Science in Society Archive

Horizontal Gene Transfer - The Hidden Hazards of Genetic Engineering

Dr. Mae-Wan Ho

An earlier version of this paper has been published in the Biosafety series of the Third World Network, Penang in 2001. Still an earlier version (2000) is on the website of SCOPE – a National Science Foundation-funded research project involving Science Journal and groups at the University of California at Berkeley and the University of Washington in Seattle.

Abstract

Genetic engineering involves designing artificial constructs to cross species barriers and to invade genomes. In other words, it enhances horizontal gene transfer – the direct transfer of genetic material to unrelated species. The artificial construct (transgenic DNA) typically contains genetic material from bacteria, viruses and other genetic parasites that cause diseases as well as antibiotic resistance genes that make infectious diseases untreatable. Horizontal transfer of transgenic DNA has the potential to create new viruses and bacteria that cause diseases, spread antibiotic resistance genes to pathogenic bacteria and trigger cancer in mammalian cells. There is an urgent need to establish effective regulatory oversight to prevent the escape and release of these dangerous constructs into the environment, and to consider whether some of the most dangerous experiments should be allowed to continue at all.

Key words: antibiotic resistance genes, dormant viruses, CaMV promoter, cancer, naked DNA, transgenic DNA, T-DNA, conjugation.

GM pollen and baby bees

Two years ago, Prof. Hans-Hinrich Kaatz from the University of Jena, was reported to have new evidence, as yet unpublished, that genes engineered into genetically modified (GM) crops have transferred via pollen to bacteria and yeast living in the gut of bee larvae. [1]

The finding was not unexpected, however. The question one has to ask is why so few experiments of this kind were carried out before large-scale releases of GM crops and products have been allowed to take place.

Prof. Katz’ work has yet to appear in print after it was initially rejected by the journal Nature. Judging from the furore created by the publicity at the time, it would not be surprising if the work has been suppressed, either by the author himself or by the pro-biotech scientific establishment. The same journal is currently embroiled in ‘retracting’ undesirable findings on transgenic contamination of Mexican landraces that it has published (see "A brief history of the CaMV 35S promoter controversy").

Scientists have drawn attention to the possibility of horizontal gene transfer – the transfer of genetic material across species barriers - in connection with the release of GM organisms in recent years [2,3], but the first warnings actually date back to the mid-1970s when genetic engineering began.

Some of us have argued that the hazards of ‘horizontal’ gene transfer to unrelated species are inherent to genetic engineering [4]. The gene-constructs created in genetic engineering have never existed in billions of years of evolution. They consist of new combinations of genetic material originating from dangerous bacteria, viruses and other genetic parasites, including genes coding for antibiotic resistance, ‘antibiotic resistance marker genes’ (See Box 1). The artificial constructs are designed to cross all species barriers and to invade genomes; in the course of which, new viruses and bacteria that cause diseases may be created, and antibiotic resistance genes spread to bacterial pathogens, making infectious diseases untreatable.

Horizontal gene transfer is not just a theoretical possibility. As microbial geneticists and virologists studying the resurgence of infectious diseases within the past 25 years are all too aware. There is overwhelming evidence that horizontal gene transfer and recombination were responsible for creating the new pathogens and spreading drug and antibiotic resistance genes among the pathogens. But more relevant to the present discussion is the horizontal transfer of GM constructs from GM crops. There is empirical evidence for that too, which I shall describe towards the end of this paper, despite attempts to dismiss and misinterpret the findings.

Box 1

What are antibiotic resistance marker genes?

Antibiotics are chemicals that kill bacteria and cells. There are many kinds of antibiotics. An antibiotic resistance gene codes for a protein that make the bacteria or cell resistant to, ie, survive treatment with specific kinds of antibiotics. An antibiotic resistant marker gene is one that accompanies foreign genes to be transferred into cells. It is placed next to the foreign genes in the construct, so that those cells that have taken up the construct will also be resistant to the antibiotic. This gives a convenient way to select for transformed cells, simply by using the antibiotic(s) to kill off the rest.

Horizontal gene transfer may spread transgenes to the entire biosphere

Horizontal gene transfer is the transfer of genetic material to cells or genomes belonging to unrelated species, by processes other than usual reproduction. In the usual process of reproduction, genes are transferred vertically from parent to offspring; and such a process can occur only within a species or between closely related species.

