11:11 AM Page 282

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CADAnorak Swab D A C worn by a bank robber, a cigarette-butt discarded at the scene of a crime or the back of a stamp on an envelope used to send a ‘poison-pen’ or blackmail letter.

Historical Just as multilocus probes have been used for many applications other than in crime testing, so too have single-locus probes. A good example is the determina- tion of the parentage of grapevines used for wine making. Grapevines are propagated vegetatively, so that individual vines of a cultivar are genetically identical to each other and to the single original seedling from which the cultivar originated. Most of the cultivars in existence in north-eastern France are centuries old and their provenance was not known. However, using 17 microsatellite loci, 4 kb Bowers et al. (1999) were able to show that 16 of the common cultivars have genotypes consistent with their being progeny of a single pair of grape cultivars that were widespread in the region in the Middle Ages. VNTRs can be found in mitochondrial DNA, as well as nuclear DNA, and these have particular applications. The reasons for this are threefold. First, mitochondrial sequences are passed from mother to child in the egg. Thus, brothers and sisters have identical mitochondrial DNA. Secondly, the small size of mitochondrial DNA (16–20 kb) means that Fig. 14.8 Use of a single locus probe to determine the identity of a rapist. Semen was extracted from an anorak there is less scope for variability, but this is more and a vaginal swab. The victim’s profile is in track D and than compensated for by the copy number (~10 000 that of two suspects in tracks A and C. The profile matches copies per cell). That is, mitochondrial DNA is individual A. (Photo courtesy of Dr. P. Gill.) naturally amplified. Thirdly, in very old or degraded specimens, the nuclear DNA may be totally de- composed, but mitochondrial DNA can still be that it is possible to convert the DNA profile into a recovered. For example, mitochondrial-DNA ana- numerical format. This enables a database to be lysis was used to confirm that skeletons found in established and all new profiles can be matched to Ekaterinburg, Russia, were the remains of the last that database. tsar and his family (Gill et al. 1994). A similar ana- Detection of VNTR polymorphisms requires that lysis showed that an individual living in Cheddar an adequate amount of DNA be present in the test Gorge in the UK was related to a Stone Age indi- sample. This is not a problem in paternity disputes vidual whose skull was found nearby. Since bones but can be an issue in forensic testing. With the are more likely than soft tissue to survive in the advent of single-locus probes, the amount of DNA event of major accidents that involve fire, mito- required is much less of an issue, since the test loci chondrial DNA analysis will play an increasingly in the sample can be amplified by PCR. As a result, important role in identifying victims. Indeed, such it now is possible to type DNA from a face-mask an analysis was done in the UK following the 1999 POGC14 9/11/2001 11:11 AM Page 283

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Paddington train crash, in which one carriage was The first generation of protein drugs were exact completely incinerated. copies of the human molecules but protein engineer- Just as the mitochondrion is transmitted maternally, ing is now being used to develop second-generation the Y chromosome is transmitted only through male molecules with improved properties (see theme 4, descendants. Because there is only a single copy of p. 299). More recently, macromodifications have the Y chromosome in normal diploid cells, recom- been made to proteins, as exemplified by the recently bination between different Y chromosomes does not approved drug (Table 14.3) for rheumatoid arth- occur. Any changes that do occur in the Y chromo- ritis, which consists of the tumour necrosis factor some from generation to generation must arise receptor fused to the Fc portion of human IgG1. from DNA rearrangements or by accumulation of random mutations. That is, the Y chromosome Transgenic animals and plants as should be highly conserved. Sykes and Irven (2000) bioreactors: ‘pharming’ obtained proof of this. They probed a randomly ascertained sample of males with the surname ‘Sykes’ ‘Pharming’ is the play on words that refers to the use with four Y-chromosome microsatellites and found of transgenic animals and plants to produce recom- that half of them had the same Y haplotype. This binant therapeutic proteins. As discussed in earlier suggests that all those with the same haplotype have chapters, recombinant-protein synthesis in animal a common ancestor, even though conventional cells has a number of advantages over microbial genealogical analysis suggests otherwise. expression systems, the most important of which is the authentic post-translational modifications that are performed in animal cells. However, large- Theme 2: New drugs and new therapies scale culture of animal cells is expensive, due to for genetic diseases the amount of medium and serum required and the necessity for precise and constant growth condi- Introduction to theme 2: tions. The production of in the proteins as drugs serum of transgenic mice (Palmiter et al. 1982a) (see One of the earliest commercial applications of p. 209) provided the first evidence that recombinant gene-manipulation techniques was the production proteins could be produced, continuously, in the in bacteria of human proteins with therapeutic body fluids of animals. Five years later, several groups applications. Not surprisingly, the first such prod- reported the secretion of recombinant proteins in ucts were recombinant versions of proteins already mouse milk. In each case, this was achieved by join- used as therapeutics: human growth hormone and ing the to a mammary-specific , insulin. Prior to the advent of , such as that from the casein gene. The first proteins human growth hormone was produced from pitu- produced in this way were sheep β-lactoglobulin itary glands removed from cadavers. Not only did (Simons et al. 1987) and human tissue-plasminogen this limit the supply of the hormone but, in some activator (tPA) (Gordon et al. 1987, Pittius et al. cases, it resulted in recipients contracting Creutzfeld– 1988). There have been over 100 such reports since Jakob syndrome. The recombinant approach resulted these early experiments, and a selection is listed in in unlimited supplies of safe material. This safety Table 14.4. aspect has been extended to various clotting factors Although proteins can be produced at high con- that were originally isolated from blood but now centrations in mouse milk (e.g. 50 ng/ml for tPA), carry the risk of HIV infection. As the methods for the system is not ideal, due to the small volume of genes became more and more sophisticated, milk produced. Therefore, other animals, such as an increasing number of lymphokines and sheep and goats, have been investigated as pos- were identified and significant amounts of them sible bioreactors. Such animals not only produce produced for the first time. A number of these were large volumes of milk, but the regulatory practices shown to have therapeutic potential and found their regarding the use of their milk are more accept- way into clinical practice (Table 14.3). able. An early success was Tracy, a transgenic ewe POGC14 9/11/2001 11:11 AM Page 284

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Table 14.3 Some recombinant proteins that are used therapeutically.

Year Product Clinical indication

1982 Human insulin 1985 Human growth hormone Pituitary dwarfism 1986 Prevention of hepatitis B infection

Interferon-a2a Hairy-cell leukaemia 1987 Tissue plasminogen activator Acute myocardial infarction 1989 Erythropoietin Anaemia associated with chronic renal failure

1990 Interferon-g1b Chronic granulomatous disease 1991 Granulocyte–macrophage-colony-stimulating factor Bone-marrow transplant Granulocyte-colony-stimulating factor Chemotherapy-induced neutropenia 1992 Human interleukin-2 Renal-cell carcinoma Factor VIII A 1993 Human DNase Cystic fibrosis 1994 Glucocerebrosidase Gaucher’s disease

1996 Interferon-b1a Multiple sclerosis 1997 Factor IX Haemophilia B Consensus interferon Chronic HCV infection Platelet growth factor Chemotherapy-induced thrombocytopenia Platelet-derived growth factor b Lower-extremity diabetic ulcers 1998 Tumour necrosis factor receptor linked to Fc portion Rheumatoid arthritis of human IgG1 Glucagon Hypoglycaemia 1999 Factor VIIa Haemophilia

Table 14.4 Some recombinant proteins produced in the secretions of animal bioreactors.

System Species Product Reference

Milk Mouse Sheep b-lactoglobulin Simons et al. 1987 Human tissue-plasminogen activator Gordon et al. 1987 Human urokinase Meade et al. 1990 Human growth hormone Devinoy et al. 1994 Human fibrinogen Prunkard et al. 1996 Human nerve growth factor Coulibaly et al. 1999 Spider silk Karatzas et al. 1999 Rabbit Human erythropoietin Massoud et al. 1996

Sheep Human a1-antitrypsin Wright et al. 1991 Goat Human tissue-plasminogen activator Ebert et al. 1991

Blood serum Rabbit Human a1-antitrypsin Massoud et al. 1991 Pig Recombinant Lo et al. 1991, Weidle et al. 1991 Urine Mouse Human growth hormone Kerr et al. 1998 Semen Mouse Human growth hormone Dyck et al. 1999 POGC14 9/11/2001 11:11 AM Page 285

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producing extremely high levels (30 g/l) of human Chapter 10). The use of animals as bioreactors has α 1-antitrypsin (AAT) in her milk (Wright et al. 1991). been extensively reviewed (Clark 1998, Rudolph Artificially inseminated eggs were microinjected 1999, Wall 1999, Houdebine 2000). with a DNA construct containing an AAT gene fused to a β-lactoglobin promoter. These eggs were Plants as bioreactors implanted into surrogate mothers, of which 112 gave birth. Four females, including Tracy, and one Plants are a useful alternative to animals for male were found to have incorporated intact copies recombinant- because they are of the gene and all five developed normally. Over the inexpensive to grow and scale-up from laboratory lactation period, sheep can produce 250–800 l of testing to commercial production is easy. Therefore, milk, so the production potential is significant. there is much interest in using plants as production Using similar protocols, Ebert et al. (1991) have systems for the synthesis of recombinant proteins demonstrated the production of a variant of human and other speciality chemicals. There is some con- tPA in goat milk. Of 29 offspring, one male and one cern that therapeutic molecules produced in animal female contained the transgene. The transgenic expression systems could be contaminated with female underwent two pregnancies and one out of small quantities of endogenous viruses or prions, a five offspring was transgenic. Milk collected over her risk factor that is absent from plants. Furthermore, first lactation contained only a few milligrams of tPA plants carry out very similar post-translational modi- per litre, but improved expression constructs have fication reactions to animal cells, with only minor since resulted in an animal generating several differences in glycosylation patterns (Cabanes- grams per litre of the protein. Recombinant human Macheteau et al. 1999). Thus plants are quite III, which is used to prevent blood suitable for the production of recombinant human clots forming in patients that have undergone heart- proteins for therapeutic use. bypass operations, was the first protein expressed A selection of therapeutic proteins that have been in transgenic animal milk to reach commercial expressed in plants is listed in Table 14.5. The first production, and is currently marketed by Genzyme such report was the expression of human growth Transgenics Corporation. hormone, as a fusion with the nopal- The production of foreign proteins in secreted ine synthase enzyme, in transgenic and body fluids has the obvious advantage that trans- sunflower (Barta et al. 1986). Tobacco has been the genic animals can be used as a renewable source of most frequently used host for recombinant-protein the desirable molecule. In addition to milk, other expression although edible crops, such as rice, are production systems have been investigated, includ- now becoming popular, since recombinant proteins ing serum (Massoud et al. 1991), semen (Dyck et al. produced in such crops could in principle be admin- 1999) and urine (Kerr et al. 1998). In each case, an istered orally without purification. The expression of important consideration is whether the protein is human antibodies in plants has particular relevance stable and whether it folds and assembles correctly. in this context, because the consumption of plant The assembly of complex proteins comprising up to material containing recombinant antibodies could three separate polypeptides has been demonstrated provide passive immunity (i.e. immunity brought in milk, e.g. fibrinogen (Prunkard et al. 1996), colla- about without stimulating the host immune system). gen ( John et al. 1999) and various immunoglobulins production in plants was first demon- (e.g. Castilla et al. 1998). Other abundantly secreted strated by Hiatt et al. (1989) and During et al. fluids that are likely to be exploited for recombinant- (1990), who expressed full-size immunoglobulins in protein expression in the future include the albumen tobacco leaves. Since then, many different types of of hens’ eggs and silkworm cocoons. There has antibody have been expressed in plants, predomin- already been some success with the latter, using antly tobacco, including full-size immunoglobulins, both (e.g. Nagaraju et al. 1996) and Fab fragments and single-chain Fv fragments (scFvs). infection of silkworm larvae with baculovirus For example, a fully against vectors (Tamura et al. 1999, Yamao et al. 1999) (see herpes simplex virus-2 (HSV-2) has been expressed POGC14 9/11/2001 11:11 AM Page 286

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Table 14.5 A selection of pharmaceutical recombinant human proteins expressed in plant systems.

Species Recombinant human product Reference

Tobacco, sunflower (plants) Growth hormone Barta et al. 1986 Tobacco, potato (plants) Serum albumin Sijmons et al. 1990 Tobacco (plants) Higo et al. 1993 Rice (plants) a-Interferon Zhu et al. 1994 Tobacco () Erythropoietin Matsumoto et al. 1995 Tobacco (plants) Haemoglobin Dieryck et al. 1997 Tobacco (cell culture) Interleukins-2 and 4 Magnuson et al. 1998 Tobacco (root culture) Placental alkaline phosphatase Borisjuk et al. 1999

Rice (cell culture) a1-Antitrypsin Terashima et al. 1999 Tobacco (seeds) Growth hormone Leite et al. 2000 Tobacco (chloroplasts) Growth hormone Staub et al. 2000

in soybean (Zeitlin et al. 1998). Even secretory IgA 2001). These genes will then become targets for new (sIgA) antibodies, which have four separate poly- small-molecule drugs. Already drugs have been peptide components, have been successfully expressed developed based on such genotype– in plants. This experiment involved the generation correlations. For example, Wettereau et al. (1998) of four separate transgenic tobacco lines, each identified a molecule that normalizes atherogenic expressing a single component, and the sequential lipoprotein levels caused by a genetic deletion of a crossing of these lines to generate plants in which microsomal triglyceride transfer protein. all four were stacked (Ma et al. 1995). The identification of genetic changes associated Plants producing recombinant sIgA against the oral with particular disease offers a number pathogen Streptococcus mutans have been generated of novel approaches to the development of therapies. (Ma et al. 1998), and these plant-derived antibodies As well as using such changes as novel targets for (‘plantibodies’) have recently been commercially small-molecule drug design, there is an opportunity produced as the drug CaroRxTM, marketed by Planet to use the techniques described in Chapter 11 to gen- Biotechnology Inc. A number of other biotechnology erate animals with the exact same genetic defect and companies are bringing antibody-expressing trans- which can be used as models to test new drug candid- genic plants into commercial production (see Fischer ates (disease modelling). Furthermore, where drugs & Emans 2000). cannot be developed to treat a particular disorder, there might be an opportunity to correct the disease by further modification to the genome (gene therapy). The impact of Finally, it is likely that, in the near future, transgenic Many of the drugs currently on the market treat the animals could be used to provide healthy organs for symptoms of the disease rather than the cause of the humans requiring transplants (xenotransplantation) disease. This is analogous to reversing a mutant (Box 14.2). These topics are discussed in more detail phenotype by selecting a mutation at a second below. site. Not surprisingly, many of these drugs have side-effects quite separate from those (e.g. toxicity) Transgenic animals as models caused by their metabolism. Now that the first drafts of human disease of the human genome sequence are available, it will be possible to convert disease phenotypes into Mammals have been used as models for human nucleotide changes in specific genes (Bailey et al. disease for many years, since they can be exploited to POGC14 9/11/2001 11:11 AM Page 287

