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11 Molecular Genetics and Genetic Engineering Fundamental advances in biology during the past 12 years have brought scientists to an understanding of inheritance at the molecular level. Two technically straightforward and basic techniques--molecular cloning and DNA sequencing--are valuable and precise methods in themselves that can be used to learn about the structure and function of genes. These two techniques demonstrate an overwhelming synergistic effect: Cloning has made possible the isolation of pure DNA segments, and sequencing of the nucleotide bases that comprise a DNA molecule has made possible the analysis and characterization of those isolated segments. Thus, scientists now can routinely dissect the set of genes possessed by a particular organism and define location, arrangement, and struc- ture. From this point any number of creative manipula- tions can be employed to learn more about the transfer of desirable genes and the enhancement of traits, including those of food animals and crop plants. Combined with conventional plant and animal breeding techniques and the knowledge provided through the collab- orations of geneticists, biochemists, immunologists, molecular biologists, pathologists, and virologists, the two techniques create a solid foundation for basic research and for application in treatment and in the diagnosis of both inherited and pathogenic disease. Endless numbers of basic questions await answers: What are the precise mechanisms of expression of a gene? What prompts a gene to switch on or off? How does loca- tion of a gene affect its expression? The DNA-based 1 1

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12 technologies only now are being used in earnest to address such basic questions. These questions should become major preoccupations for the most talented researchers. Structure, Organization, and Expression of Genes Estimates of the total number of genes--the genome--in the nucleus of each cell of a crop plant or food animal range from 10,000 to 100,000. It is indeed remarkable that methods can be devised to isolate one single gene from among the thousands in the genome and manipulate it in ways that result in the expression of the gene trait in a recipient organism. The techniques leading to such gene expression are isolation, cloning, and transfer. Isolation The first step in a genetically engineered manip- ulation is to locate a single gene from among the thou- sands comprising the genome. Currently, researchers most often work with one of the few genes that have been char- acterized through past studies, for searching out the location of a gene not yet studied is much like trying to find a citation in a book without the aid of an. index. It is an arduous task that researchers have rendered somewhat easier by the creation of gene libraries for organisms. To prepare a gene library the DNA of the organism is cut, using selected restriction enzymes that recognize a specific sequence of bases and then snip the strands between particular bases. A series of different restric- tion enzymes can be used to snip the DNA until it is re- duced to lengths of approximately one to several genes. These smaller segments are sorted using a process called electrophoresis and then cloned to produce a quantity of the genetic material sufficient for further analysis. Each of these segments of DNA--the gene library--can then be searched, one at a time, to locate the desired gene. me tool used to pinpoint the gene is called a probe. The ordered pairing of nucleotide bases in the double helix renders each DNA strand complementary to the other. The ability of separate strands to bind to their complementary strand, a process called hybridization,

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13 provides a powerful probe for locating specific genes. A probe is a length of DNA or RNA, usually containing a radioactive tag, that has a sequence complementary to that of the desired gene. The radioactive tag makes the probe easily identifiable after it has paired with the nucleotide bases of the gene. Probes can be made when the sequence of a protein is known--the protein that is the end product of a particular gene. Working backward through the steps of gene expression, the researcher can determine the nucleotide base sequence of the gene and then synthesize the probe. In addition, chromosomes or segments of chromosomes can now be identified by various molecular and cytogenetic techniques as being carriers of specific genes. Use of these methods reduces the size of the gene library that must be searched to locate a gene. Cloning ~ ~ ~ _ _ Following isolation the gene is cloned, or duplicated, and inserted into its new host cell. To date, the method most often used to accomplish both is insertion of the gene into a bacterial plasmid. A plasmid is a small circle of DNA that exists separately from an organism's main chromosomal complement. A plasmid carries its own - DNA replication sequence and usually maintains itself in multiple copies within the cell. To clone a gene, the ring-shaped plasmid is cleanly cut open using a restriction enzyme. me restriction enzyme is also used to prepare a length of DNA containing an isolated gene. When the cut plasmid and the isolated gene are mixed together in the presence of DNA ligase--an enzyme that rejoins cut ends of DNA molecules--the iso- lated gene fragment is incorporated into the plasmid ring. Now as the repaired plasmid replicates, the cloned gene is also replicated. In this manner copious amounts of the cloned gene may be produced within the bacterial host cell. Cloned genes have four major uses: (1) as research tools to study the structure and function of the gene, (2) in the manufacture of the protein product coded for by the gene, transfer of a specific trait into a new organism, and (4) as diagnostic test probes for the detection of specific viral diseases in medicine. (3) in the Production of aene conies for the

