-Caveat Lector- Library: Biosafety of selectable marker genes Keith Harding Crop Genetics, Scottish Crop Research Institute, Invergrowrie, Dundee DD5 2DA, UK ABSTRACT This article reviews the use of selectable marker genes in genetically modified organisms (GMOs), and considers their biosafety in plants likely to be released into the environment. It examines those marker genes currently used in constructs, appraises their possible hazards and explores alternatives to undesirable markers. Selectable marker genes are important for the production of GMOs, and they are essential for the selection of modified cells at relatively low frequencies. Marker genes are used to "tag" genes of interest and comprise several classes. Antibiotic and herbicide resistance genes are the most commonly used markers in the modification process. They confer resistance or tolerance to an organism by overexpressing protein molecules sensitive to a selective agent, or an altered insensitive target protein or express detoxifying enzymes. The hazards associated with selectable markers refer to the toxicity of genes and their gene products, the potential for transfer of resistance genes to undesirable organisms and the potential of detoxification reactions to cause damage to the environment. There are concerns relating to the transfer to pathogenic organisms of some genes conferring resistance to antibiotics, such as chloramphenicol and streptomycin, which are reserve drugs for life-threatening diseases. Detoxification of antibiotics by phosphorylation may well have an environmental impact should these marker gene products chemically modify cellular metabolites which themselves become pharmacologically active. Transfer of herbicide resistance genes into non-modified crops and feral relatives could well have a significant impact on agriculture and the environment. Selectable markers are needed for genetic modification, and new negative and positive alternative marker technologies for selection are available including markers for root inhibition, induced toxicity, nitrate utilization, and amino acid biosynthesis. In those organisms where there are no appropriate alternatives, selectable markers may be removed or inactivated or other techniques may be used without the need for marker genes. INTRODUCTION Since the report on biotechnology by Spinks (1982) to the Royal Society, advances in recombinant DNA technology and the implications for the release of GMOs are making an international impact. Biotechnological benefits have the realistic potential to be transferred to other industries for the production of dairy products, and chemicals of industrial and pharmaceutical importance. Commercial enterprise and the potential benefits have to be balanced against the risk of introducing potential hazards into the environment. The purpose of this article is to appraise the hazards and risks associated with the use of selectable marker genes in the release of GMOs, and their possible implications for the environment and crop management to provide a safe and confident basis for the development of GMO-related biotechnology in agriculture. SELECTION PROCEDURES AND MARKER GENES Selectable marker genes are essential for the production of GMOs. In the process of transformation where cells are modified genetically, DNA from a donor organism is transferred into another thereby modifying the recipient organism. The DNA is usually but not invariably in the form of a synthesised DNA molecule, a plasmid (often termed a construct or DNA vector) in which a "gene of commercial interest" is linked closely to the selectable marker. After transformation, the marker gene enables the selection of transformed events from a high background of non-transformed cells. Selectable markers act as genetic tags to identify transformed cells, enabling their growth in the presence of chemicals which normally inhibit non-transformed cells. They are not an end-product in themselves, but without them selection would be very difficult. There are several categories of selectable markers and other marker genes, often referred to as scorable or assayable genes (Table 1): this review will focus on antibiotic and herbicide resistance marker genes. Genetic modification of plants To generate genetically modified plants, the construct containing the selectable marker gene must be introduced into recipient plant cells. This is normally done in the laboratory using a variety of techniques involving: chemical treatments, liposomes, electroporation, micro- and macro-injection, particle bombardment, silicon carbide fibre and micro-laser. These methods of modification have been critically reviewed (Potrykus, 1991). The development of a wide range of selectable marker systems along with various genetic transfer techniques has accelerated progress in the genetic modification of plants (Klee et al., 1987). The major sources of DNA fragments for constructs have been plant pathogenic microorganisms and viruses, especially DNA sequences derived from Agrobacterium and the caulimoviruses (Howell, 1982; Zambryski, 1992). There have been a large number of releases of genetically modified crops in field trials (Dale et al., 1993). The number of trials is likely to increase rapidly with the growth and expansion of biotechnology. Definitions and hazards In identifying the hazards and appraising the risks associated with the use of selectable marker genes in releasing GMOs into the environment, it is important to consider the following definitions (Royal Society, 1992): 1. Hazard is a characteristic that, in particular circumstances, could lead to harm. 2. Risk is the chance, in quantitative terms, of harm occurring from a defined hazard, and the expression "risk of (a specific event or set of events)" must refer to the hazard. 3. Harm occurs when a hazard is realised and damage is done to a human being (or other organisms or to a population or to the environment). 