Agro/ANSC/Biol/Hort/Gene 305

Agro/ANSC/Biol/Hort/Gene 305 Fall, 2016 Biotechnology. Chpt 20 Genetics by Brooker. (# 15)

Biotechnology is broadly defined as technologies that involve the use of living organisms, or their products, to benefit humans. It is not a new topic. It began about 12,000 years ago when humans began to domesticate animal and plants for the production of food. Since the 1970s, molecular genetics has provided new, improved ways to make use of organisms to benefit humans. An organism that has integrated recombinant DNA into its genome is called transgenic

Microorganisms in Biotechnology: (Table 21.1) Molecular genetic tools are very important in influencing and improving our use of microorganisms. Overall, the use of recombinant microorganisms is an area of great research interest and potential. However, there are problems such as safety concerns and negative public perception. Insulin regulates several physiological processes, particularly the uptake of glucose into fat and muscle cells. It is produced by the B cells of the pancreas. Persons with insulin-dependent diabetes have a defect in their B cells. Therefore, they cannot synthesize enough insulin. Sources of insulin included: Cows and human cadavers. But now, patients can use insulin made by recombinant bacteria (Figure 21.1)

Organismal cloning refers to methods that produce two or more genetically identical individuals. Identical twins are genetic clones from one fertilized egg. - Cloning is an easier undertaking in plants. Plants can be cloned from somatic cells. For several decades scientists believed that mammalian somatic cells were unsuitable for cloning. But in 1997, Ian Wilmut and his colleagues at the Roslin Institute created Dolly! Evidence suggested that Dolly may be “genetically older” than her actual age would have indicated. In recent years, cloning from somatic cells has been achieved in several mammalian species: Sheep, cows, mice, goats and pigs. With regard to livestock, farmers can use somatic cells from their best individuals to create genetically homogeneous herds. This may be advantageous with regard to agricultural yield. However, such a herd may be more susceptible to rare diseases. - People have become greatly concerned with the possibility of human cloning. To some, it is morally wrong and threatens the basic fabric of parenthood and family. To others, it offers a new avenue of reproduction. - In the public sector, the sentiment toward human cloning has been generally negative. Indeed, many countries have issued an all-out ban. While others permit limited research in the area. In the future our society will have to wrestle with the legal and ethical aspects of cloning. (Figures 21.8, 21.9)

1 Stem cells supply the cells that construct our bodies from a fertilized egg. In the adult, stem cells also replenish damaged cells. Stem cells have two common characteristics: 1. They have the capacity to divide 2. They have the capacity to differentiate into one or more specialized cell types (Figure 21.10, 21.11, 21.12) - Interest in stem cells centers around two main areas 1. They may help us understand the basic genetic mechanisms that underlie the process of development 2. They offer the potential to treat human diseases or injuries that cause cell and tissue damage. This application has already become a reality. For example, bone marrow transplants are used to treat patients with certain forms of cancer. As shown in Table 21.4, embryonic stem cells could potentially be used to treat a wide variety of diseases

Agrobacterium tumefaciens can Be Used to Make Transgenic Plants - The production of transgenic plants is somewhat easier than transgenic animals. Plant cells are totipotent. An entire organism can be regenerated from a somatic cell. Agrobacterium tumefaciens is a bacterium that naturally infects plants causing crown gall tumor. (Figure 21.15) - The A. tumefaciens T DNA can be used as a vector to introduce cloned genes into plants. First, the Ti plasmid needs to be modified. The genes that cause tumors are deleted. Selectable marker genes are inserted into the T DNA. KanR is commonly used. Unique restriction sites are added for the convenient insertion of any gene (Figure 21.16) - A. tumefaciens does not infect all plant species. Other methods are available: Biolistic gene transfer is the second most common way to produce transgenic plants. A “DNA gun” is used to shoot DNA-coated microprojectiles into the cells Microinjection: Microscopic-sized needles are used to inject DNA into the cells Electroporation: An electric current is used to create transient pores in the plasma membrane through which DNA can enter.

APPLICATIONS OF TRANSGENIC PLANTS AND ANIMALS - Transgenic organisms can provide important information regarding the functional role of genes. - Transgenic animals are also becoming invaluable tools for studying the mechanisms and treatment of human disease - Gene replacements and gene knockouts have become powerful tools for understanding gene function - In some cases, gene knockouts produce phenotypic effect. This shows that a gene is critical in a certain tissue or during a specific stage of development - In other cases, gene knockouts produce no detectable phenotypic effect at all. This led to the conclusion that mammalian genomes have a fair amount of gene redundancy. - A particularly exciting avenue of gene replacement research is its application in the study of human disease: Cystic fibrosis (CF) - Researchers have produced mice that are homozygous for the same type of mutation that is found in humans with CF.