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14 Transfer Plasmids are not the only vectors, or vehicles, used to transport a gene into a new organism. A virus pos- sessing natural gene transfer capabilities or a trans- posable element (a DNA sequence that has the ability to move from place to place within the genome and affect the expression of neighboring genes) also can carry the genetically engineered gene into its host. In addition, vector systems can be based on other means of moving genes such as microinjection of DNA into the cell nucleus or direct uptake of DNA by cells from their culture medium. Expression One of the key uncertainties in gene transfer is whether or not the foreign gene will be transcribed to RNA and the RNA translated into the protein product in its new environment. me goal of these manipulations, gene isolation, cloning, and transfer, is gene expres- sion. To be successful, an appropriate level and timing of expression must be achieved during the lifetime of the recipient organism. mat is, function of the genetic process governing the periods when the gene is off (when no protein is produced) and when it is on (when protein is produced) is critical. Only moderate success has been achieved thus far in- transferring cloned genes into test plants and animals. Progress is hampered by a lack of vectors that can effectively carry recombinant DNA into a new host and of the regulation of expression in the transferred foreign genes. In vitro analyses can yield much basic informa- tion on factors contributing to successful genetic manip- ulations; however, in viva studies ultimately must be conducted in both plants and animals as well as in micro- organisms. Opportunities in the Plant Sciences me knowledge base supporting genetic engineering technology for the transfer and expression of foreign genes in crop species is limited. Relatively few im- portant plant genes have been cloned and sequenced. In part this extends from a lack of knowledge of the

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15 biochemical pathways in plants; few important gene products have been isolated and purified to the extent that they can be used in developing probes for isolating the gene. Gene Isolation There is a major need for increased understanding of the genetic basis of important plant traits. This know- ledge will come only through a concerted effort by plant geneticists, cytogeneticists, biochemists, and develop- mental biologists to search the germ plasm of major crop species and their relatives for agriculturally important traits. m ese traits then must be defined, in both genetic and biochemical terms. Traits controlled by one or more major genes amenable to genetic engineering include selectivity for herbicidal action, some cases of disease resistance, and synthesis and regulation of plant growth substances, such as in dwarfism. Other traits might include the key regulatory steps in metabolic pathways, such as assimilation of nu- trients and partitioning of photosynthate (the combined products of photosynthesis), tolerance to toxic metals, and possibly tolerance to various physical environmental stresses. In several cases where plant and bacterial metabolic pathways are similar and where mutants are available or can more efficiently be induced in bacteria, genes from bacterial sources may well be used in the ge- netic engineering of plants. Fatty acid synthesis, aro- matic amino acid synthesis, biological nitrogen fixation, and carbon fixation are traits currently under investiga- tion in a number of laboratories. Transposable elements, bits of mobile genetic information, were first recognized in maize and are now known to be present in many different organisms. Because these elements can move from one location in the genome to another, they may be very effective vectors for recom- binant DNA. Transposable elements can cause phenotypic instability; they turn off or otherwise alter the expression of neighboring genes. m is ability makes transposable elements unique tools for the isolation and characterization of genes. Specific transposable elements may be able to function in species other than those in which they occur. There are certain structural similarities of transposable elements in organisms as divergent as the fruit fly

