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
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.
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
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.
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.
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
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
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)
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
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
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
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
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.
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.
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.
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.
Amabilecuevas, C.F. and Chicurel, M.E. (1993). Horizontal gene
transfer. Am. Sci. 81:332-341.
Barinaga, M. (1996). A shared strategy for virulence. Science 272:1261-1263.
Bik, E.M., Bunschoten, A.E., Gouw, R.D. and Mooi, F.R. (1995). Genesis of novel epidemic vibrio-cholerae-0139 strain-evidence for horizontal transfer of genes involved in polysaccharide synthesis. Embo J. 14:209-216.
Coffey, T.J., Dowson,C.G., Daniels, M. and Spratt, B.G. (1995). Genetics and molecular-biology of b-lactam-resistant pneumococci. Microbial Drug Resistance-Mechanisms Epidemiology and Disease 1:29-34.
Bootsma, J.H., Vandijk, H. Verhoef, J., Fleer, A. and Mooi, F.R. (1996). Molecular characterization of the bro b-lactamase of moraxella (Branhamella) catarrhalis. Antimicrobial Agents and Chemotherapy 40:966-972.
Coghlan, A. (1996). Gene shuttle virus could damage the brain. New Scientist May 11:6.
Cohen, J. (1996). New role for HIV:a vehicle for moving genes into cells. Science 272:195.
Cohen, P. (1996). Doctor, there's a fly in my genome. New Scientist March 9:16.
Colman, A. (1996). Production of proteins in the milk of transgenic livestock - problems, solutions and successes. Am. J. Clin. Nutrition 63:S639-S645.
Commandeur, P. and Komen, J. (1992). Biopesticides: Options for biological pest control increase. Biotech Develop. Monitor 13 (Dec):6-7.
Costerton, J.W., Lewandowski, Z., DeBeer, D., Caldwell, D., Korber, D. and James, G. (1994). Biofilms, the customized microniche. J. Bacteriol. 176:2137-2142.
Creamer, R. and Falk, B.W. (1990). Direct detection of transcapsidated barley yellow dwarf luteoviruses in doubly infected plants. J. Gen. Virol. 71:211-217.
Damency (1994). The impact of hybrids between genetically modified crop plants and their related species:introgression and weediness. Mol. Ecol. 3:37-40.
Doerfler, W. (1991). Patterns of DNA Methylation - evolutionary vestiges of foreign DNA inactivation as a host defense mechanism. Biol. Chem. Hoppe-Seyler 372:557-564.
Doerfler, W. (1992). DNA methlation:eukaryotic defense against the transcription of foreign genes? Microbial Pathogenesis 12:1-8.
Doucet-Populaire, F. (1992). Conjugal transfer of genetic information in gnotobiotic mice, in Microbial Releases (Ed. M.J. Gauthier), Springer Verlag, Berlin, 345 pp.
Dover, G. A. and Flavell, ed. (1982). Genome Evolution, Academic Press, London, 382 pp.
Eber, G., Chevre, A.M. Baranger, A., Vallee, P., Tanfuy, X. and Renard, M. (1994). Spontaneous hybridization between a male-sterile oilseed rape and two weeds. Theor. App. Gene. 88:362-368.
Finnegan H. and McELroy (1994). Transgene inactivation plants fight back! Bio/Techology 12:883-888.
Frischer, M.E., Stewart, G.J. and Paul, J.H. (1994). Plasmid transfer to indigenous marine bacterial-populations. FEMS Microbiol. Ecol. 15:127-135.
Green, A.E. and Allison, R.F. (1994). Recombination between viral RNA and transgenic plant transcripts. Science 263:1423.
Guillot, J.F. and Boucaud, J.L. (1992). In vivo transfer of a conjugative plasmid between isogenic Escherichia coli strains in the gut of chickens, in the presence and absence of selective pressure. Pp. 167-174, in Microbial Releases (Ed. M.J. Gauthier), Springer Verlag, Berlin, 345 pp.
Hama, H., Suzuki, K. and Tanaka, H. (1992). Inheritance and stability of resistance to Bacillus thuringiensis formulations in diamondback moth, Plutella xylostella (Linnaeus) (Lepidoptera:Yponomeutidae). Appl. Entomol. Zool. 27:355-362.
Heaton, M.P., and Handwerger, S. (1995). Conjugative mobilization of a vancomycin resistance plasmid by a putative enterococcus-faecium sex-pheromone response plasmid. Microbial Drug Resistance-Mechanisms Epidemiology and Disease 1:177-183.
Heitman, D. and Lopes-Pila, J.M. (1993). Frequency and conditions of spontaneous plasmid transfer from E. coli to cultured mammalian cells. BiosSystems 29:37-48.
Ho, M.W. (1987). Evolution by process, not by consequence: implications of the new molecular genetics for development and evolution. Int. J. comp. Psychol. 1:3-27.