Bacteria have long been known to exchange genes across species barriers in nature. There are three ways in which that is accomplished. In conjugation, genetic material is passed between cells in contact; in transduction, genetic material is carried from one cell to another by infectious viruses; and in transformation, the genetic material is taken up directly by the cell from its environment. For horizontal gene transfer to be successful, the foreign genetic material must become integrated into the cell’s genome, or become maintained in the recipient cell in some other form. In most cases, foreign genetic material that enters a cell by accident, especially if it is from another species, will be broken down before it can incorporate into the genome. Under certain ecological conditions which are still poorly understood, certain foreign genetic material escapes being broken down and become incorporated in the genome. For example, heat shock and pollutants such as heavy metals can enhance horizontal gene transfer; and the presence of antibiotics can increase the frequency of horizontal gene transfer 10 to 10 000 fold. [5]

While horizontal gene transfer is well known among bacteria, it is only within the past 12 years that its occurrence has become increasingly recognized among higher plants and animals [6]. The scope for horizontal gene transfer is essentially the entire biosphere, with bacteria and viruses serving both as intermediaries for gene trafficking and as reservoirs for gene multiplication and recombination (the process of making new combinations of genetic material) [7].

There are many potential routes for horizontal gene transfer to plants and animals. Transduction is expected to be a main route as there are many viruses that infect plants and animals. Recent research in gene therapy indicates that transformation is potentially very important for cells of mammals including human beings. A great variety of ‘naked’ genetic material are readily taken up by all kinds of cells, simply as the result of being applied in solution to the eye, or rubbed into the skin, injected, inhaled or swallowed. In many cases, the foreign gene constructs become incorporated into the genome. [8]

Direct transformation may not be as important for plant cells, which generally have a protective cell wall. But soil bacteria belonging to the genus Agrobacterium are able to transfer the T (tumour) segment of its Tumour-inducing (Ti) plasmid (see below) into plant cells in a process resembling conjugation. This T-DNA is widely exploited as a gene transfer vehicle in plant genetic engineering. Foreign genetic material can also be introduced into plant and animal cells by insects and arthropods with sharp mouthparts. In addition, bacterial pathogens that enter plant and animal cells may take up foreign genetic material and carry it into the cells, thus serving as vectors for horizontal gene transfer [9] . There are almost no barriers preventing the entry of foreign genetic material into the cells of probably any species on earth. The most important barriers to horizontal gene transfer operate after the foreign genetic material has entered the cell [10].

Most foreign genetic material, such as those present in ordinary food, will be broken down to generate energy and building blocks for growth and repair. There are many enzymes (proteins that act as catalysts for chemical reactions in living organisms) that break down foreign genetic material; and in the event that the foreign genetic material is incorporated into the genome, chemical modification can still put it out of action and eliminate it.

However, viruses and other genetic parasites such as plasmids and transposons, have special genetic signals and probably overall structure to escape being broken down. A virus consists of genetic material wrapped in a protein coat. It sheds its overcoat on entering a cell and can either hi-jack the cell to make many more copies of itself, or it can jump directly into the cell’s genome. Plasmids are pieces of ‘free’, usually circular, genetic material that can be indefinitely maintained in the cell separately from the cell’s genome. Transposons, or ‘jumping genes’, are blocks of genetic material which have the ability to jump in and out of genomes, with or without multiplying themselves in the process. They can also land in plasmids and be propagated there. Genes hitch-hiking in viruses, plasmids and transposons, therefore, have a greater probability of being successfully transferred into cells and genomes. They are vectors for horizontal gene transfer.

Natural genetic parasites are restricted by species barriers, so for example, pig viruses will infect pigs, but not human beings, and cauliflower viruses will not attack tomatoes. It is the protein coat of the virus that determines host specificity, which is why naked viral genomes (the genetic material stripped of the coat) are generally found to have a wider host range than the intact virus [11]. Similarly, the signals for propagating different plasmids and transposons are usually specific to a limited range of host species, although there are notable exceptions.

As more and more genomes have been sequenced, it is becoming apparent that gene trafficking or horizontal gene transfer has played an important role in the evolution of all species [12], although the extent has been grossly exaggerated in early report [13]. Horizontal gene trafficking is regulated by as yet ill-understood internal constraints in the organisms in response to ecological conditions [14].

Genetic engineering is unregulated horizontal gene transfer

Genetic engineering is a collection of laboratory techniques used to isolate and combine the genetic material of any species, and to multiply the constructs in convenient cultures of bacteria and viruses in the laboratory. Most of all, the techniques allow genetic material to be transferred between species that would never interbreed in nature. That is how human genes can be transferred into pig, sheep, fish and bacteria; and spider silk genes end up in goats. Completely new, exotic genes are also being introduced into crops livestock and fish for a variety of purposes, from the production of food and textiles to pharmaceuticals and industrial chemicals.