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16 Drosophila and the flax plant Linum, for example. me discovery and characterization of transposable elements in leading crop species,could be very important in ad- vancing the technology of gene isolation, the develop- ment of vectors, and the control over suppression of undesirable genes. Because of their enormous potential for use in genetic engineering, the search for trans- posable elements in important crop plants and the study of their structure and function are extremely important. Transposable elements can be used to isolate genes when other methods, such as screening in bacteria, will not work. m e strategy is illustrated by recent success in cloning maize genes. First, the progenies of a plant that contains identifiable transposable elements are screened for the absence of a trait possessed by the original plant, such as resistance to a disease. m e absence of the trait suggests that the transposable element has moved to a position adjacent to, or in the middle of, the gene responsible for that trait. me DNA of such an altered plant is then isolated and cut with restriction enzymes. file transposable element, which has . . . . a specific and unique nuCleotlae sequence, IS usea as a probe to locate DNA segments that contain the transpos- able element's DNA. m ese segments are then isolated, cloned, and sequenced. me DNA flanking the element is suspected of being a part of or perhaps the entire gene responsible for the trait in question. Transposable elements have potential for use, in a similar fashion, in turning off undesirable genes. Such a naturally occurring case of gene dysfunction caused by the presence of DNA sequences in the middle of a gene has been described in soybeans. Gene Transfer In animal and bacterial systems the availability and early characterization of viruses and bacteriophages that naturally integrate into the genome of the host aided in the development of viral vectors that carry recombinant DNA into these host organisms. Most plant viruses are RNA viruses; the genetic information is carried by RNA rather than DNA. Only two groups of plant viruses con- tain DNA as their genetic material. No plant virus, to the best of current knowledge, is capable of being inte- grated into a host's chromosome.

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17 Research is under way to develop a number of vector systems for use in transferring recombinant DNA into plants. Plasmids as Vectors Two naturally occurring systems in plants do involve insertion of DNA sequences into chromo- somes. The megaplasmids, Ti (tumor inducing) and Ri (root inducing), are carried into host plant cells in nature by the soil bacteria Agrobacterium tumefaciens and A. rhizogenes, respectively. - _ mey produce the diseases crown gall (Ti) and hairy root (Ri). These megaplasmids contain a small region of DNA called T DNA (transfer DNA), which is transferred by an unknown mechanism into the chromosome of the host plant. After researchers understood that the disease caused by these bacteria was the result of insertion of plasmid T DNA into the plant chromosome, these plasmids were adapted for use in the first-generation plant genetic engineering experiments. More sophisticated use of vectors, based on the ability of T DNA to insert into chromosomes, will be possible once the molecular mech- anism of the transfer is understood. While the diseases caused by these bacteria are found only in dicotyledons, the transfer mechanism also might be made to work in monocotyledons, including some economically important grain crops as well as in those dicots that are not sus- ceptible to crown gall. Little is known about the target site for insertion of T DNA. The limited evidence available suggests that there is not a specific insertion site--a potential disadvantage because of the importance of gene location for expression. This problem might be solved by modify- ing the T DNA or adding other sequences to the T DNA to make it specific for a single insertion site. Transposable Elements as Vectors Transposable elements also have the ability to insert DNA into plant chromo- somes. m e expression of a gene adjacent to a trans- posable element on the chromosome is either stimulated or suppressed by the presence of the element. A transposable element also may carry its own functional genes that might encode an enzyme for transfer of the element itself. Further research is needed to assess the potential of transposable elements as vectors for plants. Important research goals within the next few years are to understand differences between active and

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18 vestigial elements; element interaction and movement; circumstances governing the target site; and the meaning of the large, complex DNA sequences in the interior of some of these elements. Viruses as Vectors As previously noted, plant viruses l have been of marginal use thus far in plant genetic engineering. A better understanding of the genome structure of the few DNA-containing viruses and the many RNA plant viruses may lead to new and more promising possibilities. Such viruses might be developed as suitable vectors for in vitro assays that can quickly indicate the expression of a transferred alien gene. In addition, viruses might be used as cloning vectors to produce large amounts of a particular gene product. For example, as an economical alternative to the production of high-value biochemicals via cell cultures in fer- menters, genetically engineered viruses might be devel- oped to infect the crop in a farmer's field with the ability to increase the synthesis of necessary biochem- icals prior to harvest. Viruses or viral sequences might be used to increase the efficiency of gene transfer. After entering the cell the recombinant DNA-containing viral sequence could replicate, increasing the probability that one or more copies of the gene would be integrated into~the genome. Attempts to insert DNA into the cauliflower mosaic virus, thought to have potential as a replicating vector, have had little success. me virus is apparently too small to accommodate most genes. Cauliflower mosaic virus commonly attacks members of the cabbage family and causes banding of veins in the leaves of the plant. Very recently a small bacterial gene encoding the enzyme, dihydrofolate reductase (dhfr) was inserted into cauliflower mosaic virus. Turnip plants became systemically infected, following inoculation with the recombinant virus, and acquired resistance to methotrexate. This resistance is a trait conferred by the activity of the dhfr enzyme. Other Vectors. _Microinjection and Direct DIVA Uptake Other vector approaches in plants are currently under investigation. Chief among these are microinjection and direct DNA uptake. Microinjection, as a means of introducing DNA into the cell nucleus, has been successful in animal embryo sys- tems. A few picoliters of fluid containing recombinant

