Unregulated Hazards ‘Naked’ and ‘Free’ Nucleic Acids

ISIS Report -Produced for the Third World Network

Mae-Wan Ho, Angela Ryan Biology Department, Open University, Walton Hall, Milton Keynes MK7 6AA, UK
J.Cummins Department of Plant Sciences, University of Western Ontario Ontario, Canada
T. Traavik Dept. of Virology, Institute of Medical Biology,MH-Breivika And Norwegian Institute of Gene Ecology, N-9037 Tromso, Norway

Executive Summary

A huge variety of naked/free nucleic acids are being produced in the laboratory and released unregulated into the environment. They are used as research tools, in industrial productions and in medical applications such as gene therapy and vaccines. These nucleic acids range from oligonucleotides consisting of less than 20 nucleotides to artificial constructs thousands or millions of basepairs in length, typically containing a heterogeneous collection of genes from pathogenic bacteria, viruses and other genetic parasites belonging to practically every kingdom of living organisms. Most of the nucleic acids and constructs have either never existed in nature, or if they have, not in such large amounts. They are, by definition, xenobiotics – substances foreign to nature - with the potential to cause harm. Some, such as gene therapy vectors and vaccines, have already been shown to elicit toxic and other harmful reactions in preclinical trials.

Nucleic acids are now known to persist in all environments, including the digestive system of animals. Transformation by the uptake of DNA is recognized to be a significant route of horizontal gene transfer among bacteria, and there is overwhelming evidence that horizontal gene transfer and recombination have been responsible for the recent resurgence of drug and antibiotic resistant infectious diseases.

Recent investigations associated with gene therapy and vaccines leave little doubt that naked and free nucleic acids are readily taken up by the cells of all species including human beings, and may become integrated into the cell’s genetic material. There is also abundant evidence that the extraneous nucleic acids taken up can have significant and harmful biological effects including cancers in mammals.

The need to establish regulatory oversight of naked/free nucleic acids at both national and international levels is long overdue. It is irresponsible to continue to exclude them from the scope of the International Biosafety Protocol.

‘Naked’ and ‘free’ nucleic acids

‘Naked’ nucleic acids are DNA/RNA produced in the laboratory and intended for use in, or as the result of genetic engineering (1).‘Free’ nucleic acids refer to the laboratory-produced nucleic acids transfected into cells or organisms, whether incorporated as transgenic DNA or not, and subsequently released into the environment by secretion, excretion, waste disposal, death, industrial processing, or carried by liquid streams, or in airborne dust and pollen.

A huge variety of naked nucleic acids are being produced in the laboratory (see Box 1), which are used as research tools, in industrial productions, and in medical applications such as gene therapy and vaccines. They range from oligonucleotides consisting of less than 20 nucleotides to artificial constructs thousands of basepairs in length, and artificial chromosomes millions of basepairs long. The constructs typically contain antibiotic resistance marker genes plus a heterogeneous array of genes from pathogenic bacteria, viruses and other genetic parasites belonging to practically every kingdom of living organisms on earth (2).Most of the naked nucleic acids and constructs have either never existed in nature, or if they have, not in such large amounts. They are, by definition, xenobiotics – substances foreign to nature -(3) with the potential to cause harm.

There is no regulation governing the release of naked nucleic acids into the environment. Many novel constructs are incorporated into transgenic micro-organisms and animal cell cultures for commercial drug production, and into crops, livestock, fish and other aquatic organisms for food, animal feed, and other purposes. These constructs are therefore greatly amplified, and at the same time introduced into foreign genomes where recombination with host genes and the genes of the host’s viral pathogens may readily occur. Transgenic wastes containing large amounts of free or potentially free transgenic DNA are being released unregulated into the environment, including those from microorganisms and cell cultures supposed to be in ‘contained use’ (see Box 2)(4).Under the current EU Directive for Contained Use, contained users are allowed to release certain live transgenic microorganisms in liquid waste, and all killed microorganisms and cells containing transgenic DNA as solid waste.