In order to overcome natural species barriers restricting gene transfer and maintenance, genetic engineers have made a huge variety of artificial vectors (carriers of genes) by combining parts of the most infectious natural vectors – viruses, plasmids and transposons - from different sources. These artificial vectors generally have their disease-causing functions removed or disabled, but are designed to cross wide species barriers, so the same vector may now transfer, say, human genes spliced into the vector, to the genomes of all other mammals, or of plants. Artificial vectors greatly enhance horizontal gene transfer (see Box 2) [15].

Box 1

Artificial vectors enhance horizontal gene transfer

  • They are derived from natural genetic parasites that mediate horizontal gene transfer most effectively.
  • Their highly chimaeric nature means that they have sequence homologies (similarities) to DNA from viral pathogens, plasmids and transposons of multiple species across Kingdoms. This will facilitate widespread horizontal gene transfer and recombination.
  • They routinely contain antibiotic resistance marker genes that will enhance horizontal transfer in the presence of antibiotics, either intentionally applied, or present as pollutant in the environment. Antibiotics are known to enhance horizontal gene transfer between 10 to 10 000 fold.
  • They often have ‘origins of replication’ and ‘transfer sequences’, signals that facilitate horizontal gene transfer and maintenance in cells to which they are transferred.
  • Chimaeric vectors are well-known to be structurally unstable, ie, they have a tendency to break and rejoin incorrectly or join up with other DNA, and this will increase the propensity for horizontal gene transfer and recombination.
  • They are designed to invade genomes and to overcome mechanisms that breakdown or disable foreign DNA, thus increasing the probability of horizontal transfer.

Although different classes of vectors are distinguishable on the basis of the main frame genetic material, practically every one of them is chimaeric, being composed of genetic material originating from the genetic parasites of many different species of bacteria, animals and plants. Important chimaeric ‘shuttle’ vectors enable genes to be multiplied in the bacterium E. coli and transferred into species in every other Kingdom of plants and animals. Simply by creating such a vast variety of promiscuous gene transfer vectors, genetic engineering biotechnology has effectively opened up highways for horizontal gene transfer and recombination, where previously the process was tightly regulated, with restricted access through narrow, tortuous footpaths. These gene transfer highways connect species in every Domain and Kingdom with the microbial populations via the universal vessel used in genetic engineering, E. coli.

Despite the now copious evidence for the potential of horizontal gene transfer, there is still no legislation in any country to prevent the escape and release of artificial vectors and other artificial constructs into the environment [16].

What are the hazards of horizontal gene transfer?

Most artificial vectors are either derived from viruses or have viral genes in them, and are designed to cross species barriers and to invade genomes. In the case of plants, the most common, Agrobacterium T-DNA vector, though not a virus, is nonetheless derived from the Ti plasmid that codes for proteins involved in invading cells and genomes. They have the potential to recombine with the genetic material of other viruses to generate new infectious viruses that cross species barriers. Such viruses have been appearing at alarming frequencies recently, although good epidemiological data are still wanting. The antibiotic resistance genes carried by artificial vectors can spread to bacterial pathogens, and there appears to have been an accelerated rate at which bacteria become resistant to new antibiotics in recent years.

Has the growth of commercial-scale genetic engineering biotechnology contributed to the resurgence of drug and antibiotic infectious diseases within the past 25 years? [17]. There is already overwhelming evidence that horizontal gene transfer and recombination have been responsible for creating new viral and bacterial pathogens and for spreading drug and antibiotic resistance among the pathogens. One way that new viral pathogens may be created is through recombination with dormant, inactive or inactivated viral genetic material that are in all genomes, plants and animals without exception. Recombination between external and resident, dormant viruses has also been implicated in many animal cancers [18].

As stated earlier, the cells of all species including our own can take up foreign genetic material. Artificial constructs that are designed to invade genomes may well invade our own. These insertions may lead to inappropriate inactivation or activation of genes (insertion mutagenesis), some of which may lead to cancer (insertion carcinogenesis [19]). The hazards of horizontal gene transfer are summarized in Box 3.