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19 DNA can be injected into a plant cell, and even into the nucleus, with fine glass pipettes. me cells then can be cultured. To date, no confirmed transformation of a plant species by this approach has been reported, but results are expected soon. Microinjection technology will be important in the transfer of chromosomes in advanced cytogenetic manipu- lations and possibly also for the transfer of genes into organelles. Investigations in these areas offer oppor- tunities for research collaboration among molecular biol- ogists, cell biologists, and biophysicists. In direct DNA transfer, DNA is taken up by cells from their culture medium and is integrated, by unknown mech- anisms, into the chromosome. Such methods work in bac- teria and animals. Similar approaches have so far proved less successful in plants, but the situation may be changing. It has long been known that plant viral RNA s and DNAs can be taken up in a biologically active form. me same has been shown for T DNA, but at a lower effi- ciency. It is possible, but not yet widely accepted, that lipid vesicles or analogous vesicular structures made from plant membranes might increase the efficiency of delivery of DNA as they fuse with the recipient cell membrane. mese latter methods are attractive and important areas for further investigation. They should be appli- cable to all plants and they avoid incorporation of the accompanying DNA of a potentially pathogenic vector. Cell Culture and Plant Regeneration As important and exciting as the recent advances have been in developing vectors for use in plant gene trans- fer, major challenges remain. A useful gene transfer system requires the ability to manipulate the cells of a species so that alien DNA can be inserted in a wav that does not kill the cell. In addition, the cell must de- velop into a viable, functioning plant that has not been altered in undesirable ways. Plant organ and tissue culture is a well-established technology that originated in the early part of the twentieth century. In certain ornamental and woody species, use of tissue culture for propagating new plants is a small but important agricultural industry. Progress in manipulating cultures of major food crops, particu- larly the cereals and legumes, however, has been much slower. Chapter 4 of this report addresses the

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20 rather thin scientific basis supporting the current know- ledge of organogenesis and plant developmental biology. It is important to note here, however, that the current inability to successfully regenerate, at will and at high frequency, whole plants from individual cells of major crop species severely limits use of even current gene transfer technology. Much of the sophisticated cell culture and related technologies required to undertake state-of-the-art gene transfer research in major crop plants is largely in the hands of a small number of in- dustrial laboratories. m e deficiencies in fundamental knowledge of plant development will become even more serious in the future unless a major research commitment is made by the public sector. An alternative to the use of single somatic cells for genetic transformation is the insertion of genes into pollen nuclei, ovules, or recently fertilized embryos. By using gametes or developing embryos instead of somatic cells, both the potential for unwanted mutations from prolonged in vitro culture and the problem of regenera- ting a whole plant containing the new genes would be avoided. Nevertheless, the development of a firm scien- tific and experimental basis in the physiology, topology, biochemistry, and genetics of plant morphogenesis, in- cluding normal and somatic embryogenesis, will make an important contribution to several areas of agricultural biology, not least of which is the area of gene transfer Gene Expression me comparison of gene structures has yielded some insights into the factors governing expression of plant genes. What is known about expression, however, is greatly exceeded by what remains unknown. me recent success of gene transfer experiments using T DNA as a vector will dramatically quicken the pace of research on factors affecting gene expression in different plants. Further experiments will enable scientists to dissect the DNA regulatory sequences that flank the coding region of a gene--that segment providing the on and off signals for the transcription of DNA. After making changes in the nucleotide base sequence of these regulating, flanking regions, scientists can study the consequences by mea- suring the expression of the gene when it is put into the chromosomes of different plants. m is type of study,

