Transgenic Transgression of Species integrity and Species Boundaries

Mae-Wan Ho

Biology Department, Open University, Walton Hall, Milton Keynes, MK7 6AA, U.K.
tel: 44-1908-653113
fax: 44-1908-654167

Beatrix Tappeser

Oeko-Institute e. V., Institute of Applied Ecology, P.O. Box 6226, D-79038, Freiburg, Germany
fax: 49-761-475437


Biodiversity and Species Integrity
What is Transgenic Technology?
Transgressing Species Integrity Results in Unpredictable Physiological Effects
Transgressing Species Barriers
Horizontal Gene Transfer Links the Whole Biosphere
Vectors Mediate Horizontal Transfer of Antibiotic Resistance Genes
Vectors Mediate Genetic Recombination to Generate New Pathogens
The Natural Microbial Populations Form a Major Thoroughfare and Reservoir for Horizontal Gene Transfer
Viral Resistance Transgenes Can Generate Live Viruses by Recombination
Vectors Resist Breakdown in the Gut and Can Infect Mammalian Cells
Genes Carried by Vectors Can Persist Indefinitely in the Environment

Key words: allergenity, antibiotic-resistance, biodiversity, recombination, replication, vectors, virulence.


Biodiversity and species integrity are inextricably linked. Transgenic technology transgresses both species integrity and species boundaries, leading to unexpected, systemic effects on the physiology of the transgenic organisms produced as well as the balanced ecological relationships on which biodiversity depends. Allergenic and toxic products have arisen in transgenic organisms and recent evidence suggests that transgenic resistance to pests and diseases may be associated with increased allergenity. Vectors for multiplying and transferring genes are chimaeric recombinations of parts of different genetic parasites so as to increase their host range, thus allowing them to transgress species barriers. They are now also designed to overcome endogenous cellular mechanisms which help to maintain species integrity. The vectors, carrying transgenes and antibiotic resistance marker genes, form potentially infectious units for further transgressions of species barriers by horizontal gene transfer, i.e., by infection.

Recent evidence also suggests that vectors carrying transgenes may spread horizontally via microorganisms, animals and human beings in an uncontrolled and uncontrollable manner. The teeming microbial populations in the terrestrial and aquatic environments serving as a horizontal gene transfer highway and reservoir, facilitating the multiplication, recombination of vectors and infection of all plant and animals species.

Vector-mediated horizontal gene transfer and recombination have been shown to be responsible for the rapid evolution of multiple antibiotic resistance and for the emergence of new and old pathogens. Horizontal gene transfer can effectively create new LMOs across national boundaries. It is a runaway process that cannot be regulated. This makes it paramount to control what is released in the first place. We shall discuss the implications of the findings for biosafety risk assessment and the biosafety protocol.

Biodiversity and species integrity

Biosafety cannot be considered apart from the biodiversity that we are concerned to protect, nor from the human beings who have actively maintained and generated biodiversity past and present, in the course of making their livelihood. Thus, socioeconomic impacts cannot be excluded from biosafety considerations.

What is biodiversity? It is a dynamically balanced ecology of multiple, interdependent, interconnected species that are nevertheless autonomous and distinct. Each species is a complex developmental system that has evolved in concert with its ecological environment while maintaining its integrity for tens, if not hundreds of millions of years.