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Box 14.2 Xenotransplantation

Transplantation is widely used to treat organ failure the simplest strategy would be to knock out the gene but there is a shortage of organ donors resulting in by . has long waiting times and many unnecessary deaths. not yet been achieved in pigs, although the success In the future, transgenic animals could be used to of gene targeting/nuclear transfer in sheep (Chapter supply functional organs to replace failing human 11) suggests that knockout pigs could be produced ones. This process is termed xenotransplantation. in the next few years. Alternative procedures include There is vigorous debate concerning the ethics of introducing genes encoding other carbohydrate- xenotransplantation but, ethics aside, the technique metabolizing enzymes, so that the Gal-a(1,3)- remains limited by the phenomenon of hyperacute Gal groups are modified into some other less rejection, which is caused by the host immune immunogenic moiety (e.g. Sandrin et al. 1995, system. Hyperacute rejection is dependent both on Cohney et al. 1997, Osman et al. 1997). Once antibodies raised against the foreign organ and the hyperacute rejection has been overcome, there activation of the host complement system. In both may be further problems, including delayed rejection, cases, the major trigger for rejection appears to be a involving natural killer cells and macrophages (Bach disaccharide group (Gal-a(1,3)-Gal) which is present et al. 1996), and the requirement for T-cell tolerance in pigs but not in primates (Cooper et al. 1994, (Bracy et al. 1998, Kozlowski et al. 1998, Yang et al. Sandrin et al. 1994). 1998). There are also concerns that endogenous Transgenic strategies have been investigated to pig retroviruses could be activated following avoid hyperacute rejection, including the expression transplantation, perhaps even recombining with of complement-inactivating protein on the cell human retroviruses to produce potent new hybrids surface (Cozzi & White 1995, reviewed by Pearse with unknown properties. Detailed studies have et al. 1997), the expression of antibodies against so far shown no evidence of such a phenomenon (e.g. the disaccharide group (Vanhove et al. 1998) Heneine et al. 1998, Patience et al. 1998). For recent and attempts to inhibit the expression of a(1,3) reviews on the prospects of xenotransplantation galactosyltransferase, the enzyme that forms this (particularly porcine-to-human), the reader should particular carbohydrate linkage, an enzyme that is consult Lambrigts et al. (1998), Sandrin and present in pigs but not in primates. In the latter case, McKenzie (1999) and Logan (2000).

carry out detailed analyses of the molecular basis of oncogenes (e.g. Sinn et al. 1987). This exemplifies disease and to test newly developed therapeutics so-called gain-of-function diseases, which are caused prior to clinical trials in humans. Before the advent by a dominantly acting allele and can be modelled of transgenic animal technology, however, models simply by adding that allele to the normal genome, of inherited diseases (i.e. diseases with a genetic basis) e.g. by microinjection into eggs. Other gain of func- were difficult to come by. They could be obtained as tion diseases that have been modelled in this way spontaneously occurring mutants, suitable mutant include the Gerstmann–Straussler–Scheinker (GSS) animals identified in mutagenesis screens and sus- syndrome, a neurodegenerative disease caused by a ceptible animal strains obtained by selective breeding. dominantly acting mutated prion protein gene. In Gene manipulation now offers a range of alternative one patient suffering from this disease, a mutation strategies to create specific disease models (see was identified in codon 102 of the prion protein reviews by Smithies 1993, Bedell et al. 1997, Petters gene. Transgenic mice were created carrying this & Sommer 2000). mutant form of the gene in addition to the wild-type Some of the earliest transgenic disease models locus and were shown to develop a similar neuro- were mice predisposed to particular forms of cancer, degenerative pathology to their human counter- because the germ line contained exogenously derived parts (Hsiao et al. 1990). Other examples of POGC14 9/11/2001 11:11 AM Page 288

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gain-of-function disease models include Alzheimer’s symptoms (Blouin et al. 2000). Similarly, crossing disease, which was modelled by overexpression of the transgenic mice overexpressing the anti-apoptotic amyloid precursor protein (Quon et al. 1991), and protein Bcl-2 to rds mutants, which show inherited the triplet-repeat disorder spinocerebellar ataxia slow retinal degeneration, resulted in hybrid off- type 1 (Burright et al. 1995). Simple transgene addi- spring in which retinal degeneration was strikingly tion can also be used to model diseases caused by reduced. This indicates that Bcl-2 could possibly dominant negative alleles, as recently shown for be used in gene therapy to treat the equivalent the premature ageing disease, Werner’s syndrome human retinal-degeneration syndrome (Nir et al. (Wang et al. 2000). 2000). Recessively inherited diseases are generally caused The most complex diseases involve many genes, by loss of function, and these can be modelled by and transgenic models would be difficult to create. . The earliest report of this strategy However, it is often the case that such diseases can was a mouse model for hypoxanthine-guanine be reduced to a small number of ‘major genes’ with phosphoribosyltransferase (HPRT) deficiency, gen- severe effects and a larger number of minor genes. erated by disrupting the gene for HRPT (Kuehn et al. Thus, it has been possible to create mouse models of 1987). A large number of genes have been modelled Down’s syndrome, which in humans is generally in this way, including those for cystic fibrosis (Dorin caused by the presence of three copies of chromo- et al. 1992, Snouwaert et al. 1992), fragile-X syn- some 21. Trisomy for the equivalent mouse chro- drome (Dutch–Belgian Fragile X Consortium, 1994), mosome 16 is a poor model because the two β-thalassaemia (Skow et al. 1983, Ciavattia et al. chromosomes do not contain all the same genes. 1995) and mitochondrial cardiomyopathy (Li et al. However, a critical region for Down’s syndrome has 2000). Gene targeting has been widely used to been identified by studying Down’s patients with model human cancers caused by the inactivation partial deletions of chromosome 21. The generation of tumour suppressor genes, such as TP53 and of yeast artificial chromosome (YAC) transgenic RB1 (reviewed by Ghebranious & Donehower 1998, mice carrying this essential region provides a useful Macleod & Jacks 1999). model of the disorder (Smith et al. 1997) and has While the studies above provide models of single- identified increased dosage of the Dyrk1a (minibrain) gene defects in humans, attention is now shifting gene as an important component of the learning towards the modelling of more complex diseases, defects accompanying the disease. Animal models of which involve multiple genes. This is a challenging Down’s syndrome have been reviewed (Kola & area of but there have been some encour- Hertzog 1998, Reeves et al. 2001). aging early successes. In many cases, the crossing of different modified mouse lines has led to interesting Gene transfer to humans – gene therapy discoveries. For example, undulated mutant mice lack the gene encoding the factor Pax-1, The scope of gene therapy and Patch mutant mice are heterozygous for a null allele of the platelet-derived growth-factor gene. Gene therapy is any procedure used to treat disease Hybrid offspring from a mating between these two by modifying the genetic information in the cells of strains were shown to model the human birth defect the patient. In essence, gene therapy is the antithesis spina bifida occulta (Helwig et al. 1995). In other of the disease modelling discussed above. Whereas cases, such crosses have pointed the way to possible disease modelling takes a healthy animal and uses novel therapies. For example, transgenic mice over- gene-manipulation techniques to induce a specific expressing human α-globin and a mutant form of disease, gene therapy takes a diseased animal (or the human β-globin gene that promotes polymeriza- human) and uses gene-manipulation techniques tion provide good models of sickle-cell anaemia in an attempt to correct the disorder and return (Trudel et al. 1991). However, when these mice are the individual to good health. Gene transfer can be crossed to those ectopically expressing human fetal carried out in cultured cells, which are then reintro- haemoglobin in adulthood, the resulting transgenic duced into the patient, or DNA can be transferred hybrids show a remarkable reduction in disease to the patient in vivo, directly or using viral vectors. POGC14 9/11/2001 11:11 AM Page 289

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X Mutant (disease) gene – loss of function

D Mutant (disease) gene – dominant gain of function

1 Gene augmentation therapy Functional gene

X X

2 Gene inhibition therapy Antisense gene etc.

D D

3 Gene targeting Homologous functional gene

Xor D

4 Assisted killing Antigen, etc.

D Death D Immune system stimulated

5 Prodrug therapy

TK etc Ganciclovir

D Death D Fig. 14.9 Overview of gene-therapy Confers strategies. sensitivity

The ex vivo approach can be applied only to certain • Gene-augmentation therapy (GAT), where DNA is tissues, such as bone marrow, in which the cells are added to the genome with the aim of replacing a amenable to culture. Gene therapy can be used to missing gene product. treat diseases caused by mutations in the patient’s • Gene targeting to correct mutant alleles. own DNA (inherited disorders, cancers), as well as • Gene-inhibition therapy, using techniques such infectious diseases, and is particularly valuable in as antisense RNA expression or the expression of cases where no conventional treatment exists or intracellular antibodies to treat dominantly acting where that treatment is inherently risky. Strategies diseases. include the following (Fig. 14.9): • The targeted ablation of specific cells. POGC14 9/11/2001 11:11 AM Page 290

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Therapeutic gene transfer effectively generates met. Tumour-infiltrating lymphocytes (cells that transgenic human cell clones and, for this reason, naturally seek out cancer cells and then kill them by only somatic cells can be used as targets. The prospect secreting proteins such as tumour necrosis factor of germ-line transgenesis in humans raises serious (TNF)) were isolated from patients with advanced ethical concerns and, with the rapid advances in cancer. The cells were then genetically marked with technology allowing germ-line transformation and a neomycin-resistance gene and injected back into nuclear transfer in numerous mammals, these the same patient (Rosenberg et al. 1990). concerns will need to be addressed in the very near The first clinical trial using a therapeutic gene- future ( Johnson 1998). As an alternative to perman- transfer procedure involved a 4-year-old female ent gene transfer, transient gene therapy can be patient, Ashanthi DeSilva, suffering from severe com- achieved using oligonucleotides, which can disrupt bined immune deficiency, resulting from the absence at many levels but do not per- of the enzyme adenosine deaminase (ADA). This dis- manently change the genetic material of the cell ease fitted many of the ideal criteria for gene-therapy (Pollock & Gaken 1995). experimentation. The disease was life-threatening The tools and techniques for gene therapy are (therefore making the possibility of unknown treat- essentially similar to those used for gene transfer to ment-related side-effects ethically acceptable), but any animal cells. , direct delivery or the corresponding gene had been cloned and the (see Chapter 11) can be used to intro- biochemical basis of the disease was understood. duce DNA into cells. Viral vectors are most popular Importantly, since ADA functions in the salvage because of their efficiency of gene transfer in vivo. pathway of nucleotide biosynthesis (p. 177), cells However, extreme precautions need to be taken to in which the genetic lesion had been corrected had ensure the safety of such vectors, avoiding potential a selective growth advantage over mutant cells, problems, such as the production of infectious viruses allowing them to be identified and isolated . by recombination and the pathological effects of Conventional treatment for ADA deficiency involves viral replication. A number of viral vectors have bone-marrow transplantation from a matching donor. been developed for gene therapy, including those Essentially the same established procedure could be based on oncoretroviruses, lentiviruses, adenovirus, used for gene therapy, but the bone-marrow cells adeno-associated virus, herpes virus and a number would be derived from the patient herself and would of hybrid vectors combining advantageous elements be genetically modified ex vivo (Fig. 14.10). Cells of different parental viruses (Robbins et al. 1998, from the patient were subjected to leucophaeresis Reynolds et al. 1999). The risks associated with viral and mononuclear cells were isolated. These were vectors have promoted research into other delivery grown in culture under conditions that stimulated methods, the most popular of which include direct T-lymphocyte activation and growth and then injection of DNA into tissues (e.g. muscle), the injec- transduced with a retroviral vector carrying a nor- tion of liposome–DNA complexes into the blood and mal ADA gene as well as the neomycin-resistance direct transfer by particle bombardment. Although gene. Following infusion of these modified cells, both inherently much safer than viruses, such procedures this patient and a second, who began treatment in show a generally low efficiency (Scheule & Cheng early 1991, showed an improvement in their clin- 1996, Tseng & Huang 1998). ical condition as well as in a battery of in vitro and in vivo immune-function studies (Anderson 1992). However, the production of recombinant ADA in Gene-augmentation therapy for recessive diseases these patients is transient, so each must undergo The first human genetic-engineering experiment was regular infusions of recombinant T lymphocytes. one of gene marking, rather than gene therapy, and Research is ongoing into procedures for the trans- was designed to demonstrate that an exogenous formation of bone-marrow stem cells, which would gene could be safely transferred into a patient and provide a permanent supply of corrected cells. that this gene could subsequently be detected in Gene-augmentation therapies for a small number cells removed from the patient. Both objectives were of recessive single-gene diseases are now undergoing POGC14 9/11/2001 11:11 AM Page 291

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1 Remove bone 2 Culture marrow from mononuclear cells patient ex vivo

3 Transfect (or transduce) with functional ADA gene

Fig. 14.10 Procedure for ex vivo gene 5 Return modified 4 Select stable therapy, based on the treatment for cells to patient transformants ADA deficiency.