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21 which ideally would include experiments with the same gene and flanking sequences in differing plant species, requires a major commitment of time and expertise. Effect of Location on Gene Expression Experimental evidence indicates that factors involved in directing gene expression reside in the immediate flanking sequences. Equally important signals, however, may be present in the coding region of the gene itself and also in sequences some distance from the gene, or even on different chromosomes. m e transformation technology currently available is insufficiently precise for use in targeting an insertion to a specific location in the chromosome. Emus, the possibility that location may be an important factor in governing gene expression must be addressed by repeated experiments in which several dif- ferent insertions of the same gene are made at various locations. The same gene inserted in a single copy at one location may be regulated quite differently than when inserted in multiple copies at the same locus or in multiple copies at different loci. Regulatory Sequences The regulatory signals con- trolling gene expression in bacteria differ from those in plants. Results of limited work to date indicate that sequences regulating gene expression in animals and ani- mal viruses do not function in plants. Whether such sequences in one plant genus or family will always work in others is not yet known. Regulatory sequences in T DNA do function throughout a wide range of plant species that span many families. To a more limited extent, the same is true for cauliflower mosaic virus; regulatory sequences from this virus, when used in a T DNA-based transformation system, have been demonstrated to function as a regulatory signal in genera that are not considered to be hosts for the virus. The regulatory sequence flanking the nuclear gene that encodes a small subunit of the photosynthetic enzyme ribulose-l,S-bisphosphate carboxylase/oxygenase in peas also functions in the petunia. In other cases, however, regulatory sequences fail to correctly control gene expression in unrelated species. Failure is tentatively attributed to an as yet poorly understood species specificity of the regulatory sequences. Most genes are turned on and off at specific times in development or under special conditions. In various laboratories the expression of such genes is now

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22 beginning to be studied. Regulatory sequences flanking important genes that are known to be triggered by light, heat, or growth hormones, for example, can be isolated and fused to a reporter gene. me reporter gene, usually a microbial gene carrying the trait for resistance to an antibiotic, provides a tag that can be used for screening and locating cells or plants that have incorporated the regulated gene sequence. The regulation of the trans- ferred gene can then be tested by looking for its ex- pression in the appropriate tissue or by triggering its expression using the appropriate environmental stimulus. This work, however, is in its most preliminary stages. Transient Expression Assays Gene expression research would be greatly aided by a system in which genes could be expressed and assayed quickly within plant cells. The current system using the Ti plasmid requires weeks to months to obtain results from a gene transfer experi- ment. A so-called transient expression assay system might be developed by using modified plant viruses as promoter vectors for individual plant cells. The ability of an inserted gene to be transcribed and translated could be quickly assayed in a single cell by using sensi- tive hybridization and antibody probes to look for the messenger RNA (mRNA) and protein product of the inserted gene. The mRNA carries the code for a particular protein from the DNA in the nucleus to the cytoplasm. m ere it acts as a template for the formation of that protein. Such an assay system would significantly advance the science of plant genetic engineering, because even small adjustments to sections of the transferred gene could be tested within a matter of days to find the nucleotide sequence that will be expressed in the host plant. The stability and function of foreign gene products, in- cluding enzymes and other proteins, could be tested quickly using such a system. Multiple Gene Traits For many years plant breeders and cytogeneticists have obtained novel gene combinations by crossing certain distantly related species of the same or a closely related genus. Often such wide crosses involve an increase in the ploidy level to include duplication of the chromosomes from both parents. An example from nature is wheat. It has been shown that wheat is a hexa- ploid resulting from crosses among three genera:

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23 Agropyron, Aegilops, and Triticum. Much has been learned using these breeding and cytogenetic methods. me development of microinjection and other such vector technologies, improvement in fluorescence- activated sorting technology to refine methods for iso- lating chromosomes, and the construction of artificial chromosomes, so far only achieved in yeast, may provide future means for the transfer and expression of agri- culturally significant complex genetic traits to yield new genotypes. As experimental tools, these methods will lead to advances in our understanding of coordinated gene regulation; as practical tools, they will lead to more rapid product development. These methods also will make possible the genetic engineering of plants for complex quantitative traits such as yield, disease resistance, and production of important secondary products such flavors, fragrances, and pharmaceuticals. Research Status Basic research of a multidisciplinary nature is required to isolate, analyse, transfer, and express plant genes using modern biotechnology methods. ~ . .: ~ . _ _ ~ _ _ _ m e research requires expensive materials and some expensive equip- ment. Optimal use of resources and the multidisciplinary nature of the work dictate a concentration of effort and resources rather than a diffuse, decentralized organization. The ARS must take a strong lead in both basic and applied research in plant genetics to sustain agri- cultural growth and prosperity in the United States. m e agency must be particularly committed to focused research on important crop plants, the maintenance and use of germ plasm collections, and the high-risk, multidisciplinary research that is essential in bringing newer biotech- nologies into practice. To improve the available technology and the efficiency of gene isolation and molecular cloning in plants, spe- cial attention should be directed toward the following: 0 Characterization of the biochemical basis and genetic traits involved in important plant processes such as photosynthesis, carbohydrate partitioning, yield, heterosis, stress tolerance, and morphogenesis; Molecular characterization of mobile genetic elements, such as transposable elements, plant viruses,

