Report on horizontal gene transfer - Department of Public Prosecution versus Gavin Harte and others, New Ross, Ireland

Mae-Wan Ho, March 22, 1999

Personal qualifications

Mae-Wan Ho, Reader in Biology at the Open University, B. Sc. (First Class) 1964, and Ph. D. 1967, H K University; more than 30 years in research and 25 years teaching experience; nearly 200 publications covering human biochemical genetics, molecular genetics, evolution, developmental biology, and biophysics. Awards include, Chan Kai Ming Prize for Biological Sciences (HK) 1964: Fellow of the National Genetics Foundation (USA) 1971-1974; Vida Sana Award (Spain) 1998; Guest of Honour in Women of the Year Luncheon & Assembly (UK) 1998. From 1994, Scientific Advisor to the Third World Network and other public interest organizations on biotechnology and biosafety. Debated issues in the United Nations, the World Bank, the European Parliament, in the UK, USA and many other countries all over the world. Author of many papers and reports for the public and for policy-makers, frequent broadcaster and public lecturer. Recent publications relevant to genetic engineering:
Genetic Engineering Dream or Nightmare? Mae-Wan Ho, Gateway Books, Bath, UK, 1998; (revised, 2nd. edition, 1999).
Gene Ecology and Gene Technology of Infectious Diseases, Mae-Wan Ho et al, Microbial Ecology in Health and Disease 10, 33-59, 1998.
Genetic Engineering and Infectious Diseases, Mae-Wan Ho et al, Third World Network, Penang, 1998.
Fatal Flaw in Food Safety Assessment: The FAO/WHO Joint Biotechnology & Food Safety Report, Mae-Wan Ho and Ricarda A. Steinbrecher, Third World Network, Penang, 1998.
Fatal Flaw in Food Safety Assessment: The FAO/WHO Joint Biotechnology & Food Safety Report, Mae-Wan Ho and Ricarda A. Steinbrecher, Environmental and Nutritional Interactions 2, 51-84.

1. Horizontal gene transfer and genetic engineering (1)

1.1 Horizontal gene transfer refers to the transfer of genes or genetic material directly from one individual to another by processes similar to infection. It is distinct from the normal process of vertical gene transfer - from parents to offspring - which occurs in reproduction. Genetic engineering bypasses reproduction altogether by exploiting horizontal gene transfer, so genes can be transferred between distant species that would never interbreed in nature. For example, human genes are transferred into pig, sheep, fish and bacteria. Toad genes are transferred into potatoes. Completely new, exotic genes, can therefore be introduced into food crops.

1.2 Natural agents exist which can transfer genes horizontally between individuals. These are viruses, many of which cause diseases, and other pieces of parasitic genetic material, called plasmids and transposons, many of which carry and spread antibiotic and drug resistance genes. These are able to get into cells and then make use of the cell's resources to multiply many copies or to jump into (as well as out of ) the cell's genome. The natural agents are limited by species barriers, so that for example, pig viruses will infect pigs, but not human beings, and cauliflower viruses will not attack tomatoes. However, genetic engineers make artificial vectors (carriers of genes) by combining parts of the most infectious natural agents, with their disease-causing functions removed or disabled, and design them to overcome species barriers, so the same vector may now transfer, say, human genes, which are spliced into the vector, into the cells of all other mammals, or cells of plants.

1.3 Typically, foreign genes are introduced with strong genetic signals - called promoters or enhancers - to boost the expression of the genes to well above the normal level that most of the cell's own genes are expressed. The most commonly used promoters are from plant viruses which are related to animal viruses (see below). There will also be selectable "marker genes" introduced along with the gene(s) of interest, so that those cells that have successfully integrated the foreign genes into their genome can be selected. The most commonly used marker genes are antibiotic resistant genes originally isolated from bacterial plasmids and transposons, which enable the cells to be selected with antibiotics. These marker genes often remain in the genetically engineered organisms.

1.4 One viral promoter which is in practically all transgenic plants is from the cauliflower mosaic virus (CaMV), which is closely related to human hepatitis B virus, and less so, to retroviruses such as the AIDS virus. The CaMV promoter can drive the synthesis of these other viruses;(2) it is active in most plants, in yeast, insects (3) and E. coli.(4) As all genomes contain dormant viruses, there is a potential for the CaMV promoter to reactivate them. Its strong promoter activity causes introduced genes to be overexpressed, and may also have effects on host genes far away from the site of foreign gene insertion. The promoter from another virus - the figwort mosaic virus, is similar to CaMV in many respects, and therefore equally hazardous. Recombination between the figwort and CaMV promoters in the same plant is bound to take place with untoward consequences for the crop plant, and also in creating new, broad host range viruses.