Ho, M.W. (1993). The Rainbow and The Worm, The Physics of Organisms, World Scientific, Singapore, 202 pp.
Ho, M.W. (1995). Unravelling gene biotechnology, Soundings 1:77-98.
Ho, M.W. (1996a). Why Lamarck won't go away. Ann. Human Genetics 60:81-84.
Ho, M.W. (1996b). Natural being and coherent society. Pp. 286-307, in Gaia in Action, Science of the Living Earth (Ed. P. Bunyard), Floris Books, Edinburgh, 351 pp.
Ho, M.W., Goodwin, B.C., Metcalf, J., Murphy, P., and Rose, S. P.R. (1987). S298 Genetics, A Second Level Science Course, Open University Press, Milton Keynes, 820 pp.
Höfle, M.G. (1994). Auswirkungen der Freisetzung bakterieller Monokulturen auf die naturliche Mikroflora aquatischer Okosysteme. Pp. 795-820, in Biologische Sicherheit/Forschung Biotechnologie BMFT (Ed Germany) vol.3, 1003 pp.
Hoffman, T., Golz, C. and Schieder, O. (1994). Foreign DNA sequences are received by a wild-type strain of Aspergillus niger after co-culture with transgenic higher plants. Curr. Genet. 27:70-76.
Holmes, M.T. and Ingham, E.R. (1994) Abstract for 79th Annual Ecological Society of America meeting. Bull. Ecol. Soc. Am. 75:2.
Inose, T. and Kousaku, M. (1995). Enhanced accumulation of toxic compounds in yeast cells having high glycolytic activity:a case study on the safety of genetically engineered yeast. Int. J. Food Science Tech. 30:141-146.
Jablonka, E. and Lamb, M. (1995). Epigenetic Inheritance and Evolution. The Lamarckian Dimension, Oxford University Press, Oxford, 301 pp.
Jäger, M.J. and Tappeser, B. (1995). Risk Assessment and Scientific Knowledge. Current data relating to the survival of GMOs and the persistence of their nucleic acids:Is a new debate on safeguards in genetic engineering required? - considerations from an ecological point of view. Preprint circulated and presented at the TWN-Workshop on Biosafety, April 10, New York.
Jorgensen, R.B. and Andersen, B. (1994). Spontaneous hybridization between oilseed rape (Brassica napus) and weedy B. campestris (Brassicaceae): a risk of growing genetically modified oilseed rape. Am. J. Botany 12:1620-1626.
Kapur, V., Kanjilal, S., Hamrick, M.R., Li, L.L., Whittam, T.A., Sawyer, S.A. and Musser, J.M. (1995). Molecular population genetic-analysis of the streptokinase gene of Streptococcus-pyogenes-mosaic alleles generated by recombination. Mol. Microbiol. 16,:509-519.
Kell, C.M., Hordens, J.Z., Daniels, M., Coffey, T.J., Bates, J., Paul, J., Gilks, C. and Spratt, B.G. (1993). Molecular epidemiology of penicillin-resistant pneumococci isolated in Nairobi, Kenya. Infection and Immunity 61:4382-4391.
Kendrew, J., ed. (1995). The Encyclopedia of Molecular Biology, Blackwell Science, Oxford, 1165 pp.
Lee, H.S., Kim, S.W., Lee, K.W., Ericksson, T. and Liu, J.R. (1995). Agrobacterium-mediated transformation of ginseng (Panax-ginseng) and mitotic stability of the inserted beta-glucuronidase gene in regenerants from isolated protoplasts. Plant Cell Reports 14:545-549.
Lewis, D.L. and Gattie, D.K. (1991). The ecology of quiescent microbes. ASM News 57:27-32.
Lin, S., Gaiano, N., Culp, P., Burns, J.C., Friedmann, T., Yee, J.-K. and Hopkins, N. (1994). Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265:666-669.
Meister, I. and Mayer, S. (1994). Genetically engineered plants:releases and impacts on less developed countries, A Greenpeace inventory, Greenpeace International.
Mihill, C. (1996). Killer diseases making a comeback, says WHO. Guardian 10/5/96 p. 3.
Mikkelsen, T.R., Andersen, B. and Jorgensen, R.B. (1996). The risk of crop transgene spread. Nature 380:31.
Neilson, J.W., Josephson, K.L., Pepper, I.L., Arnold, R.B., Digiovanni, G.D. and Sinclair, N.A. (1994). Frequency of horizontal gene-transfer of a large catabolic plasmic (PJP4) in soil. App. Environ. Microbiol. 60:4053-4058.
Nordlee, J.A., Taylor, S.L., Townsend, JA., Thomas, L.A. and Bush, R.K. (1996). Identification of a brazil-nut allergen in transgenic soybeans. The New England Journal of Medicine March 14:688-728.
Osbourn, J.K., Sarkar, S. and Wilson, M.A. (1990). Complementation of coat protein-defective TMV mutants in transgenic tobacco plants expressing TMV coat protein. Virology 179:921-925.
