By Dr. Mae-Wan Ho, Director, Institute of Science in Society, PO Box 32097, London NW1 0XR, UK.

David Schubert, cell biologist from the Salk Institute in La Jolla, California argued [1] that "GM food is not a safe option, given our current lack of understanding of the consequences of recombinant technology". He called for "all GM plant products destined for human consumption to be tested for long-term toxicity and carcinogenicity before being brought to the market."

This is yet another independent scientist whose warnings are dismissed [2] in a manner similar to the way the UK Advisory Committee on Releases to the Environment (ACRE) dismisses safety concerns raised in the public hearing on T25 (Chardon LL) GM maize, in its recently published response [3].

I will address some specific points in that document under three headings, horizontal gene transfer, the hazards of CaMV 35S promoter and transgenic instability.

On horizontal gene transfer

ACRE claims there is "no evidence" that transgenic DNA can spread from GM crops to unrelated species, such as bacteria and mammalian cells, but covers its tracks with the following statement (p.7): "This is not to say that that horizontal gene transfer between plants and bacteria could not happen, but the evidence suggests that if it does, it is a very rare event."

That's false reassurance. A rare event is sufficient to trigger a catastrophe, as, in contrast to chemicals that degrade and disappear, genes and genetic material multiply and recombine. All major disease epidemics are due to such "very rare" events.

ACRE has ignored and omitted to mention most of the evidence we sent [4], and concentrated on two key papers by Gebhard and Smalla [5, 6]: "The authors of these publications did not demonstrate, nor did they claim to provide evidence of horizontal gene transfer under laboratory conditions or in field studies."

ACRE is selectively referring to the authors' apologies in interpreting their own results, but as I have shown, in a detailed review of the work submitted to ACRE [7], the results offered prima facie evidence of horizontal gene transfer. The authors themselves had indeed considered that possibility, first and foremost. ACRE is guilty of misrepresentation, and failing to interpret the experimental results correctly by excluding obvious explanations. At the very least, ACRE should have called for further investigations, as that was the only field monitoring experiment ever carried out.

ACRE continues,

"Horizontal gene transfer between plant and bacterial cells can be achieved by using forced conditions in the laboratory and by supplying pressure to select for the gene product and consequently the transgene itself."

That is pure obfuscation. So-called "forced conditions" and "pressure to select", are nothing of the sort, they are laboratory techniques to enable us to detect rare events, much as antibiotic resistance and other marker genes (eg, green fluorescent protein) are used to detect rare transformed cells or track their fate, in making GMOs, or following the developmental fate of stem cells.

Among the many ignored research reports on horizontal gene transfer submitted to ACRE [8] are some commissioned by the UK government showing that transgenic DNA in food has transferred to gut bacteria after only a single meal, and that the Agrobacterium vector system used in making most GM plants can be a vehicle for gene escape in the soil. Agrobacterium was found to transfer genes into human cells in much the same way that it transfers genes into plant cells. This work has not been followed up, nor has ACRE called for further research.

ACRE has also omitted to mention that transgenic DNA can pass through the gut and placenta into the blood stream ending up in some blood cells, liver and spleen cells, and some cells of the foetus and newborn. This work goes back to the early 1990s and is still continuing [9, 10]. ACRE has not called for similar studies to be carried out. Tracking the passage of transgenic DNA into the blood stream and blood cells could have been part of the experiment that monitored the fate of transgenic DNA in food eaten by human volunteers, but it was not [11].

By not calling for more definitive experiments to follow up positive findings, and by not acknowledging the restrictive scope of the investigations, ACRE continues to take the absence of evidence as evidence of absence.

The hazards of CaMV 35S promoter

ACRE 's continuing denial appears on pp.14-15 of their response, which focuses on a paper we wrote [12], and according to ACRE, "no new data or direct experimental evidence is presented to support the authors' hypothesis that the CaMV 35S promoter, used in many GM plants, is inherently dangerous."

This bland denial can only be justified by ignoring all the papers we published subsequently that have demolished the argument - which ACRE is repeating - that people have been eating the virus for all these years without harm: "For many thousands of years CaMV and its relative have infected plants; consequently humans and animals have been eating plant material containing the 35S promoter via natural CaMV infection. No ill effects due to the activity or recombination of the virus promoter have been reported and in particular, no reports of cancer."

But people have not been eating CaMV 35S promoter plucked from its natural genetic and evolutionary context and incorporated into transgenic DNA. We published at least two further papers demolishing the criticisms [13, 14]. These were submitted to ACRE and other science advisory committees on numerous occasions, and neither ACRE nor any of our critics has ever been able to reply to them.

Roger Hull from John Innes Centre (JIC) who led the attack on us, was asked to reply to us by a local group, "Concerned Citizens of Wivenhoe".

Hull initially promised to do so, but after a long delay, wrote back to say that he had discussed the whole matter with the Director of JIC and decided not to reply, and instead, "to draw up a briefing document targeted towards decision and opinion makers".

We have pointed out to our critics that although the CaMV (virus) is specific to cruciferae, the CaMV 35S promoter is utterly promiscuous, and is active in species across the entire living kingdom, from bacteria up to human cells.