Box 3

Potential hazards of horizontal gene transfer from genetic engineering

  • Generation of new cross-species viruses that cause disease
  • Generation of new bacteria that cause diseases
  • Spreading drug and antibiotic resistance genes among the viral and bacterial pathogens, making infections untreatable
  • Random insertion into genomes of cells resulting in harmful effects including cancer
  • Reactivation of dormant viruses, present in all cells and genomes, which may cause diseases
  • Spreading new genes and gene constructs that have never existed
  • Multiplication of ecological impacts due to all of the above

Transgenic DNA may be more likely to transfer horizontally than non-transgenic DNA

Both the artificial vectors used in genetic engineering and the genes transferred to make trangenic organisms are predominantly from viruses and bacteria associated with diseases, and these are being brought together in combinations that have never existed in billions of years of evolution.

Genes are never transferred alone, but in unit-constructs, known as an ‘expression cassettes’. Each gene has to be accompanied by a special piece of genetic material, the promoter, which signals the cell to turn the gene on, ie, to transcribe the DNA gene sequence into RNA. At the end of the gene there has to be another signal, a terminator, to end the transcription and to mark the RNA, so it can be further processed and translated into protein. The simplest expression cassette looks like this:

expression cassette

Typically, each bit of the construct: promoter, gene and terminator, is from a different source. The gene itself may also be a composite of bits from different sources. Several expression cassettes are usually linked in series, or ‘stacked’ in the final construct. At least one of the expression cassettes will be that of an antibiotic resistance marker gene to enable cells that have taken up the foreign construct to be selected with antibiotics. The antibiotic resistance gene cassette will often remain in the transgenic organism.

The most commonly used promoters are from viruses associated with serious diseases. That’s because such viral promoters give continuous over-expression of genes placed under their control. The same basic construct is used in all applications of genetic engineering, whether in agriculture or in medicine, and the same hazards are involved. There are reasons to believe transgenic DNA is much more likely to spread horizontal than the organisms’ own DNA (see Box 4) [20]. Unfortunately, this hypothesis has yet to be put to direct empirical test.

Box 4

Transgenic DNA may be much more likely to spread horizontally

  • Artificial constructs and vectors are designed to invade genomes and to overcome species barriers.
  • All artificial constructs are structurally unstable [21], and hence prone to recombine and transfer horizontally.
  • The mechanisms that enable foreign gene-constructs to jump into the genome may enable them to jump out again, to re-insert at another site, or to another genome. Thus, the enzyme, integrase, which catalyzes the insertion of viral DNA into the host genome, also functions as a disintegrase catalyzing the reverse reaction. These integrases belong to a superfamily of similar enzymes present in all genomes from viruses and bacteria to higher plants and animals [22].
  • The integration sites of most commonly used artificial vectors for transferring genes are ‘recombination hotspots’, and may have an increased propensity to transfer horizontally.
  • Viral promoters, such as that from the cauliflower mosaic virus, widely used to make transgenes over-express, contain recombination hotpots, and may further enhance horizontal gene transfer [23].
  • The metabolic stress on the host organism due to the continuous over-expression of the foreign genes linked to aggressive viral promoters may also contribute to the instability of the transgenic DNA [24].
  • The transgenic DNA is typically a mosaic of DNA sequences from many different species and their genetic parasites; that means they have sequence homologies with the genetic material of many species and their genetic parasites, thus facilitating wide-ranging horizontal gene transfer and recombination [25].

Additional hazards from viral promoters

We have recently drawn attention to additional hazards associated with a promoter of the cauliflower mosaic virus (CaMV) most widely used in agriculture [26]. It is in practically all transgenic plants already commercialized or undergoing field trials, as well as a high proportion of transgenic plants under development, including the much acclaimed ‘golden rice’ [27].

CaMV is closely related to human hepatitis B virus, and less so, to retroviruses such as the AIDS virus [28]. Related viruses exchange genes much more readily than unrelated ones. Although the intact virus itself is infectious only for cruciferae plants, its promoter is promiscuous in function across Kingdoms and Domains. It is active in all higher plants, in algae, yeast, and E. coli [29], as well as frog and human cell systems [30].

All promoters of viruses and cellular genes have a modular structure, with common, interchangeable parts. The CaMV 35S promoter has been joined artificially to the cDNAs of a wide range of viral genomes, and infectious viruses were produced. Recombination between viral transgenes and infecting viruses has been already been demonstrated many times in the laboratory. In some cases, the recombinant viruses are more infectious than the original.

The CaMV 35S promoter has a recombination hotspot flanked by multiple motifs involved in recombination, similar to other recombination hotspots including the borders of the Agrobacterium T DNA vector most frequently used in making transgenic plants. The suspected mechanism of recombination requires little or no DNA sequence homologies.