The lack of regulation of naked/free nucleic acids is based largely on the assumption, now proven to be erroneous, that naked/free nucleic acids would be rapidly broken down in the environment and in the digestive system of animals(12). Another assumption is that as DNA is present in all organisms, it is not a hazardous chemical, and hence there is no need to regulate it as such(13).

DNA persists in all environments and is readily taken up by cells of all organisms

Naked or free DNA are now known to persist in all natural environments, and high concentrations are found in the soil, in marine and fresh water sediments as well as in the air-water interface, where it retains the ability to transform microorganisms(14). DNA also persists in the mouth(15) and the digestive tract of mammals(16), where it may be taken up and incorporated by the resident microbes, and by the cells of the mammalian host.

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. Human saliva contains factors that promote competence of resident bacteria to become transformed by DNA(17).

It has long been assumed that DNA cannot be taken up through intact skin, surface wounds, or the intestinal tract, or that it would be rapidly destroyed if taken up. Those assumptions have been overtaken by empirical findings. The ability of naked DNA to penetrate intact skin has been known at least since 1990. Cancer researchers found that within weeks of applying the cloned DNA of a human oncogene to the skin on the back of mice, tumours developed in endothelial cells lining the blood vessel and lymph nodes(18).

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(19). When fed to pregnant mice, the viral DNA ends up in cells of the fetuses and the new born animals, suggesting that it has gone through the placenta as well(20). The authors remark that "The consequences of foreign DNA uptake for mutagenesis and oncogenesis have not yet been investigated."(21)

Recent developments in gene therapy demonstrate how readily naked nucleic acids (see Table 2) can gain access to practically every type of human cells and cells of model mammals. Naked nucleic acids can be successfully delivered, either alone or in complex with liposomes and other carriers, in aerosols via the respiratory tract(22), by topical application to the eye(23), to the inner ear(24), via hair follicles(25), direct injection into muscle(26), through the skin(27), as well as by mouth, where the nucleic acid is taken up by cells lining the gut(28). Naked DNA can even be taken up by sperm cells of marine organisms and mammals, and transgenic animals created(29). Geneticists are contemplating using sperms as vectors in gene therapy.

High levels of foreign gene expression was observed in the liver cells of rats, mice and dogs when naked DNA was injected into blood vessels supplying the liver(30). Gene expression is observed in skin cells injected with naked DNA(31), and naked DNA was integrated into chromosomes of cells and expressed in human and pig skin(32). Researchers have found integration of a plasmid-based naked DNA malarial vaccine injected into mouse muscle in a preclinical trial, but dismissed it as "3000 times less than the spontaneous mutation rate for mammalian genome" and hence "not considered to pose a significant safety concern"(33).

Hazards of naked nucleic acids

One of the key findings is that naked viral DNA is more infectious and have a wider host range than the intact virus. Human T-cell leukaemia viral DNA formed complete viruses when injected into the bloodstream of rabbits(34). Similarly, naked DNA from the human polyomavirus BK (BKV) gave a full-blown infection when injected into rabbits, despite the fact that the intact BKV virus is not infectious(35). This hazard is particularly relevant to the entire range of virus-based gene therapy vectors and naked DNA vaccines under development(36). Modifications to viral genomes can have unexpected effects on virulence and the host range(37)

The safety of gene therapy vectors is unproven(38). The hazards include both direct toxicities and indirect effects (see Box 3) and there is a growing debate over the potential for generating infectious viruses, and harmful effects due to random insertion into the cellular genome(39), both of which are shared by naked DNA vaccines. Recombinant DNA vaccines, in both the naked and intact viral form, also tend to be more unstable and prone to recombination, increasing the likelihood of generating new viruses(40).A viral vaccine made by deleting genes from the simian immuno-deficiency virus (SIV) was found to cause AIDS in infant and adult macaques(41), raising serious safety concerns over similar AIDS viral vaccines for humans.