clinical trials. We consider cystic fibrosis (CF) as an Gene-therapy strategies for cancer example. CF is a disorder that predominantly affects the lungs, liver and pancreas. The disease is caused Cancer gene therapy was initially an extension of by the loss of a cAMP-regulated membrane-spanning the early gene-marking experiments. The tumour- chloride channel. This results in an electrolyte infiltrating leucocytes were transformed with a gene imbalance and the accumulation of mucus, often for TNF in addition to the neomycin-resistance gene, leading to respiratory failure. CF is a recessive dis- with the aim of improving the efficiency with which order suggesting that the loss of function could these cells kill tumours by increasing the amount of be corrected by introducing a functional copy of TNF they secrete. Although TNF is highly toxic to the gene. Indeed, epithelial cells isolated from CF humans at levels as low as 10 µg/kg body weight, patients can be restored to normal by transfecting there have been no side-effects from the gene ther- them with the cloned cystic-fibrosis transmembrane apy and no apparent organ toxicity from secreted regulator (CFTR) cDNA. Unlike ADA deficiency, the TNF (Hwu et al. 1993). One alternative strategy is cells principally affected by CF cannot be cultured to transform the tumour cells themselves, making and returned to the patient, so in vivo delivery strateg- them more susceptible to the immune system through ies must be applied. Targeted delivery of the CFTR the expression of cytokines or a foreign antigen. cDNA to affected cells has been achieved using Another is to transform fibroblasts, which are easier adenoviral vectors, which have a natural tropism to grow in culture, and then co-inject these together for the epithelial lining of the respiratory system. with tumour cells to provoke an immune response Recombinant viruses carrying the CFTR cDNA have against the tumour. A number of such ‘assisted been introduced into patients using an inhaler killing’ strategies have been approved for clinical (Zabner et al. 1993, Hay et al. 1995, Knowles et al. trials (see review by Ockert et al. 1999). 1995). The CFTR cDNA has also been introduced Direct intervention to correct cancer-causing genes using liposomes (e.g. Caplen et al. 1995). While such is also possible. Dominantly acting genes (oncogenes) treatments have resulted in CFTR transgene expres- have been targeted using antisense technology, sion in the nasal epithelium, there were neither con- either with antisense transgenes, oligonucleotides sistent changes in chloride transport nor reduction (see Carter & Lemoine 1993, Nellen & Lichtenstein in the severity of CF symptoms, i.e. they have been 1993) or ribozymes (Welch et al. 1998, Muotri largely ineffective. et al. 1999). An early report of cancer gene therapy POGC14 9/11/2001 11:11 AM Page 292

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with antisense oligonucleotides was that of Szczylik studies to identify genes for polygenic disorders, and et al. (1991) for the treatment of chronic myeloid success has already been achieved with hyperten- leukaemia. They used two 18-mers specific for the sion (Geller et al. 2000), non-insulin-dependent dia- BCR–ABL gene junction generated by the chromoso- betes (Deeb et al. 2000) and cardiovascular disease mal translocation that causes this particular cancer, (Mason et al. 1999). and showed that colony formation was suppressed Pharmacogenomics is the term used to describe in cells removed from cancer patients. Cancers the identification and elucidation of genetic vari- caused by loss of tumour-suppressor gene function ations that will have an impact on the efficacy of have been addressed by replacement strategies, in drugs. SNPs are essential to pharmacogenomics which a functional copy of the appropriate gene is (McCarthy & Hilfiker 2000), for they provide an easy delivered to affected cells (e.g. see Cai et al. 1993, means of determining the genotype of the patient. Harper et al. 1993, Smith et al. 1993, Hahn et al. For example, the 2-adrenergic receptor agonists 1996). A further strategy, known as prodrug activa- are the most widely used agents in the treatment tion therapy, involves the activation of a particular of asthma and several polymorphisms have been enzyme specifically in cancer cells, which converts a described within the target genes. Several studies non-toxic ‘prodrug’ into a toxic product, so killing have shown associations between SNPs in these the cancer cells. This can be achieved by driving the genes and response to therapy. One study (Buscher expression of a so-called ‘suicide gene’ selectively et al. 1999) found that homozygotes for one allele in cancer cells. An example is the HSV thymidine were up to 5.3 times more likely to respond to kinase gene, in combination with the prodrug albuterol than homozygotes for the other allele. ganciclovir. Thymidine kinase converts ganciclovir Another striking example is the response of into a nucleoside analogue, which is incorporated Alzheimer’s patients to the drug tacrine. Approx- into DNA and blocks replication by inhibiting the imately 80% of patients not carrying the ApoE4 DNA polymerase. Activation of the enzyme speci- allele improved after tacrine treatment, whereas fically in cancer cells can be achieved by preferential 60% of patients with the allele deteriorated after delivery to dividing cells through the use of onco- treatment (Poirier et al. 1995). One scenario for the retroviruses (e.g. Moolten 1986, Culver et al. 1992, future is that, before prescribing a drug, patients will Klatzmann et al. 1996). Another way is to use be genotyped by SNP analysis to determine the most transcriptional regulatory elements that are active effective therapy. only in cancer cells (e.g. Harris et al. 1994, Su et al. Another use for pharmacogenomics is the pre- 1996). vention of adverse drug reactions resulting from drug metabolism. For example, between 3 and 10% of the Caucasian population fail to metabolize the The importance of SNPs adrenergic-blocking drug debrisoquine and treatment As noted earlier (p. 277), SNPs are single-base vari- results in severe hypotension. In Afro-Americans ations that occur about once every 1000 bases the frequency of this ‘poor metabolizer’ condition is along the human genome. With the availability of 5% and in Asians it is only 1%. Affected individuals the human genome sequence, a high-resolution are homozygous for a mutant cytochrome P450 SNP map is being developed and this will facilitate gene (CYP2D6) and they also fail to metabolize over disease therapy in a number of ways (Rothberg 20% of all commonly prescribed drugs, including 2001). Two examples will be cited here: understand- codeine (Gonzalez et al. 1988). The same gene also ing polygenic disorders and pharmacogenomics. has alleles that cause an elevated-metabolizer pheno- The disease studies cited above have all focused on type, which has been correlated with increased single-gene disorders. However, many of the com- susceptibility to cancer. Clearly, before selecting mon diseases of humans are polygenic in origin and patients for clinical trials of new drugs, it would attempts to map genes for these complex conditions make sense to screen candidates for their drug- have generally failed. The availability of SNP maps is metabolizing phenotype and most large pharma- providing a new tool for use in genetic association ceutical companies now do this. POGC14 9/11/2001 11:11 AM Page 293

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cause. Finally, attenuated virus strains have the Theme 3: Combating infectious disease potential to revert to a virulent phenotype upon replication in the vaccinee. This occurs about once Introduction to theme 3 or twice in every million people who receive live The usual way of treating bacterial and infectious polio vaccine. Recombinant DNA technology offers diseases is with antibiotics. As is well known, certain some interesting solutions. microbes quickly develop resistance to the anti- Given the ease with which heterologous genes biotics in current use and this means that new anti- can be expressed in various prokaryotic and eukary- biotics are required. The traditional way of obtaining otic systems, it is not difficult to produce large quant- new antibiotics is the screening of new microbial ities of purified immunogenic material for use as a isolates from nature. An alternative way will be subunit vaccine. A whole series of immunologically described in theme 5: combinatorial biosynthesis pertinent genes have been cloned and expressed but, (p. 306). Another way is to identify new cellular in general, the results have been disappointing. For targets and screen chemical libraries for inhibitory example, of all the polypeptides of foot-and-mouth activities. A number of methods for identifying key disease virus, only VP1 has been shown to have genes involved in pathogenesis have been developed immunizing activity. However, polypeptide VP1 and these are described later in this section. In con- produced by recombinant means was an extremely trast to bacteria and fungi, viruses are not sensitive poor immunogen (Kleid et al. 1981). Perhaps it is not to antibiotics and few therapies have been available. too surprising that subunit vaccines produced in However, this could change with the development of this way do not generate the desired immune antisense drugs, as described in the previous section response, for they lack authenticity. The hepatitis B (p. 291). Where suitable therapies do exist, it can be vaccine, which is commercially available (Valenzuela advantageous to know the identity of the pathogen et al. 1982), differs in this respect, for expression of as soon as possible. Conventional laboratory proced- the surface antigen in yeast results in the formation ures take several days, but PCR methodology offers a of virus-like particles. A similar phenomenon is seen more rapid identification, as was described on p. 274. with a yeast Ty vector carrying a gene for HIV coat For many pathogens, prevention is much better than protein (Adams et al. 1987). These subunit vaccines cure, and hence vaccines are of great value. Gene- also have another disadvantage. Being inert, they manipulation techniques have greatly facilitated the do not multiply in the vaccinee and so they do not development of new vaccines, as described below. generate the effective cellular immune response essential for the recovery from infectious disease. Novel routes to vaccines Recombinant bacterial vaccines An effective vaccine generates humoral and/or cell- mediated immunity, which prevents the develop- An alternative approach to the development of ment of disease upon exposure to the corresponding live vaccines is to start with the food-poisoning pathogen. This is accomplished by presenting pertin- organism Salmonella typhimurium. This organism ent antigenic determinants to the immune system in can be attenuated by the introduction of lesions a fashion which mimics that in natural infections. in the aro genes, which encode enzymes involved Conventional viral vaccines consist of inactivated, in the biosynthesis of aromatic amino acids, p- virulent strains or live, attenuated strains, but they aminobenzoic acid and enterochelins. Whereas are not without their problems. For example, many doses of 104 wild-type S. typhimurium reproducibly viruses have not been adapted to grow to high titre kill mice, aro mutants do not kill mice when fed orally, in tissue culture, e.g. hepatitis B virus. There is a even when doses as high as 1010 organisms are danger of vaccine-related disease when using inact- used. However, the mutant strains can establish self- ivated virus, since replication-competent virus may limiting infections in the mice and can be detected remain in the inoculum. Outbreaks of foot-and- in low numbers in organs such as the liver and mouth disease in Europe have been attributed to this spleen. Such attenuated strains of S. typhimurium POGC14 9/11/2001 11:11 AM Page 294

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are particularly attractive as carriers of heterologous Recombinant viruses as vaccines antigens because they can be delivered orally and because they can stimulate humoral, secretory and Recombinant viruses can be used as vectors to cellular immune responses in the host (Charles & express heterologous antigens, and thus function as Dougan 1990). Already a wide range of heterolog- live vaccines. The first animal virus to be exploited in ous antigens have been expressed in such vaccine this way was vaccinia, which had been used pre- strains (Hackett 1993). viously as a non-recombinant vaccine providing The use of the BCG vaccine strain as an altern- cross-protection against variola virus, the causative ative vector has many advantages, including its agent of smallpox. Contributing to its success as a known safety, low cost, widespread use as a child- live vaccine were its stability as a freeze-dried pre- hood vaccine and ability of a single dose to induce paration, its low production cost and the ability to long-lasting protection. Several recombinant BCG administer the vaccine by simple dermal abrasion. strains have been constructed that stably express Vaccinia remains the most widely explored recom- foreign genes (Stover et al. 1991) and preliminary binant viral vaccine, and many antigens have been results from animal studies are very encouraging. expressed using this vector, essentially using the An alternative vector is the human oral commensal methods described on p. 197 (Ulaeto & Hruby 1994, Streptococcus gordonii (Fischetti et al. 1993). Moss 1996). The most successful recombinant-virus A different procedure for attenuating a bacterial vaccination campaign to date involved the use of pathogen to be used as a vaccine has been proposed recombinant vaccinia virus expressing rabies-virus by Kaper et al. (1984). They attenuated a pathogenic glycoprotein. This was administered to the wild popu- strain of Vibrio cholera by deletion of DNA sequences lation of foxes in central Europe by providing a bait

encoding the A1 subunit of the cholera enterotoxin. consisting of chicken heads spiked with the virus. A restriction-endonuclease fragment encoding the The epidemiological effects of vaccination were most

A1, but not the A2 or B, sequence was deleted in vitro evident in eastern Switzerland, where two decades from cloned cholera-toxin genes. The mutation of rabies came to a sudden end after only three vac- was then recombined into the chromosome of a cination campaigns (Brochier et al. 1991, Flamand pathogenic strain. The resulting strain produces the et al. 1992), and in Belgium, where the disease was immunogenic but non-toxic B subunit of cholera all but eliminated (Brochier et al. 1995). toxin but is incapable of producing the A subunit. One disadvantage of this approach in humans is This strain has been found to be safe and immu- the unacceptably high risk of adverse reactions to nogenic in carefully controlled clinical trials in a the vaccine. For use in humans, vaccinia must be fur- number of countries (Cryz 1992). ther attenuated to make it replication-deficient and Yet another approach to vaccine preparation has to minimize the likelihood of replication-competent been developed by Pizza et al. (2000), based on viruses arising by recombination (e.g. Cooney et al. whole-genome sequencing. They wanted to develop 1991, Sutter & Moss 1992, Tartaglia et al. 1992). a vaccine against group B meningococci but were Highly attenuated vaccinia derivatives (Table 14.6) hampered by the fact that there was considerable have been used to express a range of viral, bacterial sequence variation in surface-exposed proteins. and parasite antigens, with some reaching clinical Starting with the entire genome sequence of a group trials (reviewed by Moss 1996, Paoletti 1996). Other B strain of Neisseria meningitidis, they identified 350 poxviruses, such as canarypox, have also been proteins as potential protective antigens. All 350 developed as potential vaccines (e.g. see Perkus et al. candidate antigens were expressed in E. coli, purified 1995, Fries et al. 1996, Myagkikh et al. 1996). and used to immunize mice. The sera from the mice As well as vaccinia, adenovirus and alphavirus allowed the identification of proteins that are surface- vectors have also been investigated as potential exposed in meningococci, that are conserved across recombinant vaccines. Adenovirus has been used to a range of strains and that induce a bactericidal anti- express various antigens, including HSV and rabies body response, a property known to correlate with virus glycoproteins (Gallichan et al. 1993, Xiang vaccine efficacy in humans. et al. 1996). Among the alphaviruses, Semliki Forest POGC14 9/11/2001 11:11 AM Page 295

Applications of recombinant DNA technology 295

Table 14.6 Immune response to heterologous antigens expressed by vaccinia virus recombinants.

Antigen Neutralizing antibodies Cellular immunity Animal protection

Rabies virus glycoprotein +++ Vesicular stomatitis glycoprotein +++ Herpes simplex virus glycoprotein D +++ Hepatitis B surface antigen +++ Influenza virus haemagglutinin +++ Human immunodeficiency virus envelope ++not determined

Reproduced with permission from Tartaglia & Paoletti (1988).