4. Damage is the loss of inherent quality suffered by an entity (physical or biological). The following features should be considered in appraising the hazards of selectable markers: 1. Toxicity of the marker gene. 2. Toxicity of the marker gene (protein) product. 3. Transfer of the marker gene to pathogenic organisms in the environment. 4. Potential of the marker gene to cause damage to the environment. There are several strategies for the production of GMOs based on the use of resistance or tolerance to antibiotics or herbicides: 1. Overexpression of genes producing sensitive protein molecules. 2. Expression of genes to produce an insensitive target protein. 3. Expression of detoxifying enzymes. Although antibiotic and herbicide resistance genes have been invaluable in the genetic modification of plants, their expression has raised questions concerning their use and safety in food products. Support for the marketing of genetically modified plants containing an antibiotic resistance gene has been provided in a comprehensive document submitted to the Food and Drug Administration by Calgene, Inc. This documentation has been reviewed (Flavell et al., 1992), and these issues have been debated widely (Bryant and Leather, 1992; Gressel, 1992ab). The evidence supports the food and environmental safety of one marker, the kanamycin resistance gene, and its gene product (Calgene, 1990; Redenbaugh et al., 1993). Further assurance of safety comes from a report submitted to the regulatory authorities in the Netherlands (Bijvoet and Nap, 1992), the findings of which have been published (Nap et al., 1992). The report concluded that it was justifiable to use kanamycin resistance as a selection marker in genetically modified plants to be introduced into the environment for field trialing. There are health concerns relating to marker genes in genetically modified crop plants, which have been the subject of a World Health Organisation Workshop (WHO, 1994). It would appear that there is an emerging international consensus to approve the release of GMOs containing selectable marker genes on a case-by-case basis. POTENTIAL OF ANTIBIOTIC OR HERBICIDE MARKER TO CAUSE DAMAGE TO THE ENVIRONMENT Genetically modified organisms containing selectable marker genes into the environment have the potential to exchange genetic information with other organisms. These possibilities are considered below. It is quite likely that pollen will be produced from fertile, genetically modified plants and be transmitted via insects or wind to sexually compatible species; subsequent fertilization will spread the marker gene into "wild" populations leading to contamination of "native" genetic information (Raybould and Gray, 1993). The long-term environmental consequences of this "genetic pollution" are unknown. There is a paucity of information relating to the transfer distances of pollen from genetically modified plants containing marker genes, and the dynamics of its spread in a natural population. The production of hybrid seed between genetically modified plants and wild relatives in the environment will depend on many factors, including the flowering times and relative fertility. The formation and frequency of hybrid seed is likely to be affected by pre- or post-zygotic mechanisms. Horizontal gene transfer The spread of genetic information from a plant to microorganisms via non-sexual processes is generally referred to as "horizontal transfer". The residues of genetically modified crops are a potential source for such gene transfers: although there is some evidence for it in nature, the mechanisms by which it occurs are unknown. Plant cells contain DNA-degrading enzymes, which are released when cells are damaged resulting in the breakdown of the plant's DNA. Soil organisms would increase the process, and further reduce the risk of transfer from decaying material. If such DNA was available, then levels of transformation of soil microorganisms would depend upon the fraction of microorganisms that were naturally transformable. A model has been constructed to determine the proportion of antibiotic resistant organisms that could be created by such a process (Calgene, 1990). The estimated contribution at 5.4 x 10-6%, is comparable to the natural frequency of mutation. Acquisition of any new trait in an organism would be realised if it confers a selective advantage; this is likely to be low as soil microbes are known to possess genes for antibiotic resistance. Genetic modification of cereal crops has been difficult but progress is being made with the use of the particle gun (Potrykus, 1990). These crops have a different level of risk associated with horizontal gene transfer. Constructs containing both the plant and bacterial selectable markers may be present in these plant cells, and contain resistance genes for chloramphenicol and streptomycin. These are antibiotics on the list for prescribed use, which are reserved for the treatment of life threatening diseases (British National Formulary, 1993). Chloramphenicol is a reserve drug for the treatment of typhoid fever; the antibiotic streptomycin, a commonly used bacterial selectable marker is active against Mycobacterium tuberculosis, the causative agent of tuberculosis. The consumption of crops which contain such marker genes raises a further food safety issue, and their use should be considered carefully. Breakdown products The effect that marker genes have on the environment depends on the characteristics of the gene itself or on a secondary effect resulting from its action. Genetically modified crops are likely to require some level of chemical protection from certain pests or diseases. Agrochemicals are special formulations of an "active principle" with other chemicals that provide good surface contact. Some marker genes detoxify by mechanisms that include acetylation or phosphorylation (see Table 1), for example the streptomycin marker