2 Much research is underway to develop transgenic species of livestock: Fish, sheep, pigs, goats and cattle. New research involves the production of medically important proteins in the mammary glands of livestock. This approach is sometimes called molecular pharming. The production of proteins from mammals is more advantageous than the from bacteria - Certain proteins are more likely to function properly when expressed in mammals: - Post-translational modifications occur in eukaryotes - Degradation and misfolding occur in bacteria - The yield of recombinant proteins in milk can be quite large. In most cases, a transgenic cow can produce ~ 1 g/L of the transgenic protein in its milk. (Figure 21.7) - Transgenic plants can be given characteristics that are agriculturally useful. For example, the Monsanto Company has produced plants highly tolerant of glyphosate, the active ingredient in the herbicide RoundupTM. Compared to nontransgenics, these plants grow quite well in the presence of glyphosate-containing herbicides (Figure 19.16) Transgenic plants have been approved for human consumption. The first example was the FlavrSavr tomato. This transgenic plant has been given the gene that encodes an antisense RNA complementary to the mRNA for polygalactorunase (an enzyme involved in fruit ripening). The antisense RNA binds to the mRNA and prevents translation. In addition, double stranded RNA is targeted for degradation. The practical advantage of the FlavrSavr tomato is improved shelf-life. It does not spoil (overripe) as quickly as traditional tomatoes.

Genetic Testing: Genetic testing refers to the use of tests to discover if an individual has a genetic abnormality. Genetic screening refers to population-wide genetic testing (Table 24.4) - In many cases, single-gene mutations that affect proteins, can be examined at the protein level - Biochemical assays may be available for enzymes. - An alternative approach is to detect single-gene mutations at the DNA level. Researchers must have previously identified the mutant gene using molecular techniques e.g., Duchenne muscular dystrophy, Huntington disease. - The most common class of human genetic abnormality is the change in chromosome number. Most of these result in spontaneous abortions. However, about 1 in 200 live births are aneuploid or have unbalanced chromosomal alterations. Chromosomal abnormalities can be detected with a karyotype. (Table 24.4) In the U.S., genetic screening for certain disorders has become common medical practice. - Genetic testing has also been conducted on specific population in which a genetic disease is prevalent, e.g., Tay-Sachs disease in the Aschenazi Jews - Genetic testing can be performed prior to birth. There are two main types of procedures: 1. Amniocentesis: Fetal cells are obtained from the amniotic fluid. 2. Chorionic villi sampling: Fetal cells are obtained from the chorion (fetal part of the placenta). Can be performed earlier during pregnancy than amniocentesis. However, it poses a slightly greater risk of miscarriage. Genetic testing and screening are medical practices with many social and ethical dimensions.

3 Do people have the right to know about their genetic makeup? Does it do more harm than good? Another issue is privacy. In this century we will become more aware of our genetic makeup and the causes of genetic diseases. It will be necessary therefore, to establish guidelines for the uses of genetic testing.

HUMAN GENE THERAPY - Gene therapy is the introduction of cloned genes into living cells in an attempt to cure disease. Research efforts in gene therapy is aimed to: - Alleviate inherited diseases - Treat diseases such as cancer and heart disease - Combat infectious diseases such as AIDS - Human gene therapy is still at an early stage. Nevertheless, some of the initial results are promising and future prospects abound (Table 21.6) Gene Therapy Involves the Introduction of Cloned Genes into Human Cells Two transfer methods are used: 1. Nonviral approach 2. Viral approach Adenosine deaminase deficiency was the first inherited disease treated with gene therapy. Adenosine deaminase (ADA) is an enzyme involved in purine metabolism. If both copies of the gene are defective, deoxyadenosine will accumulate within the cells of the individual. Deoxyadenosine is particularly toxic to B and T cells. The destruction of these cells leads to a disease termed severe combined immunodeficiency disease (SCID). If left untreated, SCID is typically fatal at an early age. Three approaches can be used to treat adenosine deaminase (ADA) deficiency: 1. A bone marrow transplant from a compatible donor 2. Purified ADA coupled to polyethylene glycol (PEG) 3. Gene therapy - On September 14, 1990 the first human gene therapy was approved for a girl with ADA deficiency. Prior to the clinical trial, the normal ADA gene had been cloned into a retrovirus that can infect lymphocytes. - Cystic fibrosis (CF) is one of the most common recessive inherited disorders. It is caused by a defect in a gene that encodes an ion transport protein. This leads to an abnormality in salt and water balance. This, in turn, leads to accumulation of mucus in the lungs. The result is chronic lung infections which prove fatal. - CF has been the subject of much gene therapy research. To implement CF gene therapy, the normal CF gene must be delivered to lung cells The ex vivo approach used in ADA gene therapy is not possible. CF gene therapy has involved the use of an aerosol spray. - In one approach, the normal CF gene is delivered in an adenovirus - In another, the gene is delivered via liposomes When inhaled by the patient via an aerosol spray, the lung epithelial cells take up liposomes and adenoviruses. Areosol Sprays May Be Used to Treat Cystic Fibrosis