This intimate interrelationship between organism and environment is particularly high-lighted by the rapid advances in genetics within the past 20 years, as recombinant DNA technology offers powerful investigative tools. The relevant findings have been extensively reviewed beginning more than 10 years ago (Dover and Flavell, 1982; Pollard, 1984, 1988; Ho, 1987, 1996a, Rennie, 1993, Jablonka and Lamb, 1995) and have been incorporated into our Open University Genetics Course (Ho et al, 1987). They show that genes function in an extremely complex network, such that ultimately, the expression of each gene depends on that of every other. That is why an organism will tend to change in nonlinear, unpredictable ways, even when a single gene is introduced. Furthermore, the genome itself is dynamic and fluid, and engages in feedback interrelationships with the cellular and ecological environment, so that changes can propagate from the environment to give repeatable alterations in the genome (reviewed in Pollard, 1988; Ho, 1987; 1996). Conversely, as demonstrated in current transgenic experiments, introducing a single exotic gene into an organism can impact on the ecological environment. Holmes and Ingham (1994) showed that a common soil bacterium, Klebsiella planticola, engineered to produce ethanol from crop waste, drastically inhibited the growth of wheat seedlings. Similarly, the release of transgenic plants with the Bt insecticide led to the rapid evolution of Bt resistance among major insect pests (Hama et al, 1992; Commandeur and Komen, 1992).

The logical conclusion from all of the findings is that heredity does not reside solely in the constancy of DNA in the genome, but in the complex network of intercommunications extending from the socioecological environment to the genes. It is this complex, entangled network that is responsible, both for the integrity of species and for the maintenance of ecological biodiversity. Unfortunately, current practices of gene biotechnology and biosafety risk assessment are still (mis)guided by the mindset of the old reductionist paradigm in which genes are seen to be stable units, separable from each other and from the environment (see Ho, 1995).

Species integrity and biodiversity are inextricably linked, and that is why current transgenic technology poses such a threat to biodiversity. By its very nature, transgenic technology transgresses species integrity and species boundaries. This is associated with the use of genetic parasites or vectors for multiplying and transferring genes, which are designed to overcome species barriers as well as the cellular defence mechanisms that protect the organism against the invasion of foreign DNA. Let us consider transgenic technology in more detail.

What is transgenic technology?

Transgenic technology bypasses conventional breeding by using artificially constructed parasitic genetic elements as vectors to multiply copies of genes, and in many cases, to carry and smuggle genes into cells which would normally exclude them. (Parasites, by definition, require the host cell's biosynthetic machinery for replication.) Once inside cells, these vectors slot themselves into the host genome. In this way, transgenic organisms are made carrying the desired transgenes. The insertion of foreign genes into the host genome has long been known to have many harmful and fatal effects including cancer (Wahl et al, 1984; see also relevant entries in Kendrew, 1994); and this is borne out by the low success rate of creating desired transgenic organisms. Typically, a large number of cells, eggs or embryos have to be injected or infected with the vector to obtain a few organisms that successfully express the transgene(s).

The most common vectors used in gene biotechnology are a mosaic recombination of natural genetic parasites from different sources, including viruses causing cancers and other diseases in animals and plants, with their pathogenic functions 'crippled', and tagged with one or more antibiotic resistance 'marker' genes, so that cells transformed with the vector can be selected. For example, the vector most widely used in plant genetic engineering is derived from a tumour-inducing plasmid carried by the bacterium Agrobacterium tumefaciens. In animals, vectors are constructed from retroviruses causing cancers and other diseases. Unlike natural parasitic genetic elements which have varying degrees of host specificity, vectors used in genetic engineering are designed to overcome species barriers, and can therefore infect a wide range of species. Thus, a vector currently used in fish has a framework from the Moloney murine leukemic virus, which causes leukemia in mice, but can infect all mammalian cells. It has bits from the Rous Sarcoma virus, causing sarcomas in chickens, and from the vesicular stomatitis virus, causing oral lesions in cattle, horses, pigs and humans (Lin et al, 1994). Genetic engineering is also known as recombinant DNA or rDNA technology, as it uses enzymes to cut and join, and therefore recombine genetic material from different sources. Box 1 summarizes why rDNA technology differs radically from conventional breeding methods.

Box 1. rDNA Technology Differs Radically from Conventional Breeding Techniques

1. rDNA technology recombines genetic material in the laboratory between species that have very little probability of exchanging genes otherwise.

2. While conventional breeding methods shuffle different forms (alleles) of the same genes, rDNA technology enables completely new (exotic) genes to be introduced with unpredictable effects on the physiology and biochemistry of the transgenic organism.