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24 and plasmids, and properties such as host range, target sites for insertion into the chromosome, and the basis for the genetic dialogue between genes of the nucleus and organelles; Understanding of basic chromosomal structure and function underlying conventional cytogenetic manipu- lations, such as the creation of allopolyploids with wide crosses, and development of principles to guide the use of novel methods, such as microinjection and cell fusion, to manipulate chromosomes or parts of chromosomes; Understanding of the principal molecular factors and DNA sequences underlying the regulation of gene ex- pression, such as mechanisms associated with chromosomal structure, sequences flanking coding regions, signals within coding regions, and functions of introns; o Development of vector systems for transient expres- sion assays. Currently some of the strongest basic programs in plant molecular genetics are located within the research laboratories of private companies. m is is particularly true for research on gene transfer systems for plants. Research programs on plant gene isolation and structure at universities and other publicly supported research laboratories usually consist of only one or two Principal investigators. A, Public support of basic plant genetic research needs increased attention. m e creation of the Plant Gene Expression Center at Albany, California, is a first step in this direction. Aspects of Molecular Genetics of Food Animals The knowledge base supporting genetic engineering technology for animals is extensive. Much of the bio- chemical and molecular genetic understanding of mammalian systems has been achieved through research on human cell culture lines and the laboratory mouse. Discoveries made using these laboratory systems are generally applicable to food animals. me application of these new tech- niques, however, remains limited; the nucleotide sequences of most of the genes coding for valuable agri- cultural traits and regulation of the expression of such genes remain unknown or are poorly understood. Specific opportunities to apply molecular genetic techniques to the study of metabolic regulation,

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25 reproduction, and functions of the immune system and to the development of vaccines, and diagnostic and thera- peutic agents for food animals are discussed in Chapter 3. In addition, basic approaches to the study of gene isolation, transfer, and expression are covered in the previous section on plants. This discussion outlines the principal methods used to introduce recombinant genes into the genome of food animals. It presents the potential advantages offered by analysis of the nucleotide sequence of genes and the mechanisms regulating their expression in food animals for the improvement of agricultural efficiency. Gene Transfer Unlike plants, which can be propagated asexually, a whole animal cannot be regenerated from a single somatic cell. To introduce cloned genes into all cells of an animal, they must be inserted into the undifferentiated embryo. An alternative approach is the introduction of recombinant genes into the developing embryo or into somatic tissues, using retroviruses or transposons as vectors. With introduction into somatic tissues, how- ever, germ cells will usually not be genetically altered, and recombinant genes will not be passed on to the offspring. Microinjection into the Germ Line m e stable integra- tion of foreign genes into the mouse genome has been achieved by microinjecting cloned genes into the one-cell mouse embryo. me period following fertilization of the egg but prior to mixing of the genetic information of the sperm and egg appears to be an opportune time to incorpo- rate foreign genes into the genome. Successful incorpo- ration of the recombinant DNA at this one-cell stage establishes the foreign gene throughout all cells in the resulting animal, including cells of the germ line that give rise to future generations. Mouse populations have been produced that contain recombinant oncogenes or genes coding for thymidine kinase, rabbit beta-globin, human leukocyte interferon, chicken transferrin, or rat growth hormone. m ese genes have been integrated into the mouse genome, and protein products resulting from the expression of these genes have been detected. The regulatory sequence used was a