1.5 The insertion of foreign genes into the host genome is not under the control of the genetic engineer. It is entirely random, and gives rise to correspondingly random, unintended effect, including toxins and allergens in food plants, and cancer in mammalian cells.

1.6 Many of the dangers of genetic engineering are inherent to the technology, in its ability to increase the potential for horizontal gene transfer across unrelated species. Secondary, unintended horizontal gene transfer can take place from the genetically engineered crops released into the environment. The very cellular mechanisms that enable the foreign genes to insert into the genome can also mobilize them to jump out again. For example, the enzyme integrase, which catalyzes the integration of viral DNA into the host genome, also functions as a disintegrase, catalyzing the reverse reaction. These integrases are very widespread, belonging to a superfamily of similar enzymes present in all genomes, from viruses and bacteria to higher organisms.(5) The foreign genes can then reinsert into another site in the genome, or else spread, uncontrollably to other organisms.
 

2. Hazards of horizontal gene transfer from transgenic crops released into the environment

2.1 The major hazards from secondary horizontal gene transfer are as follows.
Generation of new viral and bacterial pathogens from the viral and bacterial genes introduced into the transgenic plants,
Spread of drug and antibiotic resistance marker genes among pathogens,
Random, secondary insertion of genes into cells of organisms interacting with the crop plants, with harmful effects including cancer,
Reactivation of dormant viruses that cause diseases,
Ecological impacts due to the spread of the specific exotic genes introduced.

2.2 There has been an accelerated resurgence of drug and antibiotic resistance diseases over the past 20 years, coinciding with the development of genetic engineering biotechnology on commercial scales. A group of scientists including myself have produced a report (6) on the circumstantial, deductive and inductive evidence linking genetic engineering biotechnology to the recent resurgence of infectious diseases and demanding an independent public enquiry. There is overwhelming evidence that horizontal gene transfer (and subsequent recombination) has been responsible for creating new viral and bacterial pathogens and spreading drug and antibiotic resistance genes. Many of the horizontal gene transfer events have occurred very recently.

2.3 There is as yet no direct evidence that latent viruses can be reactivated in transgenic plants, if only because the possibility has not been investigated. However, plants engineered with genes from viruses to resist virus attack actually show increased propensity to generate new, often super-infectious viruses by horizontal gene transfer and recombination with infecting viruses. (7) This suggests that other viral genes engineered into transgenic plants may also take part in horizontal gene transfer and recombination to generate new viruses.(8)

2.4 There is evidence that foreign genes introduced into plants behave differently from the plants own genes. These foreign genes may be up to 30 times more likely to escape and spread than the plants own genes.(9) One way this could happen is by secondary horizontal gene transfer via insects visiting the plants for pollen and nectar.

2.5 Secondary horizontal tranfer of transgenes and antibiotic resistant marker genes from genetically engineered crop plants into soil bacteria and fungi have been documented in the laboratory.* Successful transfers of marker genes to the soil bacterium Acinetobacter were obtained using DNA extracted from homogenized plant leaf from a range of transgenic plants: Solanum tuberosum (potato), Nicotiana tabacum (tobacco), Beta vulgaris (sugar beet), Brassica napus and Lycopersicon esculentum.(10) Despite the misleading title in one of the publications,(11) a high "optimal" gene transfer frequency of 6.2 x 10-2 was found in the laboratory from transgenic potato to a bacterial pathogen. The authors then proceeded to 'calculate' a frequency of 2.0 x 10-17 under extrapolated "natural conditions". The natural conditions, are of course, largely unknown. There is no ground for assuming that such horizontal gene transfer will not take place under natural conditions. On the contrary, other recent findings suggest that genes can transfer horizontally from transgenic plants to organisms in the soil, and in the gut and other parts of organisms feeding on the transgenic plants.

2.6 There is now abundant evidence that the genetic material, DNA, released from dead and live cells, are not readily broken down as previously supposed, but are rapidly adsorbed onto clay, sand and humic acid particles where they retain the ability to infect (transform) a range of organisms in the soil. (12) That means transgenes and marker genes will be able to spread to bacteria and viruses with the potential of creating new pathogens and spreading antibiotic resistance genes among the pathogens.

2.7 There is also recent evidence that DNA is not broken down rapidly in the gut as previously supposed.(13)That means genes can spread from ingested transgenic plant material to bacteria in the gut and also to cells of the organism ingesting the material.