will phosphorylate streptomycin at one hydroxyl group on its glucosamine ring moiety. This phosphorylating enzyme will not use other aminoglycoside antibiotics as substrates but it does phosphorylate several degradation products of streptomycin (Benveniste and Davies, 1973). These may also modify substances chemically similar to the selective agents, such as agrochemicals. The environmental consequences of such illicit reactions and the effects of their breakdown products are unknown. Non-specific catalytic activity Kanamycin resistance is one of the most widely used selectable markers for the genetic modification of plants. Its gene product detoxifies kanamycin by phosphorylation. Plant cells produce a diverse range of secondary products; many are chemically ill-defined with no known function. Some of these structures may be chemically similar to aminoglycoside antibiotics, making them possible targets for phosphorylation by the phosphotransferase encoded by the kanamycin resistance gene. Such de novo phosphorylated metabolites may well be pharmacologically active. Phosphorylation is an important step in many cellular processes, especially in cell signalling, which involves mechanisms that are elicited by G-binding protein molecules which produce secondary chemical messengers; these molecules trigger an amplification cascade to give a cellular response (Bowman, 1992). Secondary messenger systems involve multiple components, consisting of biological substances which are highly active at very low concentrations. As the range of genetically modified plant species increases the potential for non-specific phosphorylation of secondary products is also likely to increase. This may have consequences for insect-life that comes in contact with plant exudates, such as sap or pollen. Crop management It is important to consider the potential of a marker gene to confer weediness on a modified plant and on its feral or wild relatives. There is no a priori reason to suggest that plants resistant to antibiotics will become weeds as there is no evidence of cross-protection between antibiotics and commercial herbicides, although herbicide marker genes do require special consideration. The introduction of a single antibiotic marker gene into the known genetic background of a cultivated plant is most unlikely to confer a weed-like characteristic, especially as many highly bred cultivars are poor competitors against weeds. The acquisition of a single antibiotic resistance gene is unlikely to confer a selective advantage, especially as to become a weed, a plant must acquire a number of characteristics, most of which are polygenic traits (Keeler and Turner, 1991). There are serious concerns relating to the long-term ecological consequences of releasing genetically modified plants containing herbicide marker genes (Crawley et al., 1993). The differences in sensitivity of crop plants and weeds to herbicides is the basis for their selective use; erosion of these differences may well lead to loss of effective weed control (Wilkinson, 1993). It is also important to consider those genetically modified plants, which are likely to give rise to a "volunteer crop", where the crop and its infesting volunteer plants are both resistant to the same herbicide (Froud-Williams et al., 1993). The natural resistance of wheat to chlorsulfuron may cause problems when rotated with oilseed rape; the introduction of chlorsulfuron resistance into rape could result in volunteer oilseed rape becoming a major weed problem in wheat crops (Gressel, 1992b). Glyphosate is used widely to control volunteer crops such as potato, weed beet and cereals; tolerance to this pre-emergence herbicide in the volunteer crops would result in loss of weed control and disrupt established agricultural practice. Single marker genes are unlikely to alter the appearance of modified plants, so that herbicide-resistant and sensitive plants will be phenotypically indistinguishable. This may lead to management problems on farms, where grain is stored in bulk resulting in the mixing of morphologically identical seed stocks between herbicide-sensitive and -resistant varieties. A troublesome weed in cereals is wild oats: therefore, the release of genetically modified domestic oats containing the selectable marker gene for glufosinate resistance (bar) should be considered carefully, since domestic and wild oats are sexually compatible, making wild oats a worse weed in cereal rotations (Gressel, 1992a). ALTERNATIVES TO THE USE OF ANTIBIOTIC AND HERBICIDE SELECTABLE MARKERS There are alternative strategies for the genetic modification of organisms and methods are also available to remove existing marker genes from modified organisms or to repress their function. Negative selection systems The root inhibition markers use the aux2 or tms2 genes, which are derived from Agrobacterium tumefaciens and A. rhizogenes respectively. These genes encode an enzyme, amidohydrolase, which catalyses the conversion of indole acetamide and naphthalene acetamide to indole acetic acid (IAA), a natural plant growth factor or auxin, and naphthalene acetic acid (NAA), respectively. Modified plants containing these genes overproduce IAA and NAA, when grown on a medium containing indole acetamide or naphthalene acetamide, which inhibits the formation of the root system (B�clin et al., 1993). Use of the induced toxicity marker is based on the cytosine deaminase gene (codA) derived from bacteria (Stougaard, 1993). The codA gene encodes cytosine deaminase, which converts the selective agent 5-fluorocytosine to highly toxic 5-fluorouracil, which kills the modified plants. Nitrate reductase is present in most if not all plants; it catalyses the first step of the nitrate assimilation pathway by converting nitrate to nitrite. When plants are grown in the presence of ammonium instead of nitrate as a source of nitrogen, the nitrate reductase gene is expressed at very