Table 14.7 A selection of recombinant vaccines against animal viruses produced in plants. Antigen Host-plant system Reference

Herpes virus B surface antigen Tobacco Mason et al. 1992 Rabies glycoprotein Tomato McGarvey et al. 1995 Norwark virus coat protein Tobacco, potato Mason et al. 1996 Foot-and-mouth virus VP1 Arabidopsis Carrillo et al. 1998 Cholera toxin B subunit Potato Arakawa et al. 1998 Human cytomegalovirus Tobacco Tackaberry et al. 1999 glycoprotein B

virus has been used to express the gp160 protein Plants as edible vaccines of HIV (Berglund et al. 1997) and Sindbis virus has been used to express antigens from Japanese Plants have been explored as a cheap, safe and encephalitis virus (Pugachev et al. 1995). efficient production system for subunit vaccines Recombinant proteins expressed by viruses have (Table 14.7), with the added advantage that orally been shown to be immunogenetic. However, stronger administered vaccines can be ingested by eating the stimulation of the immune response can often be plant, therely eliminating the need for processing achieved by presenting the antigen on the surface of and purification (reviewed by Mason & Arntzen the virus attached to a host virus-derived carrier 1995, Walmsey & Arntzen 2000). The earliest protein. The advantage of this strategy is that the demonstration was the expression of a surface anti- recombinant antigen is presented as multiple copies. gen from the bacterium S. mutans in tobacco. This This strategy is borrowed from the surface display bacterium is the causative agent of dental caries, of foreign antigens on bacteria that are used as live and it was envisaged that stimulation of a mucosal vaccines (see above) but is much safer. Indeed, while immune response would prevent the bacteria colon- a number of conventional viral vaccines, such as izing the teeth and therefore protect against tooth vaccinia, have been developed as surface-display decay (Curtis & Cardineau, 1990). systems (e.g. see Katz & Moss 1997, Katz et al. 1997), A number of edible transgenic plants have been even plant viruses can be used for this purpose, generated expressing antigens derived from animal as discussed below (Porta & Lomonossoff 1996, viruses. For example, rabies glycoprotein has been Johnson et al. 1997). expressed in tomato (McGarvey et al. 1995), hepatitis POGC14 9/11/2001 11:11 AM Page 296

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B virus antigen in lettuce (Ehsani et al. 1997) and HIV (Wang et al. 1993, Fuller et al. 1997, Hinkula cholera antigen in potato (Arakawa et al. 1997). As et al. 1997); Ebola virus (Xu et al. 1998) ), other well as animal virus antigens, autoantigens associ- pathogens (e.g. tuberculosis (Huygen et al. 1996) ) ated with diabetes have also been produced (Ma et al. and even the human cellular prion protein in mice 1997, Porceddu et al. 1999). Plants have also (Krasemann et al. 1996). been infected with recombinant viruses expressing The DNA-vaccination approach has several addi- various antigen on their surfaces. Cowpea tional advantages. These include the following: mosaic virus (CMV) has been extensively developed • Certain bacterial DNA sequences have the innate as a heterologous antigen-presenting system (Porta ability to stimulate the immune system (see Klinman et al. 1994, Lomonossoff & Hamilton 1999). There et al. 1997, Roman et al. 1997). have been some recent successes in vaccination • Other genes encoding proteins influencing the func- trials using recombinant CMV vectors expressing tion of the immune response can be co-introduced epitopes of HIV gp41 (McLain et al. 1995, 1996a,b) along with the vaccine (e.g. Kim et al. 1997). and canine parvovirus (Dalsgaard et al. 1997). The • DNA vaccination can be used to treat diseases that first clinical trials using a plant-derived vaccine are already established as a chronic infection (e.g. were conducted in 1997 and involved the ingestion Mancini et al. 1996). of transgenic potatoes expressing the B subunit of In principle, DNA vaccination has much in the E. coli heat-labile toxin, which causes diarrhoea. common with gene therapy (discussed above), since This resulted in a successful elicitation of mucosal both processes involve DNA transfer to humans, immunity in test subjects (Tacket et al. 1994). using a similar selection of methods. However, while the aim of gene therapy is to alleviate disease, by either replacing a lost gene or blocking the expres- DNA vaccines sion of a dominantly acting gene, the aim of DNA The immune system generates antibodies in response vaccination is to prevent disease, by causing the to the recognition of proteins and other large mole- expression of an antigen that stimulates the immune cules carried by pathogens. In each of the examples system. above, the functional component of the vaccine introduced into the host is the protein responsible Selecting targets for new for the elicitation of the immune response. The intro- antimicrobial agents duction of DNA into animals does not generate an immune response against the DNA molecule, but, if In attempting to develop new antimicrobial agents, that DNA is expressed to yield a protein, that protein including ones that are active against intractable can stimulate the immune system. This is the basis pathogens such as the malarial parasite, it would be of DNA vaccination, as first demonstrated by Ulmer useful to know which genes are both essential for et al. (1993). DNA vaccines generally comprise a virulence and unique to the pathogen. Once these bacterial carrying a gene encoding the genes have been identified, chemical libraries can be appropriate antigen under the control of a strong screened for molecules that are active against the promoter that is recognized by the host cell. The gene product. Two features of this approach deserve advantages of this method include its simplicity, its further comment. First, inhibition of the gene prod- wide applicability and the ease with which large uct could attenuate the organism’s virulence but quantities of the vaccine can be produced. The DNA would not result in death of the organism in vitro; may be administered by injection, using liposomes that is, no effect would be seen in whole-organism or by particle bombardment. In the original demon- inhibition assays and so molecules only active in stration, Ulmer and colleagues introduced DNA vivo would be missed. Secondly, target genes can be corresponding to the influenzavirus nucleoprotein selected on the basis that there are no human coun- and achieved protection against influenza infection. terparts. Thus, active molecules will be less likely Since then, many DNA vaccines have been used to be toxic to humans. A number of different appro- to target viruses (e.g. measles (Cardoso et al. 1996); aches have been developed for identifying virulence POGC14 9/11/2001 05:28 PM Page 297

Applications of recombinant DNA technology 297

determinants, and details are presented below for when it infects an animal or plant host (Mahan et al. three of these: in vivo expression technology (IVET), 1993). The basis of the system is a plasmid carrying differential fluorescence induction and signature- a promoterless operon fusion of the lacYZ genes tagged mutagenesis. fused to the purA or thyA genes downstream of a unique BglII cloning site (Fig. 14.11). This operon fusion was constructed in a suicide-delivery plas- In vivo expression technology (IVET) mid. Cloning of pathogen DNA into the BglII site IVET was developed to positively select those genes results in the construction of a pool of transcrip- that are induced specifically in a microorganism tional fusions driven by promoters present in the

Bgl II thyA lacZ + IVET Salmonella DNA plasmid

Cut with Bgl II and ligate

Pl thyA lacZ

Transfer to thyA strain of Salmonella. Grow in presence of thymine

Isolate pathogen on media + thymine + Xgal

White Blue colonies colonies

thyA expressed thyA expressed from promoter from promoter Fig. 14.11 The basic principle of IVET. only active in vivo active in vitro See text for details. POGC14 9/11/2001 11:11 AM Page 298

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cloned DNA. The pool of fusions is transferred to the extracellular environment were sorted and used a strain of the pathogen carrying a purA or thyA for a second round of macrophage infection. Bacteria deletion and selection made for integration into the still capable of generating fluorescent macrophages chromosome. The recombinant pathogen is then were found to contain GFP fusions that were used to infect a test animal. Fusions that contain up-regulated by the macrophage’s intracellular a promoter that is active in the animal allow environment. transcription of the purA or thyA gene and hence bacterial survival. When the surviving pathogens Signature-tagged mutagenesis are reisolated from the test animal, they are tested in vitro for their levels of β-galactosidase. Clones that This technique is a variation on the use of trans- contain fusions to genes that are specifically induced poson mutagenesis that has been applied to the in the test animal show little lacZ expression on identification of bacterial virulence genes. The basic laboratory media and DNA sequencing can be used principle is to create a large number of different to identify the genes to which these promoters belong. transposon-generated mutants of a pathogenic The IVET system was initially developed for use in organism and to identify those mutants that can a murine typhoid model. Since it was first described, survive in vitro but not in vivo – that is, it is a whole- IVET has been used with a wide variety of Gram- genome scan for habitat-specific genes. Although positive and Gram-negative organisms, including one could test each mutant individually, this would ‘difficult’ organisms, such as Mycobacterium. One be very laborious and would use large numbers disadvantage of the original IVET system is that it of animals. By using signature-tagged mutagenesis, inherently selects for promoters that are constitut- one can test large numbers of different mutants ively and highly expressed in vivo. An alternative simultaneously in the same animal. This is achieved by IVET system uses tnpR operon fusions in place of tagging each transposon mutant with a different purA or thyA. The tnpR locus encodes a site-specific DNA sequence or tag. resolvase and is used as the selection gene. Expres- The use of sequence-tagged mutagenesis was first sion of this gene results in resolvase synthesis described for the identification of virulence genes and the deletion of a resolvase-specific reporter. from S. typhimurium in a mouse model of typhoid The advantage of this system is that it can be used fever (Hensel et al. 1995), as shown in Fig. 14.12. to identify those genes that are expressed only The tags comprise different sequences of 40 bp = = transiently during animal infection (Camilli & [NK]20, where N A, C, G or T and K G or T. The Mekalanos 1995, Camilli 1996). Handfield and arms were designed so that the amplification of the Levesque (1999) have described a number of other tags by PCR with specific primers would produce modifications to the IVET technology. probes with 10 times more label in the central region than in each arm. The double-stranded tags were ligated into a Tn5 transposon and transferred from Differential fluorescence induction E. coli to S. typhimurium by conjugation. A library of This technique represents a different way of identify- over 1500 exconjugants resulting from transposi- ing environmentally controlled promoters and was tion events was stored in microtitre dishes. Of these originally developed to facilitate identification of exconjugants, 1152 were selected and prepared as Salmonella genes that are differentially expressed 12 pools of 96 mutants. Each pool was injected into with macrophages (Valdivia & Falkow 1997). Frag- the peritoneum of a different mouse and infection ments of DNA from Salmonella were fused to a pro- allowed to proceed. Bacteria were then recovered motorless gene for green fluorescent protein (GFP) from each mouse by plating spleen homogenates and returned to the Salmonella, which was then used on culture media. DNA was extracted from the to infect macrophages. Cells that became fluorescent recovered bacteria and the tags in this DNA were were recovered using a fluorescence-activated cell amplified by PCR. Those tags present in the initial sorter (FACS) and grown on media in the absence of pool of bacteria but missing from the recovered macrophages. Bacteria that were non-fluorescent in media represent mutations in genes essential for POGC14 9/11/2001 11:11 AM Page 299

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17 1 Construct 10 oligonucleotide tags with (NK)20 Environomics the structure The techniques described above to identify bacterial virulence genes can be applied to other microbe– host interactions, e.g. protozoan infections of humans, bacterial or fungal pathogenesis of plants, or even 2 Clone tags into transposon Tn5 resident in plasmid vector. beneficial rhizosphere–microbe interactions (Rainey 1999). Essentially, these techniques are methods for scanning the entire microbial genome for genes that are expressed under particular environ- 3 Transform tagged transposon into E.coli selecting mental conditions. This approach has been termed for kanamycin resistance. The individual transformants are not isolated but kept as a pool. ‘environomics’.

Theme 4:

4 Conjugate plasmids into Salmonella and select for transposition Introduction to theme 4 of tagged transposon. Each kanamycin resistant cell should carry a different tag. One of the most exciting aspects of recombinant DNA technology is that it permits the design, devel- opment and isolation of proteins with improved operating characteristics and even completely novel 5 Arrange each individual kanamycin resistant clone, which proteins. The simplest example of protein engineer- should be result of different event and will have different tag, in 8×12 grid in 96 well microtitre plates. ing involves site-directed mutagenesis to alter key residues, as originally shown by Winter and col- leagues (Winter et al. 1982, Wilkinson et al. 1984). From a detailed knowledge of the enzyme tyrosyl- tRNA synthetase from Bacillus stearothermophilus, 6 Pool groups of 96 clones and infect mouse. After period for infection to be established, re-isolate kanamycin resistant cells including its crystal structure, they were able to from mouse. Extract DNA. predict point mutations in the gene that should increase the enzyme’s affinity for the substrate ATP. These changes were introduced and, in one case, a single amino acid change improved the affinity for

7 Screen DNA from mouse isolates and determine which tags ATP by a factor of 100. Using a similar approach, the are absent relative to those that are present in microtitre trays. stability of an enzyme can be increased. Thus Perry Tags that are missing must be attached to transposons that have inserted into a gene essential for mouse virulence. and Wetzel (1984) were able to increase the ther- mostability of T4 lysozyme by the introduction of a Fig. 14.12 The basic methodology for signature tagged disulphide bond. However, although new cysteine mutagenesis. residues can be introduced at will, they will not necessarily lead to increased thermal stability (Wetzel et al. 1988). virulence. In this way 28 different mutants with attenuated virulence were identified and some of Improving therapeutic proteins with these mutants were in genes not previously identified. single amino acid changes The principle of signature-tagged mutagenesis has been extended to the analysis of pathogenicity As noted earlier (p. 283), many recombinant determinants in a wide range of bacteria (for a review, proteins are now being used therapeutically. With see Handfield & Levesque 1999) and to fungi (Brook- some of them, protein engineering has been used to man & Denning 2000). generate second-generation variants with improved POGC14 9/11/2001 11:11 AM Page 300

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pharmacokinetics, structure, stability and bioavail- Improving enzymes: subtilisin as a ability (Bristow 1993). For example, in the neutral paradigm for protein engineering solutions used for therapy, insulin is mostly assembled as zinc-containing hexamers. This self-association Proof of the power of gene manipulation coupled may limit absorption. By making single amino acid with the techniques of in vitro (random and site- substitutions, Brange et al. (1988) were able to directed) mutagenesis as a means of generating generate that are essentially monomeric at improved enzymes is provided by the work done on pharmaceutical concentrations. Not only have subtilisin over the past 15 years (for review, see these insulins preserved their biological activity, but Bryan 2000). Every property of this serine protease they are also absorbed two to three times faster. has been altered, including its rate of catalysis, sub- Similarly, replacing an asparagine residue with strate specificity, pH-rate profile and stability to glutamine altered the glycosylation pattern of TPA. oxidative, thermal and alkaline inactivation. In the This in turn significantly increased the circulatory process, well over 50% of the 275 amino acids of half-life, which in the native enzyme is only 5 min subtilisin have been changed. At some positions in (Lau et al. 1987). Proteins can also be engineered to the molecule, the effects of replacing the usual be resistant to oxidative stress, as has been shown amino acid with all the other 19 natural amino acids with AAT (see Box 14.3). have been evaluated.