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26 metallothionein promoter sequence fused to the rat growth hormone gene. As a result the regulation of its expres- sion was not the same as in normal mice. me concen- trations of growth hormone in some of the tranagenic mice were greatly elevated, and as a result the animals grew substantially larger than normal mice. Growth hormone supplied exogenously to mice and some food animals has a dramatic effect in increasing growth rate. In addition, feed efficiency and body composition, in terms of reduced deposition of fat, often are sub- stantially improved. me extent of these effects appears to depend upon the stage of development of the animal. Younger animals do not respond to growth hormone treat- ment as markedly as do mature animals. And the effect of growth hormone on increased milk production in cows, for example, is most pronounced in low-producing dairy cattle. The results are encouraging and portend impor- tant future applications for the cattle, poultry, sheep, and swine industries. Microinjection techniques that were developed to insert cloned genes into mice embryos should be appli- cable to food animals. Specific problems in manipulating the one-cell embryo in different species must be re- solved. With poultry this may not be possible, because it will be extremely difficult to obtain and manipulate viable one-cell embryos. It may be possible, however, to insert foreign genes via the spermatozoa, which can be used in artificial insemination. Retroviral-based Vectors The genome of a retrovirus consists of single-stranded RNA that, following inocula- tion, serves as a template for reverse transcription and the production of a double-stranded DNA molecule that integrates into the chromosome of the infected cell. Integrated DNA copies of RNA retroviruses are called proviruses. Proviruses are transcribed and replicated along with the host's genes. The provirus contains special sequences at both ends of its DNA that permit it to be integrated into the cell genome in a manner similar to other movable genetic ele- ments, such as transposons. It is theorized that retro- viruses are, in fact, movable genetic elements that pos- sess genes for coat proteins, and that a virus particle is created by enveloping the RNA transcript within the coat protein. The converse is also possible; movable genetic elements or transposons might have arisen from

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27 retroviruses that lost the ability to form a virus particle. Foreign genes can be inserted into the provirus DNA. Such recombinant provirus DNAs can be cloned and used as vehicles for inserting the foreign gene into a host ani- mal cell. The advantage of proviruses as gene transfer vectors is the efficient, transposon-like mechanism by which they can be integrated into the chromosomal DNA of host cells. Other Vectors In addition to retroviral vectors, non- lytic DNA viruses, such as bovine papilloma virus (BPV), are being experimentally tested as gene transfer vec- tors. BPV does not integrate into the host cell chromo- some; it exists instead as an episome, a stable extra- chromosomal unit of DNA in the host cell nucleus. A transformed cell may contain from 20 to 100 copies of the BPV episome. It appears that some of the genes necessary for the oncogenic transformation properties of BPV are not needed for its autonomous replication in the host cell. The BPV vector appears to be an excellent candi- date for rapid assays for gene expression, because DNA from a mammalian species can be spliced into the BPV and tested for expression in cultured cells of that same species. The multiple copies of the BPV episome in each cell may amplify the expression of any intact genes included in the spliced DNA. Other methods for inserting recombinant genes have not been successful in one-cell embryos, probably because the uptake of recombinant DNA is less efficient than micro- injection and adequate testing would require enormous quantities of these embryos. These methods include the uptake of calcium phosphate-DNA precipitates; electro- poration, or uptake through the cell membrane stimulated by electrical charges; and uptake by fusion with vector- containing liposomes. Gene Identification and Cross Cloning A relatively low reproductive rate coupled with the enormous expenses involved in maintaining large populations of food animals makes it difficult to carry out the extensive breeding experiments needed for classical genetic analysis and chromosome mapping. However, mapping at the DNA level is now a reality and

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28 can be applied to food animals. One form of mapping that could be easily applied to food animals is analysis of the genome based upon restriction enzyme sites. Another is the analysis of the nucleotide sequence of genes. _, ~ Gene libraries can be obtained easily for both approaches. In addition, the discovery of restriction enzyme polymorphisms would provide exceedingly useful markers for genetic analysis in animal breeding studies. Additional information for identifying and isolating specific genes might be compiled through cross cloning, which makes use of a DNA gene probe from one species to hunt for a comparable gene in an organism belonging to another species or genus. A comparable gene should have some homology in its nucleotide sequence and therefore should hybridize with the DNA gene probe. For example, many of the identified genes available in the gene libraries of cultured human cells or the laboratory mouse could be employed as DNA probes to search for the same gene in food animals. There are many enzymes and gene products that are common to all mammals. This technique has been used extensively and successfully to locate and identify genes such as oncogenes and genes encoding globin, cytochrome, myosin, actin, tubulin, growth hor- mone, and interferons in a variety of organisms. Gene Expression The successful transfer of a functioning growth hor- mone gene into the mouse is significant in two important respects. First, it demonstrated that this gene could be cloned, microinjected into a one-cell embryo, and ex- pressed as part of the genome of the resulting tranagenic mouse. But it also emphasized the significance of types of gene regulation, because the mice grew substantially larger than a normal mouse. me DNA sequence encoding the gene product and the promoter DNA sequences encoding the regulation of the expression of the gene are both equally critical components of a recombinant gene. me second important aspect was the effect of the inserted gene on growth. A complex biological process such as growth obviously involves the expression of many --perhaps hundreds--of genes, yet growth in this case was regulated by a single gene. me ability to regulate the endogenous synthesis of this key substance offers a means to control a complex process such as growth. There are