4 DNA FINGERPRINTING - DNA fingerprinting is a technology that identifies particular individuals using properties of their DNA. It is also termed DNA profiling. - The application of DNA fingerprinting to forensics has captured the most public attention - In addition, DNA fingerprinting can also be used to determine if two individuals are genetically related. It is used routinely in paternity testing - When subjected to DNA fingerprinting, chromosomal DNA gives rise to a series of bands on a gel The order of bands is an individual’s DNA fingerprint. It is the unique pattern of these bands that makes it possible to distinguish individuals - In the 1980s, Alec Jeffries found that certain loci in human chromosomes are variable in length. These loci contain tandemly repeated sequences called minisatellites. In humans, the number of tandem repeats varies substantially: Variable Number of Tandem Repeats (VNTRs) - In the past decade, the technique of DNA fingerprinting has become automated. It is now done using PCR, which amplifies short tandem repeat sequences (STRs). Like VNTRs, STRs are found in multiple sites in human genomes and are variable among different individuals. The main difference between a VNTR and STR is size. - STRs are much shorter, usually 100–450 bp - STRs are called microsatellites, and VNTRs minisatellites - The amplified STRs are separated by gel electrophoresis. They are fluorescently labeled. A laser excites the fluorescent molecule within the STR. A detector records the amount of emission for each STR. - Within the past decade, the uses of DNA fingerprinting have expanded in many ways. In medicine, it is used to identify different species of bacteria and fungi and also different strains of the same species. This is useful for appropriate antibiotic treatment. - DNA fingerprinting is also used in forensics and relationship testing - In forensics, DNA fingerprinting can provide critical evidence that an individual was at a crime scene. Probability calculations show that a match between two samples is almost never a matter of random chance. However, factors such as mishandling of sample and misinterpretation of data must also be considered. - In relationship testing, DNA fingerprinting can provide evidence of genetic-relatedness Persons who are genetically-related will have some bands or peaks in common. The number they share depends on the closeness of their genetic relationship. For example, a child is expected to receive half of its VNTRs from one parent and the rest from the other.

GENETIC TESTING: Genetic testing refers to the use of tests to discover if an individual has a genetic abnormality. Genetic screening refers to population-wide genetic testing. (24.5) - In many cases, single-gene mutations that affect proteins, can be examined at the protein level. Biochemical assays may be available for enzymes. - An alternative approach is to detect single-gene mutations at the DNA level. Researchers must have previously identified the mutant gene using molecular techniques e.g., Duchenne muscular dystrophy, Huntington disease.

5 - The most common class of human genetic abnormality is the change in chromosome number. Most of these result in spontaneous abortions. However, about 1 in 200 live births are aneuploid or have unbalanced chromosomal alterations. Chromosomal abnormalities can be detected with a karyotype. In the U.S. genetic screening for certain disorders has become common medical practice. For example: - Pregnant women over 35 years of age are screened routinely to see if they are carriers of chromosomal abnormalities - Widespread screening for phenylketonuria - Genetic testing has also been conducted on specific population in which a genetic disease is prevalent, e.g., Tay-Sachs disease in the Aschenazi Jews - Genetic testing can be performed prior to birth. There are two main types of procedures: 1. Amniocentesis: Fetal cells are obtained from the amniotic fluid. 2. Chorionic villi sampling: Fetal cells are obtained from the chorion (fetal part of the placenta). Can be performed earlier during pregnancy than amniocentesis. However, it poses a slightly greater risk of miscarriage. (Figure 24.8) Genetic testing and screening are medical practices with many social and ethical dimensions.