Gene therapy vectors and naked DNA vaccines can cause acute toxic shock reactions (42) and severe delayed immunological reactions(43). Between 1998 and 1999, scientists from US drug companies failed to notify the FDA about six deaths that had occurred during clinical trials of gene therapy, the causes of which are yet to be determined(44). Naked DNA can also trigger autoimmune reactions, in which the body’s immune system attack and kill its own tissues and cells. New research shows that any fragment of double-stranded DNA or RNA introduced into cells can induce these reactions which are responsible for many diseases(45). Examples of autoimmune diseases are rheumatoid arthritis, insulin-dependent diabetes and Graves disease of the thyroid. Many ‘spontaneous mutations’ are due to insertions of transposable elements and other invasive DNA. Insertion mutagenesis is now found to be associated with a range of cancers, including lung(46), breast(47), colon (48) and liver (49) cancers. Finally, unintended modification of germ-cells may result from gene therapy and vaccinations(50).

Not as much is known concerning naked RNA. It is to be expected that antisense RNA, like antisense DNA, will have biological effects either in blocking the function of homologous genes or genes with homologous domains. RNA may also be reverse transcribed into complementary DNA (cDNA) by reverse transcriptase, which is present in all higher organisms as well as some bacteria(51), to become incorporated into the cell’s genome.

The direct uptake and incorporation of genetic material from unrelated species is referred to as horizontal gene transfer, or gene transfer by infection, to distinguish it from the usual vertical gene transfer from parent to offspring in reproduction.

The horizontal transfer of transgenic DNA

Many geneticists may accept that naked nucleic acids are transferred horizontally, especially to microorganisms, but dispute the transfer of transgenic DNA, which they regard to be no different from the host cell DNA.

There is evidence of secondary horizontal transfer of transgenic DNA to soil bacteria and fungi in the laboratory. In the case of fungi, the transfer was obtained simply by co-cultivation(52). Successful transfers of a kanamycin resistance marker gene 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 (oil-seed rape) and Lycopersicon esculentum (tomato)(53). 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.

Schluter et al(54) investigated horizontal gene transfer under a variety of conditions, some of which gave positive results. For example, a high gene transfer frequency of 5.8 x 10-2 per recipent bacterium was demonstrated for ampicillin resistance transgene - re-isolated from the DNA of transgenic potato - to Erwinia chrysanthem, a bacterial pathogen. This was achieved by 105 copies of the ampicillin resistance gene per potato genome, introduced into 6.4 x 108 bacteria by electroporation. When reduced to one copy of ampicillin resistance gene per potato genome, the gene transfer frequency was still significant at 4 x 10-6. The total genomic DNA from the transgenic potato, estimated to carry two copies of ampicillin resistance gene per potato genome, likewise gave a transfer frequency of 9 x 10-6. With only transgenic potato tissue, it was less than 8.7 x 10-9, effectively nil, according to the limit of sensitivity of the protocol. The same result was obtained by co-cultivation of the transgenic tuber with bacteria for 6 weeks. The negative results were not surprising, given the limited access of the bacteria to plant DNA under those conditions. The authors then ‘calculated’ an extremely low frequency of gene transfer at 2.0 x 10-17 under extrapolated "natural conditions", assuming the different factors acted independently. The natural conditions are unknown and by the authors’ own admission, synergistic effects cannot be ruled out.

Free transgenic DNA will be readily available in the rhizosphere around the plant roots, which is an ‘environmental hotspot’ for gene transfer(55). Gebbard and Smalla(56) have also 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. Many other factors, such as the density of bacteria, temperature, availability of nutrients, heavy metals and pH, can greatly influence the frequency of horizontal gene transfer in nature(57). Moreover, less than one percent of all bacteria in the environment can be isolated(58) and monitored for horizontal gene transfer, so negative results in the field must be interpreted with due caution. There is no ground to assume that horizontal transfer of transgenic DNA will not take place under natural conditions.

There are also reasons to suspect that transgenic DNA may be more likely to take part in horizontal gene transfer than the organism’s own genes (see Box 4)(59).

The hazards of horizontal gene transfer

Horizontal gene transfer is uncontrollable. Unlike chemical pollutants which break down and become diluted out, nucleic acids are infectious, they can invade cells and genomes, to multiply, mutate and recombine indefinitely.