Pollard, J. W. (1984). Is Weismann's barrier absolute? Pp. 291-315, in Beyond neo-Darwinism: Introduction to the New Evolutionary Paradigm (Eds. M.W. Ho and P.T. Saunders), Academic Press, London, 369 pp.
Pollard, J. W. (1988). The fluid genome and evolution. Pp. 63-84, in Evolutionary Processes and Metaphors (Eds. M.W. Ho and S.W. Fox), Wiley, London, 331 pp.
Prager, R., Beer, W., Voigt, W., Claus, H., Seltmann, G., Stephan, R., Bockemuhl, J. and Tschäpe, H. (1995). Genomic and biochemical relatedness between vibrio-cholerae. Microbiol. Virol. Parasitol. Inf. Dis. 283:14-28.
Reddy, S.P., Rasmussen, W.G. and Baseman, J.B. (1995). Molecular-cloning and characterization of an adherence-related operon of myocplasma-genitalium. J. Bacteriol. 177:5943-5951.
Redenbaugh, K., Hiatt, W., Martineau, B., Lindemann, J., and Emlay, D. (1994). aminoglycoside 3'-phosphotransferase-II (alph(3')II) - review of its safety and use in the production of genetically-engineered plants. Food Biotechnology 8:137-165.
Reidl, J. and Mekalanos, J.J. (1995). Characterization of vibrio-cholerae bacteriophage-K139 and use of a novel mini-transposon to identify a phage-encoded virulence factor. Molecular Microbiol. 18:685-701.
Rennie, J. (1993). DNA's new twists. Scientific American March:88-96.
Rissler, J. and Mellon, M. (1993). Perils Amidst the Promise - Ecological Risks of Transgenic Crops in a Global Market, Union of Concerned Scientists, USA.
Ripp, S., Ogunseitan, O.A. and Miller, R.V. (1994). Transduction of a fresh-water microbial community by a new Pseudomonas-aeruginosa generalized transducing phage, UTI. Mol. Ecol. 3:121-126.
Salyers, A.A. and J.B. Shoemaker (1994). Broadhost range gene transfer:plasmids and conjugative transposons. FEMS Microbiology Ecology 15:15-22.
Sandmeier, H. (1994). Acquisition and rearrangement of sequence motifs in the evolution of bacteriophage tail fibers. Mol. Microbiol. 12:343-350.
Schluter, K., Futterer, J. and Potrykus, I. (1995). Horizontal gene-transfer from a transgenic potato line to a bacterial pathogen (Erwinia-chrysanthem) occurs, if at all, at an extremely low-frequency. Bio/Techology 13:1094-1098.
Schnitzler, N., Podbielski, A., Baumgarten, G., Mignon, M. and Kaufhold, A. (1995). M-protein or M-like protein gene polymorphisms in human group-G Streptococci. J. Clin. Microbiol. 33:356-363.
Schrag, S.J. and Perrot, V. (1996). Reducing antibiotic resistance. Nature 381:120-121.
Schubbert, R., Lettmann, C. and Doerfler, W. (1994). Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice. Mol. Gen. Genet. 242:495-504.
Skogsmyr, I. (1994). Gene dispersal from transgenic potatoes to conspecifics: a field trial. Theor. Appl. Gene. 88:770-774.
Stephenson, J.R. and Warnes, A. (1996). Release of genetically-modified microorganisms into the environment. J. Chem. Tech. Biotech. 65:5-16.
Tschäpe, H. (1994). The spread of plasmids as a function of bacterial adaptability. FEMS Microbiology Ecology 15:23-32.
Upton, M., Carter, P.E., Organe, G., and Pennington, T.H. (1996). Genetic heterogeneity of M-type-3 G group-A Streptococci causing severe infections in Tayside, Scotland. J. Clin. Microbiol. 34:196-198.
Wahl, G.M., de Saint Vincent, B.R. and DeRose, M.L. (1984). Effect of chromosomal position on amplification of transfected genes in animal cells. Nature 307:516-520.
Whatmore, A.M. Kapur, V., Musser, J.M. and Kehoe, M.A. (1995). Molecular population genetic-analysis of the enn subdivision of group-A-Streptococcal emm-like genes - horizontal gene-transfer and restricted variation among enn genes. Mol. Microbiol. 15:1039-1048.
Whatmore, A.M. and Kehoe, M.A. (1994). Horizontal gene-transfer in the evolution of group-A Streptococcal emm-like genes - gene mosaics and variation in vir regulons. Mol. Microbiol. 11:363-374.
Winn, R.N., Vanbeneden, R.J. and Burkhart, J.G. (1995). Transfer, methylation and spontaneous mutation frequency of fX174am3cs70 sequences in medaka (Oryzia-latipies) and mummichog (Fundulus-heteroclitus) - implications for gene-transfer and environmental mutagenesis in aquatic species. J. Marine Environmental Research 40:247-265.