Proviral sequences – generally inactive copies of viral genomes - are present in all plant and animal genomes. As all viral promoters are modular, and have at least one module – the TATA box - in common, if not more, it is not inconceivable that the 35S promoter in transgenic constructs can reactivate dormant viruses or generate new viruses by recombination. There is evidence that proviral sequence in the genome can be reactivated [31]. In fact, this is one of the main problems encountered in human gene therapy. Viral vectors have to be ‘packaged’ in human cell lines, and replicating viruses are often generated by the viral vector recombining with proviral sequences in the cells [32].

These considerations are especially relevant in the light of the report that certain transgenic potatoes - containing the CaMV 35S promoter and transformed with Agrobacterium T-DNA - may be unsafe for young rats and that a significant part of the effects may be to "the construct or the genetic transformation (or both) [33]". The authors also noted an increase in lymphocytes in the intestinal wall, which is a non-specific sign of viral infection [34].

Evidence for horizontal transfer of transgenic DNA

It is often argued that transgenic DNA, once incorporated into the transgenic organism, will be just as stable as the organism’s own DNA. But there is both direct and indirect evidence against this supposition. Transgenic DNA may be more likely to spread, and has been found to spread by horizontal gene transfer.

Transgenic lines are notoriously unstable and often do not breed true [35]. There is a paucity of molecular data documenting the structural stability of the transgenic DNA, both in terms of its site of insertion in the genome and the arrangement of genes, in successive generations. Instead, transgenes may be silenced in subsequent generations or lost altogether. [36]

A herbicide-tolerance gene, introduced into Arabidopsis by means of a vector, was found to be up to 30 times more likely to escape and spread than the same gene obtained by mutagenesis [37]. One way this may have happened is by means of horizontal gene transfer via insects visiting the plants for pollen and nectar [38]. The reported finding that pollen can transfer transgenic DNA to bacteria in the gut of bee larvae is relevant here.

Secondary horizontal transfer of transgenes and antibiotic resistant marker genes from genetically engineered crop-plants into soil bacteria and fungi have been documented in the laboratory. Transfer to fungi was achieved simply by co-cultivation [39], while transfer to bacteria has been achieved by both re-isolated transgenic DNA or total transgenic plant DNA [40]. Successful transfers of a kanamycin resistance marker gene to the soil bacterium Acinetobacter were obtained using total DNA extracted from homogenized plant leaf from a range of transgenic plants: Solanum tuberosum (potato), Nicotiana tabacum (tobacco), Beta vulgaris (sugar beet), Brassica napus (oil-seed rape) and Lycopersicon esculentum (tomato) [41]. It is estimated that about 2500 copies of the kanamycin resistance genes (from the same number of plant cells) is sufficient to successfully transform one bacterium, despite the fact that there is six million-fold excess of plant DNA present. A single plant with say, 2.5 trillion cells, would be sufficient to transform one billion bacteria.

Despite the misleading title in one of the publications [42], a high gene transfer frequency of 5.8 x 10-2 per recipient bacterium was demonstrated under optimum conditions. But the authors then proceeded to calculate an extremely low gene transfer frequency of 2.0 x 10-17 under extrapolated "natural conditions", assuming that different factors acted independently. The natural conditions, however, are largely unknown and unpredictable, and even by the authors’ own admission, synergistic effects cannot be ruled out. Free transgenic DNA is bound to be readily available in the rhizosphere around the plant roots, which is also an ‘environmental hotspot’ for gene transfer [43]. Other workers have found evidence of horizontal transfer of kanamycin resistance from transgenic DNA to Acinetobactor, and positive results were obtained using just 100ml of plant-leaf homogenate [44].

Defenders of the biotech industry then insist that just because horizontal gene transfer occurs in the laboratory does not mean it can occur in nature. However, there is already evidence suggesting it can occur in nature.

First of all, genetic material released from dead and live cells, is now found to persist in all environments; and not rapidly broken down as previously supposed. It sticks to clay, sand and humic acid particles and retains the ability to infect (transform) a range of micro-organisms in the soil [45]. The transformation of bacteria in the soil by DNA adsorbed to clay sand and humic acid has been confirmed in microcosm experiments [46].

Reseachers in Germany began a series of experiments in 1993 to monitor field releases of transgenic rizomania-resistant sugar beet (Beta vulgaris), containing the marker gene for kanamycin resistance. They looked for persistence of transgenic DNA and for horizontal gene transfer of transgenic DNA into soil bacteria [47]. It is the first such experiment to be carried out, after tens of thousands of field releases and tens of millions of hectares have been planted with transgenic crops. It will be useful to review their findings in detail.