ACRE has selectively ignored that information, which cannot have escaped notice, as its invited experts to its open hearing confessed their ignorance that the CaMV 35S promoter was shown to be active in human cell systems in the scientific literature dating to 1989, when I challenged the panel with that information [15].

On page 7 of the Report, ACRE claims, "the expression of genes regulated by the CaMV 35S promoter in bacteria is minimal". This is obfuscation, because the claim is being justified by the base sequence modification of the gene controlled by the promoter (described following the quote), which supposedly biases it against expression in bacteria. But the case is weak, and certainly not supported by data.

ACRE goes on, "Ho et al., speculate that the 35S promoter has a tendency to recombine with other DNA which could have harmful consequences. However, there is no evidence for this, or any reason why 35S CaMV DNA would be more prone to recombination than other DNA that does not have elements associated with insertion and excision."

Its claim of "no evidence" and no reason why CaMV 35S would be more prone to recombination is false. Our first paper [12], the only one ACRE cites, was precisely provoked by a paper reporting a 'recombination hotspot' associated with the CaMV 35S promoter published by a research team in JIC [16], which ACRE has failed to cite. Not only that, a second group has confirmed the finding [17], and showed that another promoter used did not have the same propensity for recombination as the CaMV 35S promoter. That information was in one of our later papers [14] submitted to ACRE.

The JIC team that discovered the recombination hotspot in CaMV 35S promoter later admitted to the need to avoid the CaMV 35S promoter as well as other bits of DNA containing recombination hotpots. They stated in JIC's annual report in 2001 [18]:

"Analysis of junctions between genomic and transforming DNA, and between individual plasmid molecules at integration sites, demonstrates the predominance of microhomology-mediated illegitimate recombination events involving regions with secondary structure. One such region occurs in the CaMV 35S promoter, widely used to drive transgene expression in plants. The plasmid backbone provides other such regions, including the origin of replication .....The influence of transgene rearrangements on expression and silencing has been understated in the past, but our research may allow improved construct design to discourage rearrangements and improve transgene-expression stability."

This 40-strong research team has mysteriously disbanded last April, in the midst of the controversy over GM contamination of Mexican maize landraces. The most controversial finding was not the contamination itself, but molecular data suggesting that the transgenic DNA containing the CaMV 35S promoter may be "fragmenting and promiscuously scattering throughout the genome" of the landraces. Such observations would be totally consistent with our expectations given the existence of the recombination hotspot in the promoter [19]. ACRE has selectively ignored all of that too.

This is also the place to address ACRE's denial that promoters like CaMV, which shows a propensity to fragment, and could therefore jump around the genome, has the potential to cause cancer. Roger Hull has recently issued the same denial in dismissing pathologist Stanley Ewen's concern that GM crops may trigger cancer [20].

In fact, insertion of foreign DNA into genomes is known to be associated with cancer, so much so that 'insertion carcinogenesis' is a clinical entity [21]. The two recent cancer victims of gene therapy uncovered within months of each other [22, 23, 24] should make us wary about eating anything that has transgenic DNA, and more so, transgenic DNA containing CaMV 35S promoter that has an increased propensity to fragment and recombine with, ie insert into, DNA of the human cell genome [21]. GM constructs are similar, whether used for genetic modification of human cells in gene therapy or for genetic modification of plants and animals.

ACRE has ignored all of that as well, and continues its comments on our first paper,

"A prominent concern raised in this paper is that the 35S promoter in GM plants could inadvertently activate dormant viruses or non-target genes in plants or other organisms. ACRE is aware that the 35S promoter has the potential to alter the expression of host genes neighbouring the site of its insertion. This is one of the reasons why ACRE require applications for marketing consents to describe the host DNA that flanks the site into which the transgene has inserted. ...There are no reported incidences of a dormant plant virus being unintentionally activated by the insertion of a transgene with a 35S promoter. In particular, there is no evidence that the T25 insertion event has altered the maize line in any way that makes it less safe to human health or the environment than its conventional counterparts."

ACRE is admitting the 35S promoter in GM plants could alter the expression of neighbouring host genes and says it requires marketing applications to describe the site of transgene insertion. But these requirements have been very weak in the past, and do not satisfy the more stringent "event-specific" molecular characterisations that also document genetic stability in successive generations, which ISIS has demanded from the first (see below).

Similarly, the bland assurance of "no reported incidence of a dormant plant virus being unintentionally activated" and "no evidence that T25 insertion event (in Chardon LL) has altered the maize line in any way that makes it less safe" is not justified, as there has been no attempt to obtain such evidence empirically.

On the contrary, several recent papers from the JIC, co-authored by Roger Hull, have described dormant viruses discovered in the genome that can be reactivated by a variety of treatments. One of these papers [25] expressed explicit concern about new viruses emerging, "As an unforeseen hazard of plant breeding or genome manipulation, and of plant and insect movement, there must be concern that new viruses will emerge..." (italics mine).