Horizontal gene transfer is by no means unknown to our Governments. Among the scientific advice given by the UK Ministry of Agriculture, Fisheries and Food (MAFF) to the US Food and Drug Administration (FDA) at the end of 1998 (62) are the following warnings:

• Transgenic DNA can spread to farm workers and food processors via dust and pollen.

• Antibiotic resistance marker genes may spread to bacteria in the mouth, as the mouth contains bacteria that readily take up and incorporate foreign DNA (see above). Similar transformable bacteria are present in the respiratory tracts.

• Antibiotic resistance marker genes may spread to bacteria in the environment, which then serves as a reservoir for antibiotic resistance genes.

• DNA is not readily degraded during food processing nor in the silage, hence transgenic DNA can spread to animals in animal feed.

• Foreign DNA can be delivered into mammalian cells by bacteria that can enter into the cells.

• The ampicillin resistance gene in the transgenic maize undergoing ‘farm-scale’ field-trials in the UK and elsewhere is very mutable, and may compromise treatment for meningitis and other bacterial infections, should the gene be transferred horizontally to the bacteria. The potential hazards of horizontal gene transfer are unlike those we have ever experienced (see Box 5).

The dangers of generating new viruses and bacteria that cause diseases, and spreading drug and antibiotic resistance among the pathogens, were both foreseen by the pioneers of genetic engineering. That was why they called for a moratorium in the Asilomar Declaration of 1975. But commercial pressures cut the moratorium short, and guidelines were set up based on assumptions, every one of which has been invalidated by scientific findings since(63). Within the past 20 years, drug and antibiotic resistant infectious diseases have come back with a vengeance. Geneticists have confirmed that the diseases are due to new viral and bacterial strains that have been created by horizontal gene transfer and recombination. Horizontal gene transfer is now recognized to be widespread, involving the entire biosphere, with bacteria and viruses in all environments serving as reservoir and highway for gene multiplication, gene swapping and trafficking. Has genetic engineering contributed to creating the new pathogens, and will it continue to do so through the unregulated release of naked and free nucleic acids? (64) The possible links between genetic engineering biotechnology and the recent resurgence of infectious diseases are summarized in Box 6.

Dormant and relict viral sequences have been discovered in the human and other animal genomes at least 20 years ago(65). Viral sequences have also been discovered recently in plant genomes(66). Viral transgenes are found to recombine with defective viruses to generate infectious recombinants(67). Recombination between exogenous and endogenous viral sequences are associated with animal cancers(68). It is not inconceivable that the cauliflower mosaic viral promoter, which is in practically all first generation of transgenic plants, may recombine with dormant/relict viral sequences in the genome to regenerate infectious viruses(69), in view of the fact that viral promoters have modules in common. Recombination hotspots may be associated with all transcriptional promoters(70), including those of animal viruses, such as the SV40 and cytomegalovirus, used in animal and human genetic engineering(71). This possibility should be addressed by empirical investigations, particularly in view of the recent claim that a significant part of the toxicity of certain transgenic potatoes fed to young rats may be due to the transgenic construct or the transformation process, or both(72).

In the light of the existing evidence, the most dangerous naked/free DNA may be coming from the wastes of contained users of GMOs which are discharged into our environment. These include constructs containing cancer genes from laboratories in research and development of cancer and cancer drugs, virulence genes from bacteria and viruses in pathology labs and all kinds of other novel constructs and gene combinations that did not previously exist in nature, and may never have come into being but for genetic engineering.

Despite the growing body of evidence of hazards from the innumerable exotic naked nucleic acids that are created and released in increasing amounts into the environment from the burgeoning biotech industry, there is no effective regulatory oversight, nor is there any indication that our Government is prepared to establish effective regulatory oversight (see Box 7).

Conclusion

The naked/free nucleic acids created by genetic engineering biotechnology are potentially the most dangerous xenobiotics to pollute our environment. Unlike chemical pollutants which dilute out and degrade over time, nucleic acids can be taken up by all cell to multiply, mutate and recombine indefinitely. The need for regulatory oversight at both national and international levels is long overdue. It is irresponsible to continue to exclude naked/free nucleic acids from the scope of the Biosafety Protocol.