Transgenic DNA was found to persist in the soil for up to two years after the transgenic crop was planted. Though they did not comment on it, the data showed that the proportion of kanamycin resistant bacteria in the soil increased significantly between 1.5 and 2 years. Could that be due to horizontal transfer of antibiotic resistance marker gene to soil bacteria?

A total of 4000 colonies of soil bacteria were isolated, a rather small number, and none was found to have taken up transgenic DNA by the probes available. However, two out of seven samples of total bacterial DNA yielded positive results after 18 months. This suggests that horizontal gene transfer may have taken place, but the specific bacteria that have taken up the transgenic DNA cannot be isolated as colonies. That is not surprising as less than 1% of all the bacteria in the soil can be cultured by current techniques. The authors were ‘careful’ not to rule out the possibility that transgenic DNA was merely adsorbed to the surface of bacteria rather than being actually transferred into the bacteria.

The researchers also carried out microcosm experiments to which total transgenic sugar-beet DNA was added to non-sterile soil with its natural complement of microorganisms. The intensity of the signal for transgenic DNA decreased during the first days and subsequently increased. The most likely interpretation of this observation is that the transgenic DNA has been taken up by bacteria and become replicated as the bacteria multiply.

In parallel, soil samples were plated, and the total bacterial lawn allowed to grow for 4 days. After that, DNA was extracted and probed for transgenic DNA. Several positive signals were found, "which might indicate uptake of transgenic DNA by competent bacteria." But again, they were unwilling to go further.

The authors were ‘cautious’ not to claim conclusive results, because the specific bacteria carrying the transgenic DNA sequences were not isolated from the field experiment. The results do show, however, that horizontal gene transfer may have taken place both in the field and in the soil microcosm. Such selective ‘cautious’ interpretation of positive data is all too typical, and you will see more of this later. It allows the pro-biotech establishment to continue in their denial that there is any evidence of horizontal gene transfer.

DNA is not broken down sufficiently rapidly in the gut either, which is why transfer of transgenic DNA to microorganisms in the gut of bee larvae would not be surprising. A genetically engineered plasmid was found to have a 6 to 25% survival after 60 min. of exposure to human saliva. The partially degraded plasmid DNA was capable of transforming Streptococcus gordonii, one of the bacteria that normally live in the human mouth and pharynx. The frequency of tranformation dropped exponentially with time of exposure to saliva, but it was still detectable after 10 minutes. Human saliva actually contains factors that promote competence of resident bacteria to become transformed by DNA [48].

Further research with transgenic maize DNA confirmed this finding [49]. The transgenic insect-resistant maize line CG00526-176 contains three bacterial genes: the cry1A(b) specific to lepidopterans, the bar gene conferring tolerance to glufosinate, and a bla gene encoding TEM-1 b-lactamase (ampicillin resistance). The bla gene originates from the cloning vector PUC18 and is not expressed in maize, but has bacterial regulatory sequences that would allow it to become functional were it to be transferred back into bacteria. There are at least two copies of crylA(b) and bla genes integrated into the DNA of the maize line.

The researchers investigated the survival of DNA from the transgenic maize and the transfer of the antibiotic resistance bla gene to bacteria in the presence of saliva, rumen fluid and silage effluent, which are relevant to horizontal gene transfer in the oral cavity, the rumen, and in silage.

E. coli was the test micro-organism for horizontal gene transfer. Degradation of DNA was followed by gel electrophoresis as well as by polymerase chain reaction (PCR). Both pUC18 plasmid and transgenic maize DNA were used in the experiments.

On gel electrophoresis, plasmid DNA and maize DNA were shown to be degraded rapidly by rumen fluid or silage effluent within one minute, but both were incompletely degraded after at least l h exposure to saliva.

On PCR, a much more sensitive analysis, large fragments of the bla gene (>350bp) were still found in rumen fluid up to 30 mins for the plasmid and up to1 min for maize DNA. Even larger fragments (>350 and >684 bp) from plasmid and maize DNA were found up to 30 min incubation in silage effluent, and up to 24h and 2 h respectively in saliva.

PCR analysis also showed that fragments of the cryl1A(b) (>1914bp) in maize DNA could be found up to 1 min with rumen fluid, 5 min with silage effluent, and 60 min with saliva.