3. Gene multiplications and a high proportion of gene transfers are mediated by vectors which have three undesirable characteristics:
a. Many are derived from disease causing viruses, plasmids and mobile genetic elements - parasitic DNA that have the ability to invade cells and insert themselves into the cell's genome causing genetic damages.
b. They are designed to breakdown species barriers so that they can shuttle genes between a wide range of species. Their wide host range means that they can infect many animals and plants, and in the process pick up genes from viruses of all these species to create new pathogens.
c. They carry genes for antibiotic resistance, which will exacerbate a major public health problem.

The vectors used for transferring genes play the key role in transgressing species integrity and species barriers. We shall deal with each in turn.

Transgressing species integrity results in unpredictable physiological effects

Transgenic vectors themselves can cause severe immune reaction. Direct health hazard from the adenovirus vector, used in attempted gene therapy for Parkinson's disease, Alzheimer's disease and Cystic Fibrosis, has been reported (Coghlan, 1996). It caused such severe immune reaction that one patient almost died. Rats receiving injections of the virus directly into the brain and then into the foot 2 months later developed severe inflammation in the brain. These findings have to be seen in the light that not a single successful gene therapy has been documented. Some geneticists are now looking into even more aggressive gene transfer vectors: the latest one constructed from a disabled AIDS virus (J. Cohen, 1996), even though it has been pointed out that the disabled virus could recombine into a virulent form and cause AIDS.

It is not easy to transfer genes naturally between species, because there are endogenous cellular mechanisms that excise or inactivate foreign genes (Doerfler 1991, 1992). These are also responsible for the instability of transferred genes in transgenic organisms, which is posing a problem for the technology (Finnegan and McElroy, 1994). Vectors are now increasingly engineered to overcome these cellular defence mechanisms (Höfle, 1994), thus further undermine the ability of the species' developmental system to resist invasion by exotic genes carried on such transgenic vectors.

One area of major concern is the allergenicity of transgenic foods, which has become a concrete issue since the discovery of a brazil-nut allergen in a transgenic soybean (Nordlee et al, 1996). Most identified allergens are water-soluble and acid-resistant. Some, such as those derived from soya, peanut and milk, are very heat-stable, and are not degraded during cooking, while fruit-derived allergenic proteins are heat-labile (Lemke and Taylor, 1994). There are also indications that allergenicity in plants is connected to proteins involved in defence against pests and diseases. Thus, transgenic plants engineered for resistance to diseases and pests will have a higher allergenic potential than the unmodified plants (see Franck and Keller, 1995).

Another instructive case is the transgenic yeast engineered for increased glycolytic activity with multiple copies of one of its own genes, which resulted in the accumulation of a metabolite at highly mutagenic levels (Inose and Kousaku, 1995). Thus, even increasing the expression of non-exotic genes can have unpredictable toxic effects. This should serve as a warning against applying the 'familiarity principle' in risk assessment. We simply do not understand the principles of physiological regulation to enable us to categorize, a priori, those genetic modifications that will pose a risk and those that do not. It is a strong argument for the case by case approach.

Current risk assessment of transgenic foods is limited to the characterization of the introduced gene(s) and gene product(s) and known toxins. That is clearly inadequate in view of the nonlinear changes that can arise within the highly interconnected genetic network, which can only be revealed by characterizing the overall profile of expressed proteins and metabolites. Clear labelling of transgenic food products is also an integral part of biosafety so that consumers can avoid known allergens.

Transgressing species barriers

Unintended transboundary movements of LMOs, as everyone knows, can occur by cross-pollination between transgenic crop-plants and its wild relatives (see Meister and Mayer, 1994). Field trials have shown that cross-hybridization has occurred between transgenic Brassica napa and its wild relatives: B. campestris (Jorgensen and Anderson, 1994; Mikkelsen et al, 1996), Hirschfeldia incana (Eber et al, 1994; Darmency 1994) and Raphanus raphanistrum (Eber et al, 1994). Rissler and Mellon (1993) have predicted those problems arising from the introduction of exotic species, whether genetically engineered or not.