1. Traavik, T. (1999a) Too early may be too late: Ecological risks associated with the use of naked DNA as a biological tool for research, production and therapy. pp 29-31. Reported to the Directorate of Nature Management, Norway.
2. See Ho, M.W. (1998, 1999). Genetic Engineering Dream or Nightmare? The Brave New World of Bad Science and Big Business, Gateway Books, Bath 2nd ed., Gateway, Gill & Macmillan, Dublin.
3. As defined by Traavik, 1999a (note 1)
4. See Ho, 1998, 1999 (note 2); Ho, M.W., Traavik, T., Olsvik, R., Tappeser, B., Howard, V., von Weizsacker, C. and McGavin, G. (1998b). Gene Technology and Gene Ecology of Infectious Diseases. Microbial Ecology in Health and Disease 10, 33-59; Traavik, T. (1999b). An orphan in science: Environmental risks of genetically engineered vaccines. Reported to the Directorate of Nature Management, Norway.
5. See Ho et al, 1998 (note 4).
6. See Traavik, 1999b (note 4).
7. See Schindelhauer, D. (1999). Construction of mammalian artificial chromosomes: prospects for defining an optimal centromere. BioEssays 21, 76-83.
8. See Helin, V., Gottikh, M., Mishal, Z., Subra, F., Malvy, C. and Lavignon, M. (1999). Cell cycle-dependent distribution and specific inhibitory effect of vectorized antisense oligonucleotides in cell culture. Biochemical Pharmacology 58, 95-107; Campagno, D. and Toulme, J.-J. (1999). Antisense effects of ligonucleotides complementary to the hairpin of the Leishmania mini-exon RNA. Nucleosides & Nucleotides 18, 1701-1704.
9. See Hammann, C. and Tabler, M. (1999). Generation and application of asymmetric hammerhead ribozymes. Methods: a Companion to Methods in Enzymology 18, 273-380.
10. Han, Y., Zaks, T.Z., Wang, T.F., Irvine, D.R., Kammula, U.S., Marincola, F.M., Leitner, W.W. and Restifo, N.P. (1999). Cancer therapy using a self-replicating RNA vaccine. Nature Medicine 3, 823-827.
11. Beetham, P.R., Kipp, P.B., Sawycky, X.L., Arntzen, C.J. and May, G.D. (1999). A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations. PNAS 96, 8774-8778.
12. Reviewed by Lorenz, M.G. and Wackernagel, W. (1994). Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev. 58, 563-602; also, Ho, 1998,1999 (note 2); Ho, et al, 1998 (note 4); Traavik, 1999a (note 1).
13. This was said to M.W.H. by a spokesperson of the UK Health and Safety Executive when asked whether there is any recommended treatment for disposal of naked/free DNA.
14. See Lorenz and Wackernagel, 1994 (note 12); also Ho, 1998, 1999 (note 2); Ho, et al, 1998 (note 4) .
15. Mercer, D.K., Scott, K.P., Bruce-Johnson, W.A. Glover, L.A. and Flint, H.J. (1999). Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Applied and Environmental Microbiology 65, 6-10.
16. 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. Molecular and General Genetics 242, 495-504; Schubbert, R., Rentz, D., Schmitzx, 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. Nat. Acad. Sci. USA 94, 961-6.
17. Mercer et al, 1999 (note 15).
18. Brown, P. Naked DNA raises cancer fears for researchers.New Scientist 6 October, 17 (1990).
19. Schubbert et al, 1997 (note 16).
20. Doerfler, W. and Schubbert, R. (1998). Uptake of foreign DNA from the environment: the gastroinestinal tract and the placenta as portals of entry, Wien Klin Wochenschr. 110, 40-44.
21. Doerfler and Schubbert, 1998, (note 20), p. 40.
22. Yei, S., Mittereder, N., Wert, S., Whitsett, J.A., Wilmott, R.W. and Trapnell, B.C. (1994). In vivo evaluation of the safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lung. Hum. Gene Ther.l5, 731-744.