2.8 Horizontal gene transfer between bacteria in the human gut has been demonstrated since the 1970s and similar transfers in the gut of chicken and mice in the early 1990s.(14) This is confirmed in new research showing that antibiotic resistant marker genes from genetically engineered bacteria can be transferred to indigenous bacteria at a substantial frequency of 10-7 in an artificial gut. (15)

2.9 Mammalian cells are known to take up foreign DNA by many mechanisms. Studies since the 1970s have documented the ability of bacterial plasmids carrying a mammalian SV40 virus to infect cultured cells which then proceeded to make the virus. Similarly, bacterial viruses and baculovirus (of insects) can also be taken up by mammalian cells. Baculovirus is so good at gaining access that it is being engineered as a vector for human gene therapy, at the same time that it is being engineered to control insect pests in agriculture.(16)

2.10 Viral and plasmid DNA fed to mice has been found to resist digestion in the gut. Large fragments passed into the bloodstream and into white blood cells, spleen and liver cells. In some instances, the viral DNA was found attached to mouse DNA and E. coli DNA, suggesting that it has integrated into the mouse cell genome and the bacterial cell genome respectively.(17) When fed to pregnant mice, large fragments of the DNA are found in the nucleus of cells of the foetus and the newborn.(18)

2.11 Viral DNA is now known to be more infectious than the intact virus, which has a protein coat wrapped around the DNA. For example, intact human polyoma virus injected into rabbits had no effect, whereas, injection of the naked viral DNA gave a full-blown infection.(19) Viral DNA is in practically all transgenic plants especially in the form of viral promoters, which, if integrated into mammalian cells may reactivate dormant viruses or cause cancer.

2.13 Recent revelations on the results of feeding experiments performed by Dr. Arpad Puztais group in the Rowett Institute suggests that transgenic potatoes are toxic to rats, and that most of the toxicity is due to the genetic engineering process,(20) which is common to practically all transgenic plants. This same kind of transgenic potatoes was earlier shown to harm ladybirds fed on aphids that have eaten the transgenic potatoes.(21) This should raise serious concerns on the continued release of transgenic plants into the environment, whether as field trials or as commercially grown crops.

3. Specific hazards from the transgenic sugar beet released in the field-trial

3.1 I understand that genes from three bacteria and two viral promoters (from CaMV and figwort mosaic virus) known to be potentially hazardous, have been introduced into the sugar beet at the trial site in Arthurstown, New Ross.

3.2 As the insertion of these genes is a random process, and as the introduced genes will interact with host genes, there are bound to be unpredictable, unintended effects on the physiology and biochemistry of the sugar beet plant. These effects can spread very far away from the site of insertion.(22)The ecological impacts on other organisms (including insects, birds and small mammals) interacting with the transgenic sugar beet plants are largely unknown and even more unpredictable. The recent revelation that much of the toxicity of transgenic foods may be inherent to the genetic engineering technology itself suggests that the ecological impacts of the transgenic sugar beet could be devastating.

3.3 The viral promoters may reactivate dormant viruses in the transgenic sugar beet plants or create new viruses by horizontal gene transfer and recombination. These viruses could then be spread by insects and pollinators to many other plants.

3.4 Horizontal transfer of genes to bacteria and fungi in the soil may occur when the transgenic sugar beet plant material decomposes, or simply from exudates from the plant-roots. Transfer may occur to pollinators and other insects feeding on the plant, and via the insects, the genes may be transferred to unrelated species of higher plants. Transfer may also occur to gut bacteria or cells of small mammals feeding on the plants. These transfers may reactivate dormant viruses or create new viruses in all the species concerned, as the CaMV and figwort mosaic viral promoter are both active in all the species. In addition, each of these transfers will have its own ecological consequences.

3.5 Horizontal gene transfer to soil fungal and bacterial pathogens will render them resistant to Roundup (Roundup resistance being due to two bacterial genes introduced, CP4 EPSPS and gox). This can lead to an ecological disaster as it is already known that beneficial organisms such as earthworms and mycorrhizal fungi and other microorganisms involved in nutrient recycling in the soil are susceptible to glyphosate (the active ingredient in Roundup herbicide). Glyphosate is so generally toxic that it is being considered for use as an antimicrobial.(23)

3.6 Horizontal transfer of Roundup resistance genes to other species of higher plants will create weeds and superweeds resistant to Roundup.

3.7 Horizontal transfer of Roundup resistance genes to gut bacteria will create a reservoir of glyphosate resistant bacteria which may pass the genes onto pathogenic bacteria.

3.8 Horizontal gene transfer to insects, bird, and small mammals will cause many harmful effects including cancer, resulting in a loss of biodiversity.
 