Plasmid DNA exposed to saliva for 24h was still capable of transforming E. coli to ampicillin resistance, but at low efficiency: 20 cfu (colony forming units) per millilitre compared with 1.6 x103 cfu per millilitre after 24h in sterile water. Previous exposure to rumen fluid for 30s reduced transformation 5-fold. No transformants were obtained after the plasmid DNA was exposed to silage effluent or rumen fluid for longer than 1 min.

However, when E. coli and plasmid were simultaneously added to filter-sterilized silage effluent or rumen fluid, 4.75x103 cfu per ml transformants were recovered after 4.5h in rumen fluid and 11cfu per ml were recovered after 3h in silage effluent.

Thus, horizontal gene transfer can occur before the DNA is completely broken down, even when the breakdown is rapid, as in the rumen or in silage. DNA breakdown is extremely slow in saliva, and hence the oral cavity may be a very important site for horizontal gene transfer.

Those who dismiss horizontal gene transfer in GM safety often retreat to the tired old argument that it happens only between similar or homologous gene sequences. Indeed, it has been found that homology increases the frequency of horizontal gene transfer 10 million to 100 million-fold [50]. The experiments were done with DNA from transgenic potato plants carrying the nptII gene for kanamycin resistance. It transformed naturally competent cells of the soil bacteria Pseudomonas stutzeri and Acinetobacter BD413, both harboring a plasmid with an nptII gene containing a small deletion with the same high efficiency as nptII genes on plasmid DNA (3x10-5 -1x10-4), despite the presence of more than 106 fold excess plant DNA.

However, in the absence of homologous sequences in the recipient cells, the transformation dropped by at least about 108 fold in P. stutzeri and 109 fold in Acinetobacter, below the detection limit.

More than 60 bacterial species have been shown to take up and incorporate DNA (undergo transformation). Many bacteria like Bacillus subtilis and Acinetobacter sp. strain BD413, apparently take up DNA of any source into the cytoplasm. Stable maintenance and expression depends on integration into the genome by genetic recombination.

The authors state, "This indicates a very low probability of non-homologous DNA fragments to be integrated by illegitimate recombination events during transformation". Should we be reassured? Not at all.

The high frequencies of homologous recombination obtained are relevant to GM constructs released in large concentrations into the environment in GM crops and transgenic wastes. As pointed out earlier, GM constructs are highly chimerical, and contain homologies to many different species of bacteria and viruses, and hence capable of engaging in high frequencies of recombination with a wide variety of bacteria and viruses. A prominent molecular geneticist and researcher on horizontal gene transfer has recently said the same [51].

"Transgenes often contain DNA sequence homology to prokaryotes thereby increasing their likelihood of integration in bacteria significantly. Many studies have shown that DNA homology is the main barrier to HGT of chromosomal DNA (such as transgenes) in bacteria."

Thus, transgenic DNA is, in this respect, alone, optimized for horizontal gene transfer and recombination.

Still, ‘illegitimate’ recombination events should not be ignored. They may occur at lower frequencies, but will become substantial as GM constructs are released on massive scales. In particular, recombination hotspots associated with many GM constructs (such as those containing CaMV 35S promoter) may increase the frequency of illegitimate recombination.

The rhizosphere - surfaces around the plant roots - and the spermosphere - surfaces around the germinated seeds – are recognized hotspots for horizontal gene transfer between bacteria. But the frequency also depends on the location of the genes. It matters whether they are in the bacterial chromosome, or in a mobilizable plasmid, ie, a plasmid that can be transferred with helper functions supplied by other plasmids, or in a conjugative plasmid, ie, a plasmid that has its own functions for transfer during conjugation. Not surprisingly, researchers found the highest frequencies of horizontal gene transfer in both the rhizosphere and spermosphere when the GM cassette was in a conjugative plasmid, somewhat lower when it was in a mobilizable plasmid, but could not be detected when it was inserted into the bacterial chromosome [52].

However, that does not mean GM constructs located on bacterial chromosomes do not transfer. The authors were careful to point out that the main mode of horizontal gene transfer in both the rhizosphere and the spermosphere is conjugation, a process that require cell to cell contact between bacteria. Elsewhere, transformation (by direct uptake of DNA) will be more important, and there is evidence that chromosomal constructs are more efficient in transformation.

The authors warn: "On the basis of these experiments, we cannot rule out the possibility that horizontal gene transfer by transformation occurs at low frequencies in soil and that this process might have significant effect at field scale, which is an especially important point as regards risk assessment. Such rare events cannot be studied in microcosm experiments, but must be addressed in retrospective field studies."