A much more insidious, uncontrollable way for the transgenes (and associated marker genes) to spread, which is peculiar to LMOs, is by horizontal gene transfer, i.e., by infection. This process recognizes no species barriers, and is inherent to many current transgenic technologies. It is, to a large extent, why transgenic organisms are different from those obtained by conventional breeding methods.

The vectors for gene transfer are the means whereby the original species barriers are transgressed. They have the potential to infect and transgress further species boundaries in the process of horizontal gene transfer.

Horizontal gene transfer links the whole biosphere

Horizontal gene transfer is the transfer of gene by infection, between species that do not interbreed. It has been known to occur among bacteria and viruses for at least 20 years. There are three different ways for genes to be transferred. Conjugation, the mating process, requires cell to cell contact. Transduction is transfer with the help of viruses, while transformation is the direct uptake of DNA by the bacteria. As mentioned earlier, there are three kinds of genetic parasites - viruses, plasmics and mobile genetic elements. Mosaic recombinations of all classes are made and currently used by genetic engineers to multiply genes or to transfer genes. Viruses are probably the most infectious as they do not require cell to cell contact for infection and can persist in the environment indefinitely. Plasmids and mobile genetic elements are generally exchanged by cell to cell contact during conjugation or when one cell ingests (or phagocytoses) another.

It must be stressed that horizontal gene transfer has mostly been documented with specially designed plasmids in studies carried out in microcosms (Mazodier and Davies, 1991), but the spread of antibiotic resistance markers throughout bacterial communities (see later) shows that it can happen without intentional intervention. The observed correlation between the presence of antibiotics and enhanced gene transfer activities led to the speculation that low concentrations of antibiotics act like pheromones to enhance gene transfer (Davies, 1994). That has particular implications for the secondary mobility of transgenes carried in association with antibiotic resistance marker genes, as the profligate use of antibiotics is allowed to continue.

Like all other species, bacteria possess different 'restriction systeme' which degrade or silence foreign DNA. However, stressful conditions appear to reduce the effectiveness of these systems and to encourage recombination. Starving bacteria are also more competent in taking up isolated DNA. Transgenic plasmids, as mentioned earlier, are designed to overcome these restriction systems as well as to cross species barriers. So they are potentially much more effective in horizontal gene transfer, despite the 'crippling'.

For a long time, it was supposed that horizontal gene transfers did not involve higher organisms, and certainly not organisms like ourselves, because there are genetic barriers between species and genetic parasites are species-specific.

Within the past two to three years, however, the full scope of horizontal gene transfer is slowly coming to light. A search of the isi database conducted under "horizontal gene transfer" came up with 75 references published in mainstream journals between 1993 and 1996, all but 2 giving direct or indirect evidence of horizontal gene transfers. Transfers occur between very different bacteria, between fungi, between bacteria and protozoa, between bacteria and higher plants and animals, between fungi and plants, between insects ... in short, as Stephenson and Warnes (1996) remark, "The threat of horizontal gene transfer from recombinant organisms to indigenous ones is ... very real and mechanisms exist whereby, at least theoretically, any genetically engineered trait can be transferred to any prokaryotic organism and many eukaryotic ones."

The current state of our understanding is presented in Fig. 1 (not shown), where the arrows indicate transfers for which direct or circumstantial evidence already exists. If you follow those arrows, you will realize how a gene transferred to any species in a vector can reach every other species on earth, the microbial/viral pool providing the main genetic thoroughfare and reservoir. Earlier this year, a mobile genetic element, called mariner, first discovered in Drosophila, was found to have jumped into the genomes of primates including humans, where it causes a neurological wasting disease (P. Cohen, 1996). Geneticists suspect the Drosophila gene might have got into a virus which infected the primates.