1. See Ho, M.W. (1998, 1999). Genetic Engineering Dream or Nightmare? The Brave New World of Bad Science and Big Business, Gateway Books, Bath; Ho, M.W., Traavik, T., Olsvik, R., Tappeser, B., Howard, V., von Weizsacker, C. and McGavin, G. (1998a). Gene Technology and Gene Ecology of Infectious Diseases. Microbial Ecology in Health and Disease 10, 33-59; also Ho, M.W., Meyer, H. and Cummins, J. (1998b). The biotechnology bubble. The Ecologist 28(3), 146-153, and references therein.
2. Xiong, Y. and Eikbush, T. (1990). Origin and evolution of retroelements based upon the reverse transriptase sequences. The Embo Journal 9, 3363-72; see also Cummins, J. (1998). A virus promoter used in the majority of genetically engineerd crops. Available from the author at jcummins@julian.uwo.ca,
3. Smerdon, G., Aves, S. and Walton, E. (1995). Production of human gastric lipase in the fission yeast. Gene 165, 313-8; Vlack, J., Scoulten, A., Usmany, M., Belsham, G., KlingeRoode, E., Maule, A., Vanlent, M. and Zuideman, D. (1990). Expression of Cauliflower Mosaic Virus Gene I. Virology 179, 312-20.
4. Assad, F.F. and Signer, E.R. (1990). Cauliflower mosaic virus P35S promoter activity in E. coli. Mol. Gen. Genet. 223, 517-20.
5. Asant-Appiah, E. and Skalka, A.M. (1997). Molecular mechanisms in retrovirus DNA integration. Antiviral Research 36, 139-56.
6. Ho et al, 1998a (see note 1).
7. Vaden V.S. and Melcher, U. (1990). Recombination sites in cauliflower mosaic virus DNAs: implications for mechanisms of recombination. Virology 177, 717-26; Lommel, S.A. and Xiong, Z. (1991). Recombination of a functional red clover necrotic mosaic virus by recombination rescue of the cell-to-cell movement gene expressed in a transgenic plant. J. Cell Biochem. 15A, 151; Greene, A.E. and Allison, R.F. (1994). Recombination between viral RNA and transgenic plant transcripts. Science 263, 1423-5; Wintermantel, W.M. and Schoelz, J.E. (1996). Isolation of recombinant viruses between cauliflower mosaic virus and a viral gene in transgenic plants under conditions of moderate selection pressure. Virology 223, 156-64. See also Woolf, M. (1999). Super-viruses threat to farms. The Independent on Sunday, 21 March.
8. This possibility has been suggested by Cummins since 1994 (see Cummins, 1998, note 2).
9. Bergelson, J., Purrington,c.B. and Wichmann, G. (1998). Promiscuity in transgenic plants. Nature 395, 25.
10. Hoffman, T., Golz, C. & Schieder, O. (1994). Foreign DNA sequences are received by a wild-type strain of Aspergillus niger after co-culture with transgenic higher plants. Current Genetics 27: 70-76; Schluter, K., Futterer, J. & 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;
    Gebhard, F. and Smalla, K. (1998). Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA. Appl. Environ. Microbiol. 64, 1550-4.
11. De Vries, J. and Wackernagel, W. (1998). Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation. Mol. Gen. Genet. 257, 606-13; see also Gebbard and Smalla, 1998 (note 9).
12. Schlutter et al, 1995 ( note 9).
13. Reviewed by Lorenz, M.G. and Wackernagel, W. (1994). Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58, 563-602.
14. See Ho et al, 1998a (note 1) and references therein.
15. See Ho et al, 1998a (note 1) and references therein.
16. See MacKenzie, D. (1999). Gut reaction. New Scientist, 30 Jan., p.4.
17. See Ho etal, 1998a (note 1).
18. Schubbert, R., Lettmann, C. & 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; Schubbert, R., Renz, D., Schmitz, B. and Doerfler, W. (1997). Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc. Natl. Acad. Sci. USA 94, 961-6.
19. Doerfler, W., Schubbert, R., Heller, H., Hertz, J., Remus, R., Schrier. J., Kämmer, C., Hilger-Eversheim, K., Gerhardt, U., Schmitz, B., Renz, D., Schell, G. (1998)APMIS Suppl. 84, 62-8.
20. See Traavik, T. (1995). Too Early May Be Too Late. Ecological Risks Associated with the Use of Naked DNA as a Biological Tool for Research, Production and Therapy (Norwegian), Report for the Directorate for Nature Research Tungasletta 2, 7005 Trondheim. English translation, 1999; Ho et al, 1998 (see note1); also Ho, 1999 Chapter 10 ( note 1).
21. Leake, C. and Fraser, L. (1999). Scientst in Frankenstein food alert is proved right. UK Mail on Sunday, 31 Jan. ; Goodwin, B.C. (1999). Report on SOAEFD Flexible Fund Project RO818, Jan. 23, 1999.
22. Birch, A.N.E., Geoghegan, I.I., Majerus, M.E.N., Hackett, C. and Allen, J. (1997). Interaction between plant resistance genes, pest aphid-population and beneficial aphid predators. Soft Fruit and Pernial Crops. October, 68-79.
23. Doerfler, W., Schubbert, R., Heller, H. (1997). Integration of foreign DNA and its consequences in mammalian systems. Tibtech 15, 297-301.

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