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29 most likely many other single genes that code for the synthesis of the critical modulator controlling other complex, multigenic traits. The growing body of evidence on gene regulation in eukaryotes suggests that genes can be regulated at many different levels. To add to this complexity, different genes may be regulated in different ways. For example, significant progress has been made in understanding the regulation of the globin genes in humans and other animal species. It is now known that modification of the DNA may determine the switch from one hemoglobin type in the fetus to another in the adult. Methylation of the DNA seems to be an important aspect of this regulatory process. The regulation of gene expression in eukaryotes does not appear to be based on the operon system, which is the major regulatory system in prokaryotes. One problem is that the genes affecting a particular trait in eukaryotes are often not clustered according to their sequence of Furthermore, eukaryotic genes often are regulated on a long-term, irreversible basis typical of cellular differentiation and develop- ment. It is apparent, therefore, that notable strides in understanding development will go hand in hand with ad- vances in knowledge and the ability to manipulate gene regulation in food animals. expression as in orokarYotes. Research Status Studies of the fine structure of genes and the mecha- nisms regulating the expression of economically valuable traits in food animals are now possible. ~ _ gene transfer systems and methods for molecular genetic analyses that evolved from studies on laboratory mice and human cell culture should be applicable to similar studies on food animals. The ARS has a well-established research effort at Beltsville, Maryland, on gene transfer in food animals. This and related areas of molecular genetic research should be expanded during the next sev- eral years, with particular emphasis on the following: Many of the Characterization of the physiological basis and genetic traits involved in important animal processes such as disease resistance, the immune response, meta- bolic regulation of nutrient utilization, developmental biology, and other aspects of production efficiency.

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30 Development of methods to manipulate viable gametes and embryos of food animal species, and development of suitable gene transfer vehicles and methods for genetic transformation of food animals. Understanding of gene promoter sequences in food animal species and the factors and conditions that con- trol their function. This will require the development of rapid gene expression assay systems for each species. Establishment and analysis of gene libraries for tood animal genotypes. Mapping of restriction enzyme fragments, identification of DNA polymorphisms as markers, and sequencing of nucleotides of identified genes will be valuable resources for both animal breeding studies and molecular genetic research. Potential Impact on U.S. Agriculture Modern genetic technology, including recombinant DNA and the ability to isolate, transfer, and express foreign genes in crop plants and food animals, will likely have an impact on agriculture comparable to that of the dis- covery of the laws of inheritance in the late 1800s. Improved species with new capabilities might be devel- oped. Equally important will be the efficiency with which new traits can be incorporated into superior, adapted crops and food animals, and the ability to pro- duce novel combinations of traits that are difficult or impossible to create using conventional breeding methods. This technology will greatly improve current under- standing of the biochemistry and genetics of animal and plant growth, development, and reproduction. But the transfer of this knowledge to agricultural sciences is as difficult to foresee as was the development of sophisti- cated statistical models for modern plant and animal breeding from the basic gene theory of inheritance. While it is true that use can be made of a system before it is fully understood, experience shows that a mechan- istic understanding can unveil unexpected opportunities to take full advantage of a technology. A detailed understanding can also mitigate potential negative effects of a technology. A fuller understanding in the 1940s of the potency of chemical mutagens, for example, might have reduced the improper use and disposal of earlier synthetic chemicals. In the short term the new biological technologies will have a variety of important implications for agricul- ture. Interest in preserving germ plasm and in compre-

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31 hensive screening for useful traits is becoming more widespread, due in part to the influence of genetic engineering. Increasing interest is also being generated in other areas of basic plant and animal sciences, including biochemistry, physiology, pathology, and development, where genetic engineering tools serve as key adjuncts to more traditional research methods.