Box 14.3 Oxidation-resistant variants of a1-antitrypsin (AAT)

Cumulative damage to lung tissue is thought to be consequently increased exposure to neutrophil responsible for the development of emphysema, an elastase. In addition, leucocytes liberate oxygen irreversible disease characterized by loss of lung free radicals and these can oxidize methionine- elasticity. The primary defence against elastase 358 to methionine sulphoxide. Since methionine damage is AAT, a glycosylated serum protein of sulphoxide is much bulkier than methionine, it does 394 amino acids. The function of AAT is known not fit into the active site of elastase. Hence oxidized because its genetic deficiency leads to a premature AAT is a poor inhibitor. By means of site-directed breakdown of connective tissue. In healthy mutagenesis, an oxidation-resistant mutant of AAT individuals there is an association between AAT and has been constructed by replacing methionine-358 neutrophil elastase followed by cleavage of AAT with valine (Courtney et al. 1985). In a laboratory between methionine residue 358 and serine model of inflammation, the modified AAT was an residue 359 (see Fig. B14.2). effective inhibitor of elastase and was not inactivated After cleavage, there is negligible dissociation of by oxidation. Clinically, this could be important, the complex. Smokers are more prone to emphysema, since intravenous replacement therapy with plasma because smoking results in an increased concentrates of AAT is already being tested on concentration of leucocytes in the lung and patients with a genetic deficiency in AAT production.

Met 358 Ser 359

394 394

Met 358

α Fig. B14.2 The cleavage of 1-antitrypsin on binding to neutrophil elastase. POGC14 9/11/2001 11:11 AM Page 301

Applications of recombinant DNA technology 301

Many of the changes described above were made which will react with another C-protected peptide to improve the ability of subtilisin to hydrolyse pro- in the presence of the peptide ligase to form a larger tein when incorporated into detergents. However, glycosyl peptide. serine proteases can be used to synthesize peptides and this approach has a number of advantages over Methods for engineering proteins: conventional methods (Abrahmsen et al. 1991). the rational approach A problem with the use of subtilisin for peptide synthesis is that hydrolysis is strongly favoured over Given the range of modifications made to subtilisin, aminolysis, unless the reaction is undertaken in the question is not ‘what modifications are possible?’ organic solvents. Solvents, in turn, reduce the half- (for review, see Arnold 2001) but ‘how do I achieve life of subtilisin. Using site-directed mutagenesis, a the modifications that I wish to make?’ There are number of variants of subtilisin have been isolated two general techniques: the rational approach and with greatly enhanced solvent stability (Wong et al. ‘directed evolution’. There are two variations on the 1990, Zhong et al. 1991). Changes introduced rational approach. The first of these makes use of included the minimization of surface changes to reduce comparisons between related proteins. For example, solvation energy, the enhancement of internal polar barnase and binase are ribonucleases that have 85% and hydrophobic interactions and the introduction sequence identity but differ in their thermal stability. of conformational restrictions to reduce the tendency Using site-directed mutagenesis, Serrano et al. (1993) of the protein to denature. Designing these changes tested the impact on thermostability of barnase of requires an extensive knowledge of the enzyme’s all 17 amino acid differences between barnase and structure and function. Chen and Arnold (1991, binase. They observed effects between +1.1 and −1.1 1993) have provided an alternative solution. They kcal/mol and a multiple mutant combining six of utilized random mutagenesis combined with screen- the substitutions displayed a stability increase of ing for enhanced proteolysis in the presence of 3.3 kcal/mol over the wild-type enzyme. solvent (dimethyl formamide) and substrate (casein). The second variation on the rational approach The engineering of subtilisin has now gone one requires that the three-dimensional structure of the step further, in that it has been modified such that protein be known. Based on an analysis of this struc- aminolysis (synthesis) is favoured over hydrolysis, ture, particular residues are selected for mutagenesis even in aqueous solvents. This was achieved by and the biological effects then evaluated. That this changing a serine residue in the active site to cys- approach works was clearly shown by the early teine (Abrahmsen et al. 1991). The reasons for this work on lysozyme and subtilisin, but the chances of enhancement derive mainly from the increased success are not predictable. One disadvantage of this affinity and reactivity of the acyl intermediate for the approach is illustrated by the results of Spiller et al. amino nucleophile (Fig. 14.13). These engineered (1999), who were investigating the thermostability ‘peptide ligases’ are in turn being used to synthesize of an esterase using directed evolution. When they novel glycopeptides. A glycosyl amino acid is used evaluated the effect of certain mutations, they in peptide synthesis to form a glycosyl peptide ester, concluded that no amount of rational analysis of the crystal structure would have led them to predict the results obtained.

H O RCOOH + E OH 2 Protein engineering through Hydrolysis RCOOR’ +E OH RCOO E directed evolution Aminolysis Directed evolution involves repeated rounds of R–NH 2 RCOOR’ +E OH random mutagenesis, followed by selection for the improved property of interest. Any number of Fig. 14.13 The aminolysis (synthetic) and hydrolysis methods can be used to introduce the mutations, reactions mediated by an acylated protease. including mutagenic base analogues, chemical POGC14 9/11/2001 11:11 AM Page 302

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mutagenesis, error-prone PCR and spiked synthetic ing mutagenesis with phage display, up to 109 differ- oligonucleotides. The key element in the process is ent mutants can theoretically be screened in a single the ability to screen large numbers of mutants. An experiment. The constraint with this approach is example is the isolation of a more thermostable that phage display is really best suited to selecting subtilisin. Up to 1000 mutant clones are gridded proteins with altered binding characteristics, rather out on replica plates and, once they are grown, one than the other properties one might wish to engin- plate is incubated at an elevated temperature long eer. Despite this, a number of groups (Atwell & enough to inactivate the wild-type enzyme. When Wells 1999, Demartis et al. 1999, Olsen et al. 2000) an assay for hydrolytic activity is subsequently were able to select variant proteases with novel performed, only mutants with stability greater than substrate specificities. that of the wild type will display measurable activity. Once stable mutants have been identified, the replic- Gene families as aids to ate colony can be grown to identify the mutation. protein engineering Once stabilizing single amino acid changes have been identified, building a highly stable subtilisin An alternative approach to directed evolution is can be accomplished by combining individual amino ‘DNA shuffling’, which is also known as ‘molecular acids into the same molecule (Pantoliano et al. 1988, breeding’ (Minshull & Stemmer 1999, Ness et al. 1989, Zhao & Arnold 1999). 2000). This method can only be adopted if the target One of the disadvantages of the screening method protein belongs to a known protein family. If it does, described above is that it is labour-intensive and the the genes for the different family members are maximum feasible number of mutants that can be isolated and artificial hybrids created (Fig. 14.14). examined in a single screen is 104–105. By combin- As an example of this approach, Ness et al. (1999)

Ancestral species

Evolution

Species 1

Species 2

Species 3

Species 4

DNA shuffling in vitro

Hybrid genes

Fig. 14.14 Schematic representation of gene shuffling. POGC14 9/11/2001 11:11 AM Page 303

Applications of recombinant DNA technology 303

started with the genes for 26 members of the sub- had been elucidated could we even begin to under- tilisin family and created a library of chimeric stand how antibiotic yields might have been improved. proteases. When this library was screened for four Based on our current knowledge of metabolic distinct enzyme properties, variants were found that regulation we can predict that the changes in the were significantly improved over any of the parental improved strains described above will involve all of enzymes for each individual property. In a similar the following: way, Powell et al. (2000) were able to select • Removal of rate-limiting transcriptional and allo- retroviruses with increased stability for use as steric controls. gene-therapy vectors. • Kinetic enhancement of rate-limiting enzymes. As a final twist on the methods for engineering • Genetic blockage of competing pathways. proteins, Lehmann et al. (2000) have combined the • Enhanced carbon commitment to the primary use of protein families with a rational approach. metabolic pathway from central metabolism. They started with a family of mesophilic • Modification of secondary metabolic pathways to whose amino acid sequences had been determined. enhance energy metabolism and availability of Using these data they constructed a ‘consensus’ enzymatic cofactors. sequence and found that enzyme with this • Enhanced transport of the compound out of the cell. sequence was much more thermostable than any of Today, if one starts with a wild-type strain and the parent enzymes. Refinement of the approach wants to turn it into an overproducer, then the combined with site-directed mutagenesis resulted in approach would be to use recombinant DNA tech- the isolation of an enzyme whose unfolding temper- nology to make these desired changes in a rational ature was 27–34°C higher than that of the parent way, as exemplified below by phenylalanine. There enzymes. Whereas all the methods described earlier are many other examples of rational ‘metabolic tend to yield mutants with single amino acid changes, engineering’ and these have been reviewed by this method generated variants with multiple amino Chotani et al. (2000). acid changes spread throughout the molecule. Designed overproduction of Theme 5: Metabolic engineering phenylalanine Phenylalanine is a key raw material for the synthesis Introduction to theme 5 of the artificial sweetener aspartame. Phenylalanine When the large-scale production of penicillin was can be synthesized chemically but is too expensive if begun in the 1940s, the yields of penicillin were made this way. In the 1980s bacterial strains that measured in micrograms per litre of culture. Demand overproduced phenylalanine were developed, using for the antibiotic was outstripping supply and higher- the traditional mutation and selection method. At yielding strains were badly needed. Since nothing the same time, a programme of rational strain devel- was known about the biosynthetic pathway, a pro- opment was instituted at G.D. Searle, the company gramme of strain improvement was set in place that who owned the patent for aspartame. The starting- involved random mutation and screening. The best point for this programme was an analysis of the strain from each cycle of improvement then became biosynthetic pathway (Fig. 14.15). Removing feed- the starting-point for the next round of selection. In back inhibition of key steps is an essential first this way the yield of penicillin was steadily increased step. In the case of a phenylalanine producer, it is until it reached the tens of grams per litre that can be essential to knock out any feedback inhibition of the achieved today. As each new antibiotic was discov- pathway from chorismate to phenylalanine and this ered, the same process of strain improvement was was achieved by selecting strains resistant to pheny- applied. In every case, the biochemical and genetic lalanine analogues. The conversion of erythrose-4- basis of the beneficial mutations was not known. phosphate (E4P) and phosphoenol pyruvate (PEP) Only when the details of gene regulation and to DAHP is also subject to feedback inhibition, but, metabolic-pathway regulation (allosteric control) since there are three different enzymes here, each POGC14 9/11/2001 11:11 AM Page 304

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Erythrose-4-phosphate (E4P) + phosphoenol pyruvate (PEP)

DPAH

Chorismate

Prephenate Anthranilate

Indoleglycerol phosphate

Fig. 14.15 The regulation of the biosynthesis of aromatic amino acids. Note Tyrosine Phenylalanine Tryptophan that indoleglycerol phosphate is converted to tryptophan via indole but that the indole Repression normally is not released from the tryptophan Feedback inhibition synthase complex.

inhibited by a different aromatic end-product, all was confirmed when cloning transketolase (to that is necessary for a phenylalanine overproducer enhance E4P levels) and eliminating pyruvate kinase is to clone the tryptophan-sensitive enzyme and (to enhance PEP levels) enhanced yields. have it overexpressed. To overcome repression of enzyme synthesis, the existing promoters were New routes to small molecules removed and replaced with one that could be con- trolled more easily in industrial-scale fermentations. Recombinant DNA technology can be used to The above changes removed the natural control develop novel routes to small molecules. Good circuits. The next step was to remove competing examples are the microbial synthesis of the blue dye pathways, i.e. the synthesis of tyrosine and trypto- indigo (Ensley et al. 1983) and the black pigment phan. This was easily achieved by making a tyrosine melanin (Della-Cioppa et al. 1990). Neither com- and tryptophan double auxotroph. Note that stable pound is produced in bacteria. The cloning of a (non-reverting) auxotrophs can best be made by single gene from Pseudomonas putida – that encoding deleting part or all of the relevant genes. This is a naphthalene dioxygenase – resulted in the genera- task that is easy using recombinant DNA techno- tion of an E. coli strain able to synthesize indigo in a logy. Once all the control circuits and competing medium containing tryptophan (Fig. 14.16). Sim- pathways had been removed, attempts were made to ilarly, cloning a tyrosinase gene in E. coli led to increase the carbon flux through the biosynthetic conversion of tyrosine to dopaquinone, which spon- pathway. Surprisingly, overexpressing all the genes taneously converts to melanin in the presence of air. in the pathway did not enhance the yield of phenyl- To overproduce these compounds, one generates a alanine. One explanation was that the supply of pre- strain of E. coli that overproduces either tryptophan cursors (E4P and/or PEP) was rate-limiting. This or tyrosine, rather than phenylalanine, as described POGC14 9/11/2001 05:28 PM Page 305

Applications of recombinant DNA technology 305

NH2

CH2 CH COOH

N

Tryptophan

Tryptophanase (E.coli )

N

Indole

Naphthalene dioxygenase (cloned)

H OH

N HOH cis-indole-2, 3-dihydrodiol

Spontaneous O O OH OH Air oxidation Isatin hydrolase O N O N NH2 Indoxyl Isatin Isatic acid

Air oxidation Air oxidation

O O

N N N

Fig. 14.16 The biosynthesis of indigo O O N in E. coli and the formation of Indigo Indirubin alternative end products.

above. With both indigo and melanin, yields are tryptophan synthase, it was possible for the indole improved by increasing the levels of cofactors. Also, to be released for conversion by the dioxygenase in the case of indigo biosynthesis, it is necessary to (Murdock et al. 1993). engineer the tryptophan synthase gene. The reason One disadvantage of the new route to indigo is for this is that indole is an intermediate in the that one of the intermediates in its synthesis, biosynthesis of tryptophan (Fig. 14.15). However, indoxyl, can undergo an alternative spontaneous normally it is not free in the cytoplasm but remains oxidation to isatin and indirubin. The latter com- trapped within the tryptophan synthase complex. pound is an isomer of indigo with similar dyeing By modifying the trpB gene, encoding the subunit of properties, but instead of being blue it is a deep POGC14 9/11/2001 11:11 AM Page 306

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(a) Acetobacter Chemical suboxydans D-glucose D-sorbitol L-sorbose

Diacetone Chemical 2 keto-L- Chemical Ascorbic acid -2 keto-L- gulonic acid gulonic acid