The only retrospective study carried out has indeed found evidence of horizontal gene transfer from transgenic plants to soil bacteria (see above). An investigation yet to be done is horizontal transfer from GM plants to bacteria in the rhizosphere.

This is extremely relevant, due to increasing evidence that gene transfer from bacteria to eukaryotic cells (plant and animals included) may occur via conjugation [53], and that the transfer of T-DNA from Agrobacterium into plant cells has many characteristics of conjugation. Furthermore, retrotransfer of genetic material from plant cell to Agrobacterium can also take place during the process [54]. In view of the fact that Agrobacterium T DNA is the common vector for plant transformation, it will not be surprising if the bacterium may also be a main route for horizontal transgene escape.

The possibility of horizontal gene transfer from transgenic food to mammalian cells also cannot be dismissed. Viral DNA fed to mice is found to reach white blood cells, spleen and liver cells via the intestinal wall, to become incorporated into the mouse cell genome [55]. When fed to pregnant mice, the viral DNA ends up in cells of the fetuses and the new born animals, indicating that it has gone through the placenta as well [56]. The authors remark that "The consequences of foreign DNA uptake for mutagenesis [mutations] and oncogenesis [cancer] have not yet been investigated [57]." As already mentioned, recent experiments in gene therapy leave little doubt that naked nucleic acid constructs can readily enter mammalian cells and in many cases become incorporated into the cell’s genome.

In a recent experiment [58], the same team compared the fate of soybean DNA from soybean leaves, with transgenic plasmid DNA containing green fluorescent protein linked to viral promoters from the human cytomegalovirus (HCMV), the Simian virus (SV40) or Rous Sarcoma virus (RSV). Soybean DNA was tracked by probing for the presence of the plant-specific, nucleus-encoded ribulose-1,5-biphosphate carboxylase (Rubisco) gene.

They found that the plant gene, or fragments of it, can be recovered in the intestine from 2h up to 49 h after feeding, and in the caecum, for up to 121 h after ingestion. Thus, plant-associated naturally fed DNA is much more stable in the intestinal tract than naked DNA. In a total of 37 soybean leaf-fed mice, the DNA from liver and spleen were investigated for the presence of Rubisco DNA. The DNA was found in three organs in two animals.

In experiments where the plasmid containing transgenic GFP- DNA were fed to mice, however, a much higher proportion of the DNA were found in liver, spleen, kidney, blood, and in the intestinal wall, just 3 to 8 hr. afterwards. The numbers varied from a low of 3 out of 16 to 5 out of eight, the total over 8 experiments was 28 out of 87.

The researcher noted that far greater amounts of transgenic DNA were fed, typically 50 micrograms, equivalent to 1013 copies compared with about 6x108 copies of the Rubisco gene. But then, according to their findings, plant-associated DNA was much more stable and resistant to breakdown, remaining in the gut far longer.

When injected, into muscle, the green fluorescent protein DNA fragments can be amplified by PCR for up to 17 months afterwards, and from DNA from organs remote from the site of injection up to 24h after injection, and from intestinal contents 6h after injection. This indicates that transgenic DNA can circulate around the body.

Why didn’t they do the obvious experiment, which is to feed transgenic plant material to the mice and probe for both a plant gene and the transgene? Perhaps no biotech company will provide the transgenic plant material for such experiments. In fact, this obvious experiment appears to have been avoided altogether, so the denial can continue. Absence of evidence is evidence of absence.

Conclusion

Horizontal gene transfer is an established phenomenon. It has taken place in our evolutionary past and is continuing today. The signs are that natural horizontal gene transfer is a regulated process, limited by species barriers and by mechanisms that break down and inactivate foreign genetic material. Unfortunately, genetic engineering has created a huge variety of artificial constructs designed to cross all species barriers and to invade essentially all genomes. Although the basic constructs are the same for all applications, some of the most dangerous may be coming from the waste disposal of contained users of transgenic organisms [59]. These will include constructs containing cancer genes from viruses and cells from laboratories researching and developing cancer and cancer drugs, virulence genes from bacteria and viruses in pathology labs. In short, the biosphere is being exposed to all kinds of novel constructs and gene combinations that did not previously exist in nature, and may never have come into being but for genetic engineering.

There is an urgent need to establish effective regulatory oversight, in the first instance, to prevent the escape and release of these dangerous constructs into the environment, and then to consider whether some of the most dangerous experiments should be allowed to continue at all.

Article first published 01/01/01


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