low levels, and is not sensitive to chlorate which is an inhibitor of nitrate reductase. Genetically modified plants contain both their endogenous and the introduced nitrate reductase gene(s). The modified gene is constitutively expressed in the presence of ammonium, so that modified plants are killed by chlorate and only non-modified plants survive (Nussaume et al., 1991). Positive selection systems The gene sequences for glutamine synthetase, dihydrodipicolinate synthase, aspartate kinase, tryptophan synthase and histidinol dehydrogenase have been used in positive selection systems to modify genetically a variety of organisms. Glutamine synthetase catalyses the formation of glutamine from glutamate and ammonia, which is essential to the cell for growth. It has been used as a selectable marker for mammalian cells in the presence of the glutamine synthetase inhibitor, methionine sulphoximine (Bebbington et al., 1992). The branched chain amino acids, lysine, threonine, methionine and isoleucine are synthesised from aspartate via the aspartate biosynthetic pathway. Lysine biosynthesis is controlled by aspartate kinase and dihydrodipicolinate synthase, both of which are subject to feedback inhibition by lysine. Bacterial genes for these enzymes are less sensitive to lysine inhibition than their counterparts in plants, and they have been used for the genetic modification of plants (Shaul and Galili, 1992; Perl et al., 1993). Microorganisms catalyse the synthesis of tryptophan from indole and serine. The trpB gene from bacteria produces the enzyme, tryptophan synthase, and its transfer into mammalian cells allows the positive selection of such cells on medium containing indole (Hartman and Mulligan, 1988). The bacterial hisD gene produces histidinol dehydrogenase, which catalyses the oxidation of histidinol to histidine. When this gene is transferred into mammalian cells it allows the selection of cells on growth medium containing histidinol only. This is a double selection system, since histidinol is toxic and histidinol dehydrogenase detoxifies it while providing histidine for growth (Hartman and Mulligan, 1988). Removal of selectable marker genes The use of antibiotic or herbicide marker genes may be the only means to genetically modify some crop plants, in which case there is a need to remove or repress the function of these genes before their release to the environment. One method to remove a selectable marker gene is the Cre/lox recombination system, where a plant is modified using a selectable marker gene which is flanked by lox recombination sites (Dale and Ow, 1991). After a second round of modification, the cre gene is introduced accompanied by a second marker gene. The initial marker gene is excised from the genome through the action of Cre recombinase, which acts at the lox sites. After its removal, plants are left to flower and set seed, where the progeny are screened for segregation of the cre gene which is linked to the remaining marker gene. Plants without the cre and marker genes are selected and propagated. This sophisticated yet practical approach can be applied to a wide range of plants. However, it is not feasible in vegetatively propagated crops with low fertility and seed selection might scramble elite genomes in clonally propagated plants. Modulation of gene expression It has been shown that bacteria use antisense RNA to regulate messenger RNA in the production of some gene products. Antisense RNA is complementary to mRNA, it forms an RNA duplex which renders the mRNA inactive (van der Krol et al., 1988). Antisense technology could be applied to marker genes. The in vitro construction of antisense genes requires the orientation of a portion of the coding region to be reversed. When an antisense gene is introduced into a cell, it encodes antisense RNA that is complementary to the mRNA of the original marker gene and blocks its expression. Catalytic RNA cleaves either itself or other RNA molecules. Examples of catalytic RNA are ribozymes, which can act specifically on target RNA molecules to render them inactive. Catalytic RNA has created new opportunities to repress the expression of selectable marker genes. The expression of a ribozyme gene directed towards the target mRNA of the kanamycin selectable marker in plants resulted in a reduction of the kanamycin gene product (Steinecke et al., 1992). Genetically modified plants containing selectable markers could be further modified with genes that encode antibodies against the marker gene protein product (Hiatt et al., 1989). This technology is feasible, as genetically modified plants have been shown to express single (gamma and kappa) immunoglobulin chains, with functional antibodies being assembled in the progeny of sexual crosses of plants that encode these single chains. Selection without marker genes The direct introduction of DNA into a cell with the potential to develop into a whole organism, is by far the best method of modification for the release of organisms into the environment. 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WHO (1994) Health aspects of marker genes in genetically modified plants. Report of a WHO Workshop, WHO/FNU/FOS/93.6. Wilkinson, M.; Harding, K.; O'Brien, E.; Dubbels, S.; Chapters, Y.; Lawson, H. (1993) Herbicides and trangenic rape. Nature (London) 365, 114. Zambryski, P. (1992) Chronicles from the Agrobacterium-plant cell DNA transfer story. Annual Review of Plant Physiology and Plant Molecular Biology 43, 465-490. Library: Risk prevention and the political control of genetic engineering: lessons from a participatory technology assessment on transgenic herbicide-resistant crops http://binas.unido.org/binas/Library/cabi/harding.html DECLARATION & DISCLAIMER ========== CTRL is a discussion and informational exchange list. Proselyzting propagandic screeds are not allowed. Substance�not soapboxing! 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