Although horizontal gene transfers have occurred in our evolutionary past, they were relatively rare events among multicellular plants and animals (and some geneticists have disputed the involvement of horizontal gene transfer in favour of convergent evolution). However, the scope of horizontal gene transfer may have, or will be, increased because the vectors constructed for genetic engineering are chimaeras of many different vectors designed to transgress species integrity and species barriers, and therefore capable of infect many species. In the process, these vectors will recombine with a wide range of natural pathogens. That they have been 'crippled' should not lull us into a false sense of security, because it is well-known that they can be helped by other viruses and mobile genetic elements to jump in and out of genomes. Otherwise, it would have been impossible to construct any transgenic organisms at all.

Vectors mediate horizontal transfer of antibiotic resistance genes

Among the 75 references on horizontal gene transfer are documentations for the rapid spread of antibiotic resistance genes carried on plasmids among bacterial populations (Heaton and Handwerger, 1995; Coffey et al, 1995; Kell et al, 1993; Amabilecuevas and Chicurel, 1993; Bootsma et al, 1996). Multiple antibiotic resistance has spread among pathogens worldwide, and reported to be endemic in many U.K. hospitals. The rapid spread of antibiotic resistance is the result of the indiscriminate use of antibiotics which predates genetic engineering. However, using antibiotic resistance markers in transgenic vectors will exasperate the situation. The transgenic tomatoes currently marketed here and the U.S. both carry genes for kanamycin resistance. Kanamycin is used to treat tuberculosis, which is coming back all over the world, and the TB bacteria are already resistant to many antibiotics (see New Scientist, May 4 issue, 1996). One of the two out of 75 references which reported 'negative' for horizontal gene transfer is a review produced by the staff of Calgene, assuring us that the kanamycin resistance gene used in the Calgene transgenic tomato is safe (Redenbaugh et al, 1994). That study was based, not on empirical data, but on theoretical considerations.

Vectors mediate genetic recombination to generate new pathogens

As pathogens become antibiotic resistant they also exchange and recombine virulence genes by horizontal gene transfer, thereby generating new virulent strains of bacteria and mycoplasm. This has been shown for Vibrio cholerae involved in the new pandemic cholera outbreak in India (Reidl and Mekalanos, 1995; Prager et al, 1995; Bik et al, 1995), Streptococcus (Upton et al, 1996; Kapur et al, 1995; Whatmore et al, 1994, 1995; Schnitzler et al, 1995), involved in the world-wide increase in frequency of severe infections including the epidemic in Tayside Scotland in 1993, and Mycoplasma-genitalium (Reddy et al, 1995), implicated in urethritis, pneumonia, arthritis, and AIDS progression. Many unrelated bacterial pathogens, causing diseases from bubonic plague to tree blight, are now found to share an entire set of genes for invading host cells, which have almost certainly spread by horizontal gene transfer (Barinaga, 1996). Public health is approaching a major crisis everywhere in developed as well as developing countries, as, within the past twenty-five years, at least 30 new infectious diseases have appeared together with the re-emergence of old ones.

The dangers of generating pathogens by vector mobilization and recombination are real. Over a period of ten years, 6 scientists working with the genetic engineering of cancer-related oncogenes at the Pasteur Institutes in France have contracted cancer (reported in New Scientist, June 18 issue, 1987, p. 29).

The natural microbial populations form a major thoroughfare and reservoir for horizontal gene transfer

Horizontal gene transfers have been directly demonstrated between bacteria in the marine environment (Frischer et al, 1994), in the freshwater environment (Ripp et al, 1994) and in the soil (Neilson et al, 1994). It is significant that in all the experiments, horizontal gene transfers were mediated by special hybrid plasmid vectors, of the sort used in transgenic technology.