(b)

CHO COOH COOH

OH O O 2,5 DKG Endogenous reductase HO HO HO Erwinia (Corynebacterium) enzymes OH OH OH Fig. 14.17 Simplified route to vitamin OH O HO C (ascorbic acid) developed by cloning in Erwinia the Corynebacterium gene for 2,5-diketogluconic acid reductase. CH2OH CH2OH CH2OH D-glucose 2,5 diketo-D- 2-keto-L- (a) Classical route to vitamin C. (b) The gluconic acid gulonic acid simplified route to 2-ketogulonic acid, the immediate precursor of vitamin C.

burgundy colour. To make textile-quality indigo, strain, it was found that 2-ketogulonic acid was there must be no indirubin present. Screening soil converted to l-idonic acid by an endogenous 2- microorganisms with the capacity to degrade indole ketoaldonate reductase. Cloning, deletion mutagen- resulted in the identification of an enzyme, isatin esis and homologous recombination of the mutated hydrolase, that can degrade isatin to isatic acid. reductase gene into the chromosome were some After cloning the gene for isatin hydrolase in the of the several steps taken to develop an organism indigo overproducing strains, the indigo product capable of accumulating large amounts (120 g/l) of obtained performed as well as chemically produced 2-ketogulonic acid (Lazarus et al. 1990). So far, material. attempts to manufacture vitamin C directly from A slightly different approach to that above has glucose have been unsuccessful. However, enzymes yielded a new route to vitamin C. The conventional that can convert 2-ketogulonic acid to ascorbic acid process starts with glucose and comprises one have been identified and the objective now is to mirobiological and four chemical steps (Fig. 14.17). clone these activities into Erwinia (Chotani et al. By cloning in Erwinia a single gene – that from 2000). Corynebacterium encoding 2,5-diketogluconic acid reductase – the process can be simplified to a single Combinatorial biosynthesis microbiological and a single chemical step (Ander- son et al. 1985). After observations of unexpectedly A number of widely used antibiotics and immuno- low yields of 2-ketogulonic acid in the recombinant suppressants belong to a class of molecules known POGC14 9/11/2001 11:11 AM Page 307

Applications of recombinant DNA technology 307

O H C 3 CH3 OOH O OH OH H C 3 CH3 CH3 OH OH

H3C Sugar O

OCH3 OOHSugar CH3 O Sugar

CH3 Erythromycin Daunorubicin

OCH3 HO OCH3 O CH H C O 3 CH3 3 H H O O H H C O CH3 3 H O H H3C CH3 OO

OH H

O CH H 3 OCH3 Avermectin

O O CH3 HC

H3C

H3C Sugar O Sugar

O OH

CH3 Fig. 14.18 Some examples of Tylosin polyketides.

as polyketides. These molecules, which are syn- of the act gene cluster were introduced into strepto- thesized by actinomycetes, have a fairly complex mycetes making related polyketides, completely new structure (Fig. 14.18). The genes involved in the antibiotics were produced (Hopwood et al. 1985). biosynthesis of polyketides are clustered, thereby For example, introducing the actVA gene from Strep- facilitating the cloning of all of the genes controlling tomyces coelicolor into a strain that makes medermycin the synthetic pathway. The first cluster (the act genes) leads to the synthesis of mederrhodinA (Fig. 14.19). to be cloned was that for actinorhodin. When parts This approach has been repeated many times since POGC14 9/11/2001 11:11 AM Page 308

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previous theme (p. 302), and this approach has Actinorhodin H O O CH3 been widely adopted (Baltz 1998). However, novel polyketides can also be generated by simply chang- O ing the order of the different activities in type I synthases (McDaniel et al. 1999). COOH O O H Engineering metabolic control over

Medermycin recombinant pathways N(CH2)2 HO H When a recombinant cell overproduces a protein or O O CH3 the components of a biosynthetic pathway, there is a marked reduction in metabolic activity, coupled with H C 3 O O a retardation of growth (Kurland & Dong 1996). These phenotypic characteristics result from the fact that the constant demands of production placed H O O O upon the cell interfere with its changing require- ments for growth. To alleviate this problem, Farmer and Liao (2000) designed a dynamic control circuit actVA gene that is able to sense the metabolic state of the cell and thereby regulate the expression of a recombinant Mederrhodin N(CH2)2 pathway. This approach is termed ‘metabolic control HO H engineering’. O O CH3 An essential component of a dynamic controller is a signal that will reflect the metabolic state of the H C 3 O O cell. Acetyl phosphate was selected as the signal, since it is known to be a regulator of various operons influenced by nutrient availability. A component of O O O H O the Ntr regulon in E. coli, NRI, is capable of sensing the acetyl phosphate level in the cell. When phos- phorylated by acetyl phosphate, it is capable of Fig. 14.19 The formation of the new antibiotic mederrhodin from medermycin by the actVA gene product. binding to the glnAp2 promoter and activating transcription (Fig. 14.20). To reconstruct this control module, the NRI-binding site and the glnAp2 with other polyketides (for review, see Baltz 1998) promoter were inserted into a plasmid vector up- and is known as combinatorial biosynthesis. stream of a cloning site. Once a number of polyketide biosynthetic gene When the lacZ gene was placed under the control clusters had been cloned and sequenced, new of the glnAp2 promoter, there was no significant β- insights were gained on the mechanism of synthesis. galactosidase synthesis until late in the exponential In particular, two enzymic modes of synthesis were phase of growth, just as expected. A similar result discovered. In particular, polyketide synthesis takes was obtained when the lacZ gene was replaced place on an enzyme complex in a manner analogous with a construct encoding two different metabolic to fatty acid synthesis. Furthermore, there are two enzymes. Finally, as a real test of the system, an en- types of complex. In type II complexes, the different gineered construct encoding a pathway for the enzymic activities are encoded by separate subunits. synthesis of the carotenoid lycopene was placed In contrast, in type I synthesis all the different under the control of the glnAp2 promoter. This was enzyme activities are encoded by a single, very large a particularly interesting test because one of the pre- gene. Clearly, the polyketide synthases are prime cursors of lycopene, pyruvate, is also an immediate candidates for DNA shuffling, as described in the precursor of acetyl phosphate. Once again, product POGC14 9/11/2001 11:11 AM Page 309

Applications of recombinant DNA technology 309

Glucose

Glyceraldehyde 3-phosphate Lycopene

Pyruvate

NRI Acetyl phosphate

NRI-P Acetate

Expression

NRI-P glnAp2 lacZ or lycopene biosynthesis Fig. 14.20 Construction of a metabolic binding promoter genes control circuit. See text for details. region

(lycopene) formation was controlled by the meta- metabolic pathways of plants are so extensive and bolic state of the cell. Although metabolic control complex that, in most cases, such a strategy would engineering is still at an early stage, it represents a prove impossible. Fortunately, advances in plant significant degree of sophistication of control com- transformation have made it possible to carry out pared with the use of simple controllable promoters. metabolic engineering in plants themselves, and large-scale plant cell cultures can be used in the same manner as microbial cultures for the produc- Metabolic engineering in plant cells tion of important phytochemicals (reviewed by Plants synthesize an incredibly diverse array of Verpoorte 1998, Verpoorte et al. 2000). useful chemicals. Most are products of secondary The secondary metabolic pathways of most plants metabolism – that is, biochemical pathways that are produce the same basic molecular skeletons, but not involved in the synthesis of essential cellular these are ‘decorated’ with functional groups in a components but which synthesize more complex highly specific way, so that particular compounds molecules that provide additional functions. Examples may be found in only one or a few plant species. of these functions are attraction of pollinators and Furthermore, such molecules are often produced in resistance to pests and pathogens. In many cases, extremely low amounts, so extraction and purification these secondary metabolites have specific and potent can be expensive. For example, the Madagascar pharmacological properties in humans: well-known periwinkle Catharanthus roseus is the source of two examples include caffeine, nicotine, morphine and potent anti-cancer drugs called vinblastine and cocaine. vincristine. These terpene indole alkaloids are too Plants have long been exploited as a source of complex to synthesize in the laboratory and there pharmaceutical compounds, and a number of species are no alternative natural sources. In C. roseus, these are cultivated specifically for the purpose of extract- molecules are produced in such low amounts that ing drugs and other valuable molecules. We dis- over 1 ha of plants must be harvested to produce a cussed above how gene transfer to bacteria and single gram of each drug, with a commercial value of yeast can be used to produce novel chemicals, so in over $1 million. theory it would be possible to transfer the necessary It would be much more convenient to produce components from these useful plants into microbes such drugs in fermenters containing cultured plant for large-scale production. However, the secondary cells, and this has been achieved for a number of POGC14 9/11/2001 11:11 AM Page 310

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compounds, two of which (paclitaxel and shikonin) detail. Examples include the phenylpropanoid and have reached commercial production (see Verpoorte flavonoid pathways, which yield anthocyanins et al. 2000). However, cell-suspension cultures often (plant pigments) and phytoalexins (antimicrobial do not produce the downstream products made by compounds), and the terpene indole alkaloid biosyn- the parent plant. This applies to vinblastine and vin- thetic pathway, which generates important alka- cristine from C. roseus, and also to other important loids, such as vinblastine and vincristine. As more drugs, such as morphine, codeine and hyoscyamine. plant genomes are sequenced, we are likely to learn Part of the reason for this is the complexity of sec- much more about such pathways. ondary metabolism. The entire pathway is not com- All terpene indole alkaloids derive from a single pleted in a single cell, but is often segregated into universal precursor called strictosidine. This is different cell types, with consequent shuttling of formed by the convergence of two pathways, the iri- intermediates between cells. Within the cell, differ- doid pathway (culminating in secologanin) and the ent stages of the pathway are also compartmental- terpenoid pathway (culminating in tryptamine). ized, so that intermediates must be transported Strictosidine is formed by the condensation of seco- between organelles. As in bacteria, knowledge of the loganin and tryptamine, catalysed by the enzyme target biosynthetic pathway is therefore essential strictosidine synthase, and is then further modified for metabolic engineering, but in plants only a few in later steps to produce the valuable downstream secondary pathways are understood in sufficient alkaloids (Fig. 14.21). The conversion of tryptophan

COOH L-Tryptophan NH N 2 H

Tryptophan decarboxylase H O (TDC) C H 5 OGIu Tryptamine H 2 O NH2 N CH3OOC H Secologanin

Strictosidine synthase (SSS)

3 NH N H H H OGIu Strictosidine H O CH3OOC Fig. 14.21 Abbreviated pathway of terpene indole alkaloid biosynthesis, N showing the conversion of tryptophan H N into tryptamine by the enzyme N tryptophan decarboxylase, the H H H CH condensation of tryptamine and 2 secologanin into strictosidine, and the CH3O N OAc H H OH O later diversification of strictosidine into CH COOCH CH3OOC 3 2 valuable akaloids, such as ajmalicine Ajmalicine Vindoline and vindoline (a precursor of both vinblastine and vincristine). POGC14 9/11/2001 11:11 AM Page 311

Applications of recombinant DNA technology 311

to tryptamine is a rate-limiting step in the terpenoid end-point. Another example of the production of pathway, and this has been addressed by overex- novel chemicals in plants is the diversion of carbon pressing the enzyme tryptophan decarboxylase in backbones from fatty acid synthesis to the formation C. roseus cell-suspension cultures. However, while of polyhydroxyalkanoates, which form biodegrad- transformed cultures produced much higher levels able thermoplastics (Steinbuchel & Fuchtenbusch of tryptamine, no downstream alkaloids were syn- 1998). In this case, the foreign genes derive not from thesized (Goddijn et al. 1995, Canel et al. 1998). The other plants, but from bacteria. simultaneous overexpression of the next enzyme in the pathway, strictosidine synthase, did increase the Theme 6: Plant breeding in the levels of the alkaloid ajmalicine, a useful sedative, in twenty-first century some cultures, but did not result in the synthesis of vinblastine or vincristine (Canel et al. 1998). Introduction to theme 6 Thus it seems that single-step engineering may remove known bottlenecks only to reveal the posi- We have discussed several uses for transgenic plants tion of the next. The limited success of single-gene earlier in this chapter, i.e. as bioreactors producing approaches has resulted in the development of recombinant proteins (p. 285) and novel metabol- alternative strategies for the coordinated regulation ites (see previous section). Transgenic plants can of entire pathways using transcription factors. Using potentially express any foreign gene, whether that the yeast one-hybrid system (Vidal et al. 1996a), a gene is derived from bacteria, yeast, other plants or transcription factor called ORCA2 has been ident- even animals. The scope for exploitation and improve- ified that binds to response elements in the genes ment is virtually limitless, and gene-manipulation for tryptophan decarboxylase, strictosidine synthase techniques have therefore given the biotechnology and several other genes encoding enzymes in the industry a new lease of life. In the following sections, same pathway. A related protein, ORCA3, has been we consider the development of plant biotechnology identified using insertional vectors that activate in two key areas: improvement of agronomic traits genes adjacent to their integration site. By bringing and modification of production traits. the expression of such transcription factors under the control of the experimenter, entire metabolic Improving agronomic traits pathways could be controlled externally (see review by Memelink et al. 2000). The initial focus of plant biotechnology was on Apart from the modification of endogenous improving agronomic traits, i.e. the protection of metabolic pathways to produce more (or less) of a crops against pests, pathogens and weeds, and thus specific endogenous compound, plants can also be increasing yields. Major crop losses are caused every engineered to produce heterologous or entirely novel year by these so-called ‘biotic’ constraints, as well molecules. An example is the production of the as physical (or ‘abiotic’) factors, such as flooding, alkaloid scopolamine, an anticholinergic drug, in drought, soil quality, etc. The aims of the biotechno- Atropa belladonna (Hashimoto et al. 1993, Hashimoto logy industry went hand in hand with those of & Yamada 1994). Scopolamine is produced in Hyo- conventional breeders, but offered the possibility of scyamus niger but not in A. belladonna, which accu- importing useful genes from distant species that could mulates the immediate precursor hyoscyamine. H. not be used for breeding. It has been found that, in niger converts hyoscyamine into scopolamine using many cases, single genes transferred from another the enzyme hyoscyamine-6-hydroxylase (H6H), organism can provide high levels of protection. which is absent in A. belladonna. Hashimoto and colleagues isolated a complementary DNA (cDNA) Herbicide resistance encoding H6H from H. niger and expressed it in A. belladonna. The transgenic A. belladonna plants pro- Herbicides generally affect processes that are unique duced scopolamine because they were able to extend to plants, e.g. photosynthesis or amino acid biosyn- the metabolic pathway beyond its endogenous thesis (see Table 14.8). Both crops and weeds share POGC14 9/11/2001 11:11 AM Page 312

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Table 14.8 Mode of action of herbicides and method of engineering herbicide-resistant plants.