ACRE continues,

"Ho et al., have suggested that there is a 'close relationship' between CaMV and human viruses such as the hepatitis B and that CaMV 35S promoter DNA inserted into transgenic plants will recombine with DNA from these viruses. However, CaMV and human retroviruses are not members of the same genetic family and the degree of similarity between their DNA sequences is low."

CaMV belongs to the pararetrovirus supergroup that includes the Hepadnaviridae family infecting vertebrates, of which the hepatitis B virus is a member. Whether one considers that a close relationship is a moot point, as pararetroviruses share many distinctive features in their life cycle [26]. Sequence homology is also irrelevant given that the recombination mechanism of the CaMV 35S promoter depends on breaks in the double stranded DNA, which allows it to rejoin to non-homologous DNA [16, 17].

It has also become increasingly evident that the CaMV 35S promoter can substitute for the promoter of most if not all viruses, plants or animals not withstanding, and is active in all living things [13, 14]. It is truly promiscuous and dangerous. It is being quietly phased out behind the scenes, judging by its almost total absence in newer transgenic crops under development. The JIC plant geneticists claim that's because the CaMV 35S promoter compromises agronomic performance on account of the transgenic instability it causes, and has nothing to do with safety. I have just presented the arguments as to why the instability is above all a safety issue.

Transgenic DNA and transgenic instability

ACRE states, "There is also no evidence in the literature to support the idea that transgenic DNA is inherently less stable than native DNA." This statement is false.

Instability of transgenic DNA is so well known that it is a textbook topic, as I have pointed out to ACRE and other government science advisors time and again. Furthermore, there is such a vast literature on transgenic instability [27] that ACRE's denial here is embarrassing. I will only cite the report by another group in JIC [28] documenting instability arising in later generations of transgenic barley lines:

"Data from the 1998 trial showed that transgenic barley lines performed as well as non-transformed control plants and controls from tissue culture-derived parents for several agronomic traits, including yield. For other traits, a significant difference was seen between transgenic and control lines. The transgenic lines were significantly shorter and also slightly later flowering.... When we examined the next generation of the same transgenic line in the field during 1999, there was evidence that the transgenic plants were more variable compared to the controls than those in the 1998 field trial. This could be because somaclonal variation, resulting from the tissue culture and transformation procedures, and was more obvious in later generations. These results show that transgenic lines need to be examined over a number of generations under field conditions to obtain the necessary data on transgene stability and agronomic performance. Further field trials .... combined with detailed molecular and genetic analysis will allow us to increase our understanding of the transformation process so that we are better able to assess the long term effects of genetic modification." (italics mine)

The findings on transgenic barley have been replicated in different forms in every species of transgenic plants investigated with the appropriate molecular techniques.

Despite that, the only criterion on which ACRE asserts transgenic lines are stable is transgene expression, and even here, it has simply accepted the company's word, with no numerical data nor independent verification. It states on p.11,

"The transgene has been expressed under different genetic and environmental conditions through numerous generations without any evidence of instability". And in a supporting footnote 28, "Bayer CropScience estimate 23 generations of breeding, with 40 different maize varieties world-wide now containing the T25 insert."

There is still no event-specific characterisation in successive generations to document true genetic stability. If the company had submitted event-specific characterisation in its original application for commercial approval, it would be an easy test to carry out on Chardon LL maize today, to see if the insert has remained unchanged in structure and location in the plant genome.

Such a test was applied to Roundup Ready soya in 2001. Roundup Ready soya failed the test, the foreign insert was considerably scrambled compared to Monsanto's original submission [29, 30].

Will ACRE carry out such a test on Chardon LL maize and other GM crops currently under field trial or seeking commercial approval?

1. Schubert C. A different perspective on GM food. Nature biotechnology 2002, 20, 969.

2. Beachy R, Bennetzen JL, Chassy BM, Chrispeels M, Chory J, Ecker JR, Noel JP, Kay SA, Dean C, Lamb C, Jones J, Santerre CR, Schroeder JI, Umen J, Yanofsky M, Wessler S, Zhao Y and Parrott W. Divergent perspectives on GM food. Nature biotechnology 2002, 20, 1195-6.

3. The Advisory committee on Releases to the Environment's (ACRE's) response to concerns raised in written representation and submissions associated with the CHARDON LL public hearing and to statements made at ACRE's open hearing relating to the safety assessment of T25 GM maize conducted under Directive 90/220/EEC. <www.defra.gov.uk/environment/acre>, e-mail: <mailto:gm@defra.gsi.gov.uk>

4. Reviewed in many past reports, see Horizontal Gene Transfer, ISIS Reprints March 2002, ISIS Members' website <www.i-sis.org.uk>, also available in hardcopy, enquire <mailto:sam@I-sis.org.uk>

5. Gebhard, F., and Smalla, K. (1998) Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA. Appl. Environ. Microbiol. 64: 1550-1554.

6. Gebhard, F., and Smalla, K. (1999) Monitoring field releases of genetically modified sugar beets for persistence of transgenic plant DNA and horizontal gene transfer. FEMS Microbiol. Ecol. 28: 261-272.

7. "Horizontal gene transfer happens, a practical exercise in applying