An obvious route for the vectors containing transgenes in transgenic higher plants and animals as well as microorganisms to spread is via the teeming microbial populations in the soil, where transgenic plants are grown, and in aquatic environments, where transgenic fish and shellfish are currently being developed for marketing. Aquatic environments are known to contain some 108 or more virus particles per millilitre, all capable of transferring genes, of helping endogenous 'crippled' vectors move and recombining with them to generate new viruses. Microbial populations in all environments form large reservoirs supporting the multiplication of the vectors, enabling them to spread to all other species. There will also be opportunity for the genetic elements to recombine with other viruses and bacteria to generate new genetic elements and pathogenic strains of bacteria and viruses, which will, at the same time, be antibiotic resistant.

This route cannot be ignored, as transfers of transgenes and marker genes have been experimentally demonstrated: from transgenic potato to a bacterial pathogen (Schluter et al, 1995), and between transgenic plants and soil fungi (Hoffman et al, 1994). We do not know the precise frequencies for such horizontal gene transfer, as very few studies have been carried out. Similarly, there is very limited published data on the degree of stability of integrated vectors carrying trangenes and antibiotic resistance marker genes. As mentioned earlier, transgenes are often inactivated or 'silenced' by cellular mechanisms that prevent expression of foreign DNA (Finnegan and McElroy, 1994). A substantial degree of transgene instability has been reported for transgenic livestock (Colman,1996) and transgenic plants (see Lee et al, 1995), which includes non-expression of integrated genes as well as loss of integrated genes. This severely compromises the commercial viability of transgenic technology, but raises the important question of how the integrated genes are lost.

Viral resistance transgenes can generate live viruses by recombination

A major class of transgenic plants are now engineered for resistance to viral diseases by incorporating the gene for the virus' coat protein. Viruses are notoriously rapid in their mutation rate. They play a large role in horizontal gene transfer between bacteria (Reidl and Mekalanos, 1995; Ripp et al, 1994) and also exchange genes among themselves thus increasing their host range (Sandmeier, 1994). Molecular geneticists have expressed concerns that transgenic crops engineered to be resistant to viral diseases with genes for viral coat proteins might generate new diseases by several known processes. The first, transcapsidation - has been detected by Creamer and Falk (1990). It involves the DNA/RNA of one virus being wrapped up in the coat protein of another so that viral genes can get into cells which otherwise exclude them. The second, recombination, has been demonstrated in an experiment in which Nicotiana benthamiana plants expressing a segment of a cowpea chlorotic mottle virus (CCMV) gene was inoculated with a mutant CCMV missing that gene (Green and Allison, 1994). The infectious virus was indeed regenerated by recombination. A third possibility is that the transgenic coat protein can help defective viruses multiply by complementation (Osbourn et al, 1990). As plant cells are frequently infected with several viruses, recombination events will occur and new and virulent strains may be generated. Thus, the transboundary movement of the transgene will be disguised by recombination, and can only be traced with the appropriate molecular probes.

In view of the documented occurrence of transcapsidation and recombination, it is important that trial releases should include monitoring for the emergence of new viruses that may pose new threats to crop plants.

Vectors resist breakdown in the gut and can infect mammalian cells

One question which has not yet been addressed in biosafety regulations is the extent to which vector DNA can resist breakdown in the gut and infect the cells of higher organisms. In a study to test for the ability of bacterial viruses and plasmids to infect mammalian cells, it was found that plasmids of E. coli, carrying the complete poliovirus, can be transferred to cultured mammalian cells and the polioviruses recovered from the cells, even though no eukaryotic signals for reading the genes are contained in the plasmid (Heitman and Lopes-Pila, 1993). In the same paper, the authors review experimental observations made since the 1970s that the lambda phage of bacteria, and the baculovirus, supposedly specific for insect cells, are also efficiently taken up by mammalian cells; and in the case of the baculovirus, transported to the cell nucleus. Similarly, E. coli plasmids carrying the complete Simian virus (SV40) genome were also taken up simply by exposing the cell culture to a bacterial suspension. These mammalian cells accept foreign DNA parasites so well because they phagocytose bacteria and viral particles directly. Transgenic medaka and mummichog fish have even been constructed by injecting fish embryos with a bacteriophage fX174 vector carrying an oncogene, which is integrated into the fish chromosome (Winn et al, 1995). The unintended infectivity of transgenic vectors is yet another area that needs urgent investigation.