Herbicide Pathway inhibited Target enzyme Basis of engineered resistance to herbicide

Glyphosate Aromatic amino acid 5-Enol-pyruvyl shikimate- Overexpression of plant EPSP gene or introduction of biosynthesis 3-phosphate (EPSP) bacterial -resistant aroA gene synthase Sulphonylurea Branched-chain amino Acetolactate synthase Introduction of resistant ALS gene acid biosynthesis (ALS) Imidazolinones Branched-chain amino ALS Introduction of mutant ALS gene acid biosynthesis Phosphinothricin Glutamine biosynthesis Glutamine synthetase Overexpression of glutamine synthetase or introduction of the bar gene, which detoxifies the herbicide

Atrazine Photosystem II QB Introduction of mutant gene for QB protein or introduction of gene for glutathione-S-transferase, which can detoxify atrazines Bromoxynil Photosynthesis Introduction of nitrilase gene, which detoxifies bromoxynil

these processes, and developing herbicides that are to glyphosate resistance has been to introduce a selective for weeds is very difficult. An alternative gene encoding a mutant EPSP synthase. This approach is to modify crop plants so that they mutant enzyme retains its specific activity but has become resistant to broad-spectrum herbicides, i.e. decreased affinity for the herbicide. Transgenic incorporating selectivity into the plant itself rather tomato plants expressing this gene under the control than relying on the selectivity of the chemical. Two of an opine promoter were also glyphosate-tolerant approaches to engineering herbicide resistance have (Comai et al. 1985). Following on from this early been adopted. In the first, the target molecule in the research, several companies have introduced gly- cell either is rendered insensitive or is overproduced. phosate tolerance into a range of crop species, with In the second, a pathway that degrades or detoxifies soybean and the first to reach commercializa- the herbicide is introduced into the plant. An ex- tion (Nida et al. 1996, Padgette et al. 1996). Currently, ample of each strategy is considered below. nearly three-quarters of all transgenic plants in the Glyphosate is a non-selective herbicide that world are resistant to glyphosate (James 2000). inhibits 5-enol-pyruvylshikimate-3-phosphate (EPSP) Phosphinothricin (PPT) is an irreversible inhibitor synthase, a key enzyme in the biosynthesis of of glutamine synthetase in plants and bacteria. aromatic amino acids in plants and bacteria. A Bialaphos, produced by Streptomyces hygroscopicus, glyphosate-tolerant Petunia hybrida cell line obtained consists of PPT and two alanine residues. When after selection for glyphosate resistance was found to these residues are removed by peptidases the herb- overproduce the EPSP synthase as a result of gene icidal component PPT is released. To prevent self- amplification. A gene encoding the enzyme was sub- inhibition of growth, bialaphos-producing strains sequently isolated and introduced into petunia plants of S. hygroscopicus also produce the enzyme phos- under the control of a cauliflower mosaic virus phinothricin acetyltransferase (PAT), which in- (CaMV) 35S promoter. Transgenic plants expressed activates PPT by acetylation. The bar gene that increased levels of EPSP synthase in their chlo- encodes the acetylase has been introduced into roplasts and were significantly more tolerant to potato, tobacco and tomato cells using Agrobacterium- glyphosate (Shah et al. 1986). An alternative approach mediated transformation. The resultant plants were POGC14 9/11/2001 11:12 AM Page 313

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(a) (b)

Fig. 14.22 Evaluation of phosphinothricin in transgenic tobacco plants under field conditions. (a) Untransformed control plants. (b) Transgenic plants. (Photographs courtesy of Dr J. Botterman and the editor of Biotechnology.)

resistant to commercial formulations of PPT and transgenic crops are being tested, only crops resist- bialaphos in the laboratory (De Block et al. 1987) ant to glyphosate or bromoxynil are currently grown and in the field (De Greef et al. 1989) (Fig. 14.22). on a commercial scale. The benefits and risks of More recently, it has been shown that bialaphos- herbicide-resistant crops and strategies to combat resistant transgenic rice plants that were inoculated gene transfer to weeds are discussed in recent with the fungi causing sheath blight disease and reviews (Gressel 1999, 2000). subsequently treated with the herbicide were com- pletely protected from infection (Uchimiya et al. Virus resistance 1993). This agronomically important result depends on the observation that bialaphos is toxic to fungi as Major crop losses occur every year as a result of viral well as being a herbicide. PPT resistance is widely infections, e.g. tobacco mosaic virus (TMV) causes used in plants as a selectable marker (see p. 231); losses of over $50 million per annum in the tomato however, it has also been introduced into a number industry. There is a useful phenomenon known as of different crops for weed control, including sugar cross-protection, in which infection of a plant with cane and rice (Gallo-Meagher & Irvine 1996, Oard one strain of virus protects against superinfection et al. 1996). Thus far, PPT-resistant transgenic with a second, related strain. The mechanism of plants have not been commercially released. cross-protection is not fully understood, but it is The benefit of herbicide-resistant transgenic crops believed that the viral coat protein is important. is the increased yield and seed quality as competing Powell-Abel et al. (1986) developed transgenic weed species are eliminated. However, there was ini- plants that express the TMV coat protein and which tial concern that this would come with an associated had greatly reduced disease symptoms following penalty of increased herbicide use, which could have virus infection. Since that observation, the principle a serious impact on the environment. Contrary to of heterologous coat-protein expression has been these predictions, the introduction of herbicide- extended to many different plants and viruses resistant plants has actually reduced chemical use (reviewed by Beachy et al. 1990). In the case of resist- by up to 80% in many areas, as farmers adopt better ance to TMV, the coat protein must be expressed in weed-control policies and switch to herbicides with the epidermis and in the vascular tissue, through low use rates. A further risk is that transgenes for which the virus spreads systemically (Clark et al. herbicide tolerance could spread to weed species, 1990). Transgenic squash containing multiple viral resulting in a new breed of ‘superweeds’ (Kling coat-protein genes and demonstrating resistance to 1996). It is too early to say whether this will be a cucumber mosaic virus, water-melon mosaic virus problem. Although a range of herbicide-resistant 2 and zucchini yellow mosaic virus was the first POGC14 9/11/2001 11:12 AM Page 314

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virus-resistant transgenic crop to reach commercial (1993) expressed a single-chain Fv fragment (scFv) production (Tricoli et al. 1995). specific for ACMV in transgenic N. benthamiana, and While the heterologous coat-protein approach demonstrated resistance to viral infection. Other can be successful, it has been demonstrated that, in groups have generated transgenic tobacco plants many cases, the effect of transgene expression is expressing antibodies specific for TMV, resulting in mediated at the RNA rather than the protein level. reduced infectivity. Voss et al. (1995) expressed full- This can be proved by generating transgenic plants size IgGs, while Zimmermann et al. (1998) expressed carrying coat-protein genes that cannot be trans- scFv fragments. Targeting scFv fragments to the lated to yield functional protein, as first shown by plasma membrane also provides protection against Lindbo and Dougherty (1992) using the tobacco virus infection (Schillberg et al. 2001). etch virus coat-protein gene. The transgene RNA apparently interferes with viral replication (a Resistance to microbial pathogens phenomenon called RNA-mediated viral resistance (RMVR)). This requires homology between the Progress has also been made in developing resistance transgene and the target virus, and involves high- to plant-pathogenic fungi which are traditionally level transgene transcription but low-level accumu- controlled by appropriate farming practices (e.g. lation of the transcript. This has much in common crop rotation) and the application of expensive and with post-transcriptional gene silencing (discussed environmentally harmful fungicides. A straightfor- in more detail in Chapter 13), which can lead to ward approach is to engineer plants with antifungal transgene silencing and the cosuppression of homo- proteins from heterologous species. This was first logous endogenous genes, as well as viral resistance. demonstrated by Broglie et al. (1991) who showed The interested reader can consult several excellent that expression of bean chitinase can protect tobacco reviews covering this and related phenomena and oil-seed rape from post-emergent damping off (Waterhouse et al. 1998, Grant 1999, Plasterk & caused by Rhizoctonia solani. Plants synthesize a Ketting 2000, Hammond et al. 2001). wide range of so-called ‘pathogenesis-related pro- A different method of minimizing the effects of teins’ (PR proteins), such as chitinase, which are plant virus infection was developed by Gehrlach induced by microbial infection. They also synthesize et al. (1987). They generated plants that expressed antifungal peptides called defensins and other the satellite RNA of tobacco ringspot virus and such antifungal proteins. As the genes for more of these plants were resistant to infection with tobacco proteins have been cloned and characterized, the ringspot virus itself. Another potential method of number of transgenic plants constitutively express- inducing resistance to viruses is the production ing such proteins continues to rise. For example, of antiviral proteins in transgenic plants. American tobacco osmotin has been expressed in transgenic pokeweed produces an antiviral protein called potato, providing resistance to Phytophthora infes- dianthrin that functions as a ribosome-inactivating tans (Liu et al. 1994), and in transgenic rice, provid- protein. The cDNA for this protein has been cloned ing resistance to R. solani (Lin et al. 1995). Instead (Lin et al. 1991) and expressed in Nicotiana bentham- of using a protein to provide direct protection, iana (a relative of tobacco), providing resistance a metabolic-engineering strategy can be utilized. against African cassava mosaic virus (ACMV) Phytoallexins are alkaloids with antifungal activity, (Hong et al. 1996). Interestingly in this experiment, and transforming plants with genes encoding the the dianthrin gene was expressed under the control appropriate biosynthetic enzymes can increase of an ACMV promoter, such that the antiviral pro- their synthesis. Hain et al. (1993) generated tomato tein was expressed only upon viral infection. In this plants expressing the grapevine gene for stilbene manner, the toxic effects of constitutive transgene synthase, and these plants demonstrated increased expression were avoided. resistance to infection by Botrytis cinerea. Similarly, Antibodies specific for virion proteins have also Anzai et al. (1989) have used a bacterial gene been used to protect plants from viruses. In the first facilitating tabtoxin detoxification to protect tomato demonstration of this approach, Tavladoraki et al. plants against Pseudomonas syringae infection. POGC14 9/11/2001 11:12 AM Page 315

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An alternative to the use of antifungal proteins or protoxins called crystal proteins. These proteins are metabolites is to manipulate the hypersensitive potent and highly specific insecticides. The specificity response, which is a physiological defence mechan- reflects interactions between the crystal proteins ism used by plants to repel attacking pathogens. and receptors in the insect midgut. In susceptible Resistance occurs only in plants carrying a resist- species, ingested crystals dissolve in the alkaline ance gene (R) that corresponds to an avirulence conditions of the gut and the protoxins are activated (avr) gene in the pathogen. Elicitors (signalling by gut proteases. The active toxins bind to receptors molecules) released by pathogens are detected by on midgut epithelial cells, become inserted into the the plant and activate a range of defence responses, plasma membrane and form pores that lead to cell including cell death, PR-gene expression, phyto- death (and eventual insect death) through osmotic allexin synthesis and the deposition of cellulose at the lysis. Approximately 150 distinct Bt toxins have site of invasion, forming a physical barrier. Import- been identified and each shows a unique spectrum of antly, the hypersensitive response is systemic, so activity. that neighbouring cells can pre-empt pathogen Bt toxins have been used as topical insecticides invasion. A recently developed strategy is to transfer since the 1930s, but never gained widespread use, avirulence genes from the pathogen to the plant, because they are rapidly broken down on exposure under the control of a pathogen-inducible promoter. to daylight and thus have to be applied several times This has been demonstrated in tomato plants during a growing season. Additionally, only insects transformed with the avr9 gene from Chladosporium infesting the exposed surface of sprayed plants are fulvum, resulting in resistance to a range of fungal, killed. These problems have been addressed by the bacterial and viral diseases (Keller et al. 1999, expression of crystal proteins in transgenic plants. reviewed by Melchers & Stuiver 2000). Bt genes were initially introduced into tomato (Fischhoff et al. 1987) and tobacco (Barton et al. 1987, Vaeck et al. 1987) and later into cotton Resistance to insects and other pests (Perlak et al. 1990), resulting in the production of Insect pests represent one of the most serious biotic insecticidal proteins that protected the plants from constraints to crop production. For example, more insect infestation. However, field tests of these plants than a quarter of all the rice grown in the world is revealed that higher levels of the toxin in the plant lost to insect pests, at an estimated cost of nearly tissue would be required to obtain commercially US$50 billion. This is despite an annual expenditure useful plants (Delannay et al. 1989). Attempts to of approximately US$1.5 billion on insecticides for increase the expression of the toxin gene in plants by this crop alone. Insect-resistant plants are therefore use of different promoters, fusion proteins and leader desirable not only because of the potential increased sequences were not successful. However, examina- yields, but also because the need for insecticides tion of the bacterial cry1Ab and cry1Ac genes is eliminated and, following on from this, the indicated that they differed significantly from plant undesirable accumulation of such chemicals in the genes in a number of ways (Perlak et al. 1991). For environment is avoided. Typical insecticides are example, localized AT-rich regions resembling plant non-selective, so they kill harmless and beneficial introns, potential plant polyadenylation signal insects as well as pests. For these reasons, transgenic sequences, ATTTA sequences that can destabilize plants have been generated expressing toxins that mRNA and rare plant codons were all found. The are selective for particular insect species. elimination of undesirable sequences and modifica- Research is being carried out on a wide range of tions to bring codon usage into line with the host insecticidal proteins from diverse sources. However, species resulted in greatly enhanced expression of all commercially produced insect-resistant transgenic the insecticidal toxin and strong insect resistance crops express toxin proteins from the Gram-positive of the transgenic plants in field tests (Koziel et al. bacterium (Bt). Unlike other 1993). By carrying out such enhancements, Perlak Bacillus species, Bt produces crystals during sporula- and colleagues expressed a modified cry3A gene in tion, comprising one or a small number of ~130 kDa potato to provide resistance against Colorado beetle POGC14 9/11/2001 11:12 AM Page 316