23. Noisakran S, Campbell IL, Carr DJ, (1999) Ecotopic expression of DNA encoding IFN-alpha 1 in the cornea protects mice from herpes simplex virustype 1-induced encephalitis. J Immunol 162, 4184-90.
24. Yamasoba T, Yagl M, Roessler BJ, Miller JM, Rapheal Y (1999) Inner ear transgene expression after adenoviral vecotr inoculation in the endolymphatic sac. Hum Gene Ther 10, 744-69.
25. See Hoffman, R.M. (2000). The hair follicle as a gene therapy target. Nature Biotechnology 18, 20-1.
26. Budker, V., Zhang, G., Danko, I., Williams P. and Wolff, J. (1998). The efficient expression of intravascularly delivered DNA in rat muscle. Gene Therapy 5, 272-6; Han, et al, 1999 (note 12).
27. Khavari, P.A. Cutaneous gene therapy.Advances in Clinal Research 15, 27-35 (1997);
28. During, M.J., Xu, R., Young, D., Kaplitt, M.G., Sherwin, R.S., Leone, P. (1998). Peroral gene therapy of lactose intolerance using an adeno-associated virus vector. Nat. Med. 4, 1131-5.
29. Spadafora, C. (1998). Sperm cells and foreign DNA: a controversial relation. BioEssays 20, 955-64.
30. Zhang, G. Vargo, D., Budker, V., Armstrong, N., Knechtle, S. and Wolf, J., Expression of naked DNA injected into the afferent and efferent vessels of rodents and dog livers. Human Gene Ther. 8,1763-72 (1997).
31. Hengge, U., Chan, E., Foster, R.,Walker, P. and Vogel, J., Cytokine gene expression in epidermis with biological effects following injection of naked DNA,. Nat. Genet 10,161-6 (1995)
32. Hengge, U., Walker, P. and Vogel, J. Expression of naked DNA in human, pig and mouse skin. J Clin Invest 97,2911-6 (1996).
33. Martin, T., Parker, S.E., Hedstrom, R., Le, Thong, Hoffman, S.L., Norman, J., Hobart, P. and Lew, D. (1999). Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum. Gene Ther. 10, 759-68.
34. Zhaqo,T., Robinson, M., Bowers, F. and Kindt,T. Infectivity of chimeric human T-cell leukaemia virus type I molecular clones assessed by naked DNA inoculation. Proc. Natnl.Acad Sci. USA 93,6653-8 (1996).
35. Rekvig, O.P. Fredriksen, K., Brannsether, B., Moens, U., Sundsfjord, A. and Traavik, T., Antibodies to eucaryotic, including autologous, native DNA are produced during BK virus infection, but not after immunization with non-infectious BK DNA. Scand. J. Immunol. 36, 487-495 (1992).
36. Brower, V. (1998). Naked DNA vaccines come of age. Nature Biotechnology 16, 1304-5;
37. see also Traavik, 1999b (note 4). See Traavik, 1999b (note 4).
38. See Verdier, F. and Descotes, J. (1999). Preclinical safety evaluation of human gene therapy products. Toxicological Sciences 47, 9-15; Jane, S.M., Cunningham, J.M. and Vanin, E.F. (1998). Vector development: a major obstacle in human gene therapy. Annals of Medicine 30, 413-5.
39. Putnam, L. (1998). Debate grows on safety of gene-therapy vectors. The Lancet 351, 808.
40. See Ho, et al, 1998 (note 4).
41. Baba, T.W. Liska, V., Khimani, A.H., Ray, N.B., Dailey, P.J., Penninck, D., Bronson, R., Greene, M.F., McClure, H.M.,Martin, L.N. and Ruth M. Ruprecht, R.M. Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques. Nature Med. 5, 194, 203 (1999).
42. See Verdier and Desotes, 1999 (note 38).
43. See Coghlan, A. (1996). Gene shuttle virus could damage the brain. New Scientist 11 May, 6.
44. Nelson D & Weiss R. Gene research moves towards secrecy. Washington Post Nov 3, 1999
45. Suzuki, K., Mori, A., Ishii, K.J., Singer, D.S., Klinman, D.M., Krause, P.R. and Kohn, L.D. (1999). Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides. Proc. Natl. Acad. Sci. USA 96, 2285-90.