It has long been assumed that our gut is full of enzymes which can digest DNA. However, genes carried by vectors may be especially resistant to enzyme action, and much more infectious than ordinary bits of DNA. In a study designed to test the survival of viral DNA in the gut, mice were fed DNA from a bacterial virus, and large fragments were found to survive passage through the gut and to enter the bloodstream (Schubbert et al, 1994). Again, more studies of this kind are needed particularly as transgenic foods are already being marketed. Within the gut, vectors carrying antibiotic resistance may also be taken up by the gut bacteria, which would then serve as a mobile reservoir of antibiotic resistance genes for pathogenic bacteria. Horizontal gene transfer between gut bacteria has already been demonstrated in mice and chickens (Doucet-Populaire, 1992; Guillot and Boucard, 1992).

Genes carried by vectors can persist indefinitely in the environment

This subject has been extensively reviewed by Jäger and Tappeser (1996) who showed that genes carried by vectors can survive indefinitely in the environment, in dormant bacteria, or as naked DNA adsorbed to solid particles, where they are efficiently taken up by microbes. In a recent study in Eastern Germany, streptothricin was administered to pigs beginning in 1982. By 1983, plasmids encoding streptothricin resistance was found in the pig gut bacteria. This has spread to the gut bacteria of farm workers and their family members by 1984, and to the general public and pathological strains of bacteria the following year. The antibiotic was withdrawn in 1990. Yet the prevalence of the resistance plasmid has remained high when monitored in 1993 (Tschäpe, 1994), confirming the ability of microbial populations to serve as stable reservoirs for replication, recombination and horizontal gene transfer, in the absence of selective pressure. In a direct test of persistence of streptomycin-resistance, Schrag and Perrot (1996) cultured many independent lines of a streptomycin-resistant mutant of E. coli in the absence of the antibiotic. They found that all retained the resistance after 180 generations. Furthermore, they have also in the mean time, accumulated compensatory mutations in other parts of the genome that increased their competitive ability relative to the wild-type.

Bacteria and viruses can indeed, apparently disappear as they go dormant, and then reappear in a more competitive form. This has been documented for a laboratory strain of E. coli K12, which when introduced into the sewage, went dormant and undetectable for 12 days before reappearing, having acquired a new plasmid for multidrug resistance that enabled it to compete with the naturally occurring bacteria (Tschäpe, 1994). Dormant forms of bacteria and viruses can survive indefinitely as biofilms in the body and in the environment (Costerton et al, 1994; Lewis and Gattie, 1991), when they can accumulate new mutations to come back with a vengeance.

The hazards of transgenic technologies are summarized in Box 2, and the routes for transboundary movements of transgenes and marker genes via vector-mediated horizontal gene transfer in Box 3.

Box 2. Hazards of Transgenic Technologies

1. Toxic or allergenic effects due to transgene products or products from interactions with host genes.

2. Spread of transgenes to related weed species, creating superweeds (e.g. herbicide resistance).

3. Accelerating the evolution of biopesticide resistance in insect pests.

4. Adverse immune reactions caused by gene transfer vectors.

5. Vector - mediated horizontal gene transfer to unrelated species via bacteria and viruses, with the potential of creating many other weed species.

6. Potential for vector-mediated horizontal gene transfer and recombination to create new pathogenic bacteria and viruses.

7. Vector recombination to generate new virulent strains of viruses, especially in transgenic plants engineered for viral resistance with viral genes.

8. Potential for vector mediated spread of antibiotic resistance to bacteria in the environment, exacerbating an existing public health problem.