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(Perlak et al. 1993). In 1995, this crop became the soluble solids and facilitate processing. Several ex- first transgenic insect-resistant crop to reach com- amples are discussed below and Box 14.4 describes mercial production, as NewLeafTM potato marketed the recently developed technology, which by . The same company also released the provides a mechanism by which producers of trans- first commercial transgenic, insect-resistant vari- genic crops could dictate where and when their eties of cotton (BollgardTM, expressing cry1Ac and modified plants are grown. protected against tobacco bollworm) and maize (YieldGardTM, expressing cry1Ab and resistant to the Delayed ripening European corn-borer). Many other biotechnology companies have now produced Bt-transgenic crop The earliest example of quality improvement by plants resistant to a range of insects. gene transfer was the introduction of antisense Although Bt-transgenic plants currently dominate constructs targeting the polygalacturonase (pg) the market, there are many alternative insecticidal gene in tomato to delay ripening (Sheehy et al. proteins under investigation. Two types of protein 1988, Smith et al. 1988), as discussed in Chapter 13 are being studied in particular: proteins that inhibit (p. 260). Other strategies to delay ripening include: digestive enzymes in the insect gut (proteinase and cosuppression of the pg gene using sense constructs amylase inhibitors) and lectins (carbohydrate- (Smith et al. 1990), the use of antisense RNA to binding proteins). Research into these alternatives is suppress the expression of two key enzymes required driven in part by the fact that some insects are not for ethylene synthesis (Hamilton et al. 1990, Oeller affected by any of the known Bt crystal proteins. et al. 1991) and the degradation of a key intermedi- Homopteran insects, mostly sap-sucking pests such ate in the ethylene biosynthetic pathway (Klee et al. as planthoppers, fall into this category, but have 1991; see review by Fray & Grierson 1993). been shown to be susceptible to lectins such as Galanthus nivalis agglutinin (GNA). This lectin has Starch and oil modification been expressed in many crops, including potato (Shi et al. 1994, Gatehouse et al. 1996), rice (Bano- Much research has been carried out into strategies Maqbool & Christou 1999), tomato and tobacco for the modification of the starch and oil content of (reviewed by Schuler et al. 1998). seeds. In each case, it is essential to have a thorough understanding of the normal biosynthetic path- ways. Starch is the major storage carbohydrate in Modification of production traits higher plants. It consists of two components: amy- Production traits can include many characteristics, lose, which is a linear polymer, and amylopectin, such as the colour and smell of flowers and the taste, which is a branched polymer. The physicochemical, consistency and nutritional composition of food. nutritional and textural properties of food are While modifying flower colour would seem a harm- significantly influenced by the nature of the starch. less pursuit (experiments in this area are discussed For example, cooked amylose produces fibre-like on p. 261), the genetic modification of food for structures that are resistant to digestion, serve as human consumption is the subject of intensive cur- dietary fibre and require less insulin for metabolism. rent debate. Opinions as to the benefits and risks of Amylopectin, in contrast, is waxy and viscous. The the latter differ widely according to region, ethical food and other industries use a wide range of differ- standpoint and financial/food security of the nation. ent starches. These are obtained by sourcing the There is no doubt, however, that gene manipulation starch from different plant varieties coupled with has the potential to greatly increase the quality of chemical and enzymatic modification. The genetic food. Examples include modifying the starch or oil modification of plants offers a new approach to cre- content, facilitating the accumulation of vitamins ating novel starches with new functional properties. and minerals in foods that are normally poor in such The enzyme ADP-glucose pyrophosphorylase (GP) nutrients, and altering the properties of fruits and catalyses the first step in starch biosynthesis in vegetables to delay ripening, increase the levels of plants. Stark et al. (1992) cloned the gene for an E. POGC14 9/11/2001 11:12 AM Page 317

Box 14.4 Protecting your investment: terminator technology

The production of any marketable commodity In this example, the terminator gene encodes a requires a great deal of investment in terms of ribosomal-inactivating protein and is expressed research and development. Most commodities under the control of a promoter that is active in late are not self-renewing, so manufacturers can embryogenesis, such as the promoter of the lea recover their investments through long-term (late embryogenesis abundant) gene. Seeds produced sales. Transgenic plants, however, are capable of from such plants are sterile, but, since seed storage reproduction. Farmers have to make an initial outlay products (e.g. starch and oil) accumulate in early for seed purchase, but, by saving a proportion of and mid-embryonic development, the seeds are seeds from each harvest, they need never return nutritionally unimpaired. In order to maintain a stock to the manufacturer again. For producers who of fertile plants, the terminator gene is controlled by have invested heavily in development, this is inducible site-specific recombination, as described in unacceptable. So how can the producers protect detail in Chapter 13. The gene is rendered inactive by their investment? inserting a blocking element between the gene and Until recently, this was achieved through the use its promoter. This blocking element is flanked by loxP of contracts that obliged farmers not to save any sites. The transgenic plant also contains a cre seeds. However, such contracts are difficult to recombinase gene, which is controlled by a enforce, particularly in developing countries. A tetracycline-inducible promoter, and a third novel strategy is to modify the plants so that the transgene encoding the tetracycline repressor seeds are sterile, thereby forcing the farmer to return protein, which is constitutively expressed. In the to the manufacturer year after year for fresh supplies absence of tetracycline, the repressor prevents cre of seeds. This strategy, which has been termed expression, the terminator gene is not activated technology protection by the biotechnology and the plants can be grown as normal, allowing companies and terminator technology by disgruntled the producer to grow plants and produce seeds in consumers and opponents, is perhaps the most unlimited quantity. Before distribution, however, the controversial potential use of genetically modified seeds are soaked in tetracycline, which causes the plants. In essence, the technology works rather like repressor to release the cre promoter, thus inducing the anti-copying devices now incorporated into video the cre gene, leading to excision of the blocking recorders – the industry is protecting its investment fragment and activation of the terminator gene. by preventing unauthorized duplication of its Since the terminator gene is controlled by an products. The principle of terminator technology is embryonic promoter, it is not switched on until the expression of a toxic transgene, the terminator the following generation of developing seeds. gene, at a critical stage of embryonic development, The advent of terminator technology was met by thus killing the embryo and rendering the seeds a public outcry, particularly from farmers and from incapable of germination. Several variants on this environmentalists concerned that terminator genes technology were described in a patent application, could spread from the transgenic crops into wild granted in March 1998, to Pine Lands Corporation, plants, with unknown consequences. Perhaps in which has since been bought by Monsanto. One of response to this, Monsanto has since pledged not the variations is discussed below. to implement the technology now at its disposal.

(a) No tetracycline

P Tet R TETR P Cre PLCA RIP

tetO

(b) Tetracycline present TET TETR Cre

P Tet R P Cre PLCA

tetO

Oliver et al. (Pine Land Corporation) United States Patent 5 723765, ‘Control of Plant Gene Expression’, 3 March 1998. POGC14 9/11/2001 11:12 AM Page 318

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coli mutant GP which was deficient in allosteric reg- these lipids are used in detergent synthesis. Some ulation. When this gene was inserted into potato plants, such as the California bay, accumulate MCT plants, the tubers accumulated higher levels of because of the presence of a medium-chain specific starch but had normal starch composition and gran- thioesterase. When the gene for this thioesterase ule size. Since transgenic plants expressing the wild- was expressed in rape-seed and Arabidopsis, the type E. coli GP had normal levels of starch, the transgenic plants accumulated high levels of MCT allosteric regulation of GP, and not its absolute level, (Voelker et al. 1992). must influence starch levels. A different method of modulating GP activity has been used by Muller- Improving vitamin and mineral content Rober et al. (1992). They used an antisense con- struct to reduce the level of plant GP to 2–5% of wild The vitamin and mineral content of plants varies type. These plants had very low levels of starch (also from species to species, and from tissue to tissue in a 2–5% of wild type) but had a very much higher particular plant. Milled cereal grains – basically the number of tubers. These tubers had six- to eightfold endosperm component of the seed – are particularly more glucose and sucrose and much lower levels of deficient in essential vitamins and minerals, and patatin and other storage proteins. Visser et al. (1991) yet they represent the staple food for much of the have modified the starch composition of potato plants. world’s population. In developed countries, where They demonstrated that potato plants expressing an diet is varied, this is seldom a problem. However, in antisense gene to the granule-bound starch synthase poorer nations, where cereal grains are often the have little or no amylose compared with wild-type only food available, this can cause major health potato, where it can be as much as 20%. problems. Iron deficiency is the most widespread Whereas some plants accumulate starch as a nutrient deficiency in the world, reflecting the com- carbon reserve, others accumulate high levels of bination of low iron in cereal grains and the high triacylglycerols in seeds or mesocarp tissues. Higher level of phytic acid, which reduces the efficiency of plants produce over 200 kinds of fatty acids, some of iron absorption from the gut. Many cereal grains which are of food value. However, many are likely to also lack β-carotene, which is required in the diet for have industrial (non-food) uses of higher value vitamin A biosynthesis. Vitamin A deficiency in early than edible fatty acids (Kishore & Somerville 1993, childhood leads to blindness, an avoidable conse- Murphy 1999). Thus there is considerable interest quence of poor diet in many parts of Asia and Africa. in using gene-manipulation techniques to modify A number of attempts to increase the nutritional the fatty acid content of plants. As a first step towards value of cereals have been reported, and we discuss this goal, a number of genes encoding desaturases two examples of genetically engineered rice below, have been cloned, e.g. the MS desaturase, which since this cereal is the staple diet of at least two- converts linoleic acid to α-linolenic acid (Arondel thirds of the world’s population. Lucca et al. (2001) et al. 1992). described three different routes to improving the In most plants the ∆9 stearoyl acyl carrier protein iron content of rice grain, which could be used singly (ACP) desaturase catalyses the first desaturation or in combination. The first route was to transform step in seed-oil biosynthesis, converting stearoyl- rice with a gene encoding ferritin, an iron-storage ACP to oleoyl-ACP. When antisense constructs were protein. The overexpression of bean ferritin in rice used to reduce the levels of the enzyme in developing endosperm led to the accumulation of iron in a rape-seed embryos, the seeds had greatly increased bioavailable form, resulting in transgenic rice grains stearate levels (Knutzon et al. 1992). The signi- with twice the normal levels of iron. The second ficance of this result is that high stearate content is of route was to transform rice with a gene from the value in margarine and confectionery fats. fungus Aspergillus fumigatus encoding the enzyme Most oil-seed crops accumulate triacylglycerols phytase, which metabolizes phytic acid. Since the

with C16 to C22 acyl chains. However, plants that transgenic grain has lower levels of phytic acid, iron accumulate medium-chain triacylglycerols (MCT), absorption from the gut should be more efficient.

with C8 to C12 acyl chains, would be very useful, for The third approach was to transform rice with a POGC14 9/11/2001 11:12 AM Page 319

Applications of recombinant DNA technology 319

construct causing the overexpression of an endo- burden of developing and refining the tools and genous cysteine-rich metallothionein-like protein, protocols, producing high-quality enzymes, special- since cysteine peptides have been shown to have the ist molecular- kits and premade custom opposite effect to phytic acid, i.e. they enhance iron genomic or cDNA libraries. Most importantly, there absorption. The resulting rice grains had over five has been a drive to develop high-throughput versions times the normal level of cysteine residues and a of many of the techniques discussed in this book, 130-fold increase in phytase activity, theoretically allowing more and more samples to be processed in sufficient to eliminate all phytic acid from the grains. parallel, producing more data in less time than ever Burkhardt et al. (1997) described rice plants before. Sequencing has been at the forefront of this transformed with the phytoene synthase gene from paradigm shift, with the result that the sequencing the daffodil (Narcissus pseudonarcissus). The enzyme of entire genomes is now commonplace. As this sixth encoded by this gene represents the first of four steps edition of Principles of Gene Manipulation goes to press, in the β-carotene biosynthesis pathway, and the over 40 bacterial genomes plus those of four model plants were shown to accumulate the immediate eukaryotes have been completely sequenced and the product of the recombinant enzyme, phytoene. first draft of the entire human genome sequence Further work by the same group (Ye et al. 2000) has been published (International Human Genome resulted in rice plants expressing several daffodil Sequencing Consortium 2001, Venter et al. 2001). enzymes simultaneously in the endosperm, and These extraordinary achievements have ushered recapitulated the entire heterologous pathway. in a new era in molecular biology, which is analysis Hence, the transgenic rice grains were highly at the whole-genome level. More data are emerging enriched for β-carotene (provitamin A) and were from genome-sequencing projects and the random consequently golden in colour. cloning of partial cDNAs (known as expressed sequence tags (ESTs)) than can be dealt with using traditional single-gene analysis methods. A new breed Epilogue: from genes to genomes of high-throughput functional-analysis techniques Principles of Gene Manipulation tells the story of the has been developed, some of which are discussed development of genetic-engineering technology, a briefly in Chapters 6, 9 and 13 of this book. Simple story that began in the mid-1970s. When the first northern blots and reverse transcription (RT)-PCR edition of this book was published in 1980, basic experiments are being replaced by the use of DNA techniques, such as cloning, sequencing and expres- chips containing hundreds or thousands of indi- sion analysis, were all in their infancy and many vidual sequences that can be hybridized with a of today’s most widely used procedures, including suitable probe simultaneously. Instead of analysing the PCR, had not been invented. In those days, the single proteins by western blots, two-dimensional cloning and characterization of a single gene was gel electrophoresis is being used to simultaneously an elaborate task that required a great deal of in- study every single protein expressed under various ventiveness and hard work. The sequencing and conditions. The creation and functional analysis analysis of entire cellular genomes was an imposs- of individual mutants is being superseded by sys- ible goal, as far-fetched as space travel was to people tematic approaches to mutating and characterizing 100 years ago. every single gene in the genome. Techniques are Over the last 25 years, however, the technology of also being developed for the analysis of all possible gene manipulation has rapidly increased in sophist- protein–protein interactions in the cell. ication. Through the sharing of information between While the impact of the new genomics is dis- laboratories, standard molecular-biology procedures cussed in this book, as least as far as it affects trends have become more robust and reliable, new tech- in gene-manipulation technology, a full account of niques have evolved, and many of the more labori- its development, application and potential requires ous procedures that scientists had to carry out in the a book of its own. The story continues in our sister early years have been streamlined or automated. text, Principles of Genome Analysis and Genomics Biotechnology companies now take on most of the (third edition).