46. Fong KM at al (1997) FHIT and FRA3B 3p14.2 allele loss are common in lung cancer and preneoplasic bronchial lesions and are associated with cancer related FHIT cDNA splicing abberations. Cancer Res. (CNF), 57 (11) ; 2256-67.
47. Asch HL (1996) Comparative expression of the LINE-1 p40 protein in human breast carcinomas and normal breast tissues. Oncol. Res (BBN) 8 (6): 239-47.
48. Miki Y. (1992). Disruption of the ARC gene by retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res (CNF), 52 (3):643-5
49. Buendia, M.A. (1992). Mammalian hepatitis B viruses and primary liver cancer. Semin. Cancer Biol. 3, 309-20.
50. See Verdier and Descotes, 1999 (note 38); Spadafora, 1998 (note 29).
51. Mao, J.R., Inouye, M. and Inouye, S. (1996). An unusual bacterial reverse transcriptase having LVDD in the YXDD box from Escherichia coli. Biochem. Biophys. Res. Commun. 227, 489-93.
52. 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: 70-6.
53. 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.
54. 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: 1094-8.
55. Timms-Wilson, T.M., Lilley, A.K. and Bailey, M.J. (1999). A Review of Gene Transfer from Genetically Modified Micro-organisms. Report to UK Health and Safety Executive.
56. Gebhard, F. and Smalla, K. (1998). Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA. Appl. Environ. Microbiol. 64, 1550-4.
57. See Traavik, 1999a (note 1); Timms-Wilson, et al, 1999 (note 24).
58. Pace, N. (1997). A molecular view of microbial diversity and the biosphere. Science 276, 734-9.
59. See Ho, 1998, 1999 (note 1); Ho et al, 1998b(note 1); Traavik, 1999a (note 1).
60. See Kohli, A., Griffiths, S., Palacios, N., Twyman, R.M., Vain, P., Laurie, D.A. and Christou, P. (1999). Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. The Plant Journal 17, 591-601; also Ho, M.W., Ryan, A. and Cummins, J. (1999). The CaMV promoter - A recipe for disaster? Microbial Ecology in Health and Disease (in press).
61. See Ho et al, 1998b (note 4) and references therein.
62. Letter from N. Tomlinson, Joint Food Safety and Standards Group, MAFF, to US FDA, 4 December, 1998.
63. See Ho, M.W. , 1998, 1999 (note 2)
64. This was the question asked by Ho et al, 1998 (note 4) who called for an urgent public enquiry; See also Ho, M.W., Traavik, T., Olsvik, O., Midtvedt, T., Tappeser, B., Howard, C.V., von Weizsacker, C. and McGavin, G. (1998). Gene Technology in he Etiology of Drug-resistant Diseases. TWN Biotechnology & Biosafety Series 2,Third World Network, Penang.
65. See Ho, 1998, 1999 (note 2)
66. See Jakowitsch, J., Mette, M.G., van der Winden, J., Matzke, M.A. and Matzke, A.J.M. (1999). Integrated pararetrovial sequences define a unique class of dispersed repetitive DNA in plants. Proc. Nat. Acad. Sci. USA 96, 13241-6.
67. 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.
68. See Ho, 1998, 1999 (note 2) especially Chapter 13/12.
69. See Ho et al, 1999 (note 30).
70. See Robinson, W.P. and Lalande, M. (1995). Sex-specific meiotic recombination in the Prader-Willi/Angelman syndrome imprinted region. Hum. Mol. Genet. 4, 801-6;Wu,T.C. and Lichten, M. (1994). Meiosis-induced double-stranded break sites determined by yeast chromatin structure. Science 263, 515-8.
71. See Kohli , et al, 1999 (note 56).
72. Ewen, S.W.B. and Pusztai, A. (1999). Effect of diets containing genetically modified potatoes expressing Galanthus nivalis lectin on rat small intestine. The Lancet 354

The Institute of Science in Society, PO Box 51885, London NW2 9DH
telephone:   [44 20 8452 2729]   [44 20 7272 5636]

Contact the Institute of Science in Society