9. Vector-mediated spread of antibiotic resistance to gut bacteria and to pathogens.

10. Potential of vector-mediated infection of cells after ingestion of transgenic foods, to regenerate disease viruses or insert itself into the cell's genome.

11. The vectors carrying the transgene, unlike chemical pollution, can be perpetuated and amplified given the right environmental conditions. Once let loose, they are impossible to control or recall.

Box 3. Routes for Uncontrollable Transboundary Movements of LMOs Via Vector-mediated Horizontal Gene Transfer

1. Ingestion by insects, and infection via insects, of other plants and animals.

2. Ingestion by birds, and dispersal of seeds and DNA in bird droppings.

3. Release of LMOs in laboratory effluents to the general environment, and further transport by wind and water.

4. Release of vectors carrying transgene and marker genes from dead transgenic organisms, solid wastes and cells and transfer to soil bacteria and fungi where they form a long-term reservoir for replication, recombination and infecting other non LMO crops.

5. Release of vectors carrying transgene and marker genes from dead transgenic organisms, solid wastes and cells in aquatic environments and uptake by microorganisms which form a long term aquatic reservoir for replication and recombination, and also a system for long-distance dispersal.

6. Ingestion by human beings and animals, carried and deposited in sewage system or faeces in other countries.

7. Ingestion by human beings and animals, and infection of gut bacteria, creating mobile long-term enteric reservoirs for replication, recombination and dispersal of vectors.

8. Ingestion by human beings and animals, and potential infection of gut cells, which form temporary storage depots for vectors (as gut cells turnover).

9. Ingestion by human beings and animals, and passage into the bloodstream to other cells, which can form further storage depots for vectors.


According to the 1996 WHO report (see Mihill, 1996), old and new infectious diseases are coming back worldwide within the past twenty-five years, claiming the lives of 50000 men, women and children every day. Antibiotic resistance of the pathogens is identified as a major contributing factor. There is sufficient evidence that horizontal gene transfer is responsible for the emergence of both old and new pathogens, and for the evolution of multiple antibiotic resistance. We certainly do not need any more releases of transgenic organisms that would provide yet more vehicles for horizontal gene transfer. There are obvious gaps in the information required for proper risk assessment which are simply not addressed by the regulating bodies. Despite that, we know that the transboundary movement of transgene and marker genes by horizontal gene transfer cannot be controlled if current transgenic practices are allowed to continue.

Horizontal gene transfer, especially when mediated by transgenic vectors, respects neither species nor national boundaries. As Salyers and Shoemaker (1994) state, "It is probably impossible to eliminate all [horizontal] transfer capacity from a genetically engineered strain that is going to be released into the environment." This makes it paramount to control what is being released in the first place. Once transgenic organisms are released, by intent or by accident, neither they, nor the transgenes can be recalled. That is why adequate monitoring procedures must be put in place which includes tracking horizontal gene transfers at and around the site of release. Deliberate releases, as well as tolerated releases from contained uses, may indeed have unintended transboundary effects, and that must be included for consideration in the biosafety protocol.

Nature is interconnected in such a way that each and every species maintains its own integrity, and that may be the essence of biodiversity. Biodiversity may simply be a state of coherence for the ecological system akin to that which exists for an organism as a whole (see Ho, 1993, 1996b). Gene biotechnology can only be safely practised, if at all, by safe-guarding the coherence of nature's biodiversity.


We thank Beth Burrows of the Edmonds Institute, Chee Yokeling of the TWN, David Heaf, John Barrett and Peter Lund of Ifgene and an anonymous referee for valuable suggestions and information. This paper was prepared for Workshop on Transboundary movement of living modified organisms resulting from modern biotechnology: issues and opportunities for policy-makers, Aarhus, Denmark, 19-20 July, 1996. M.W.H. is particularly grateful to Dr. J.K. Moulongoy for inviting her to participate in the Workshop. She benefitted greatly from the special extended discussion session following the presentation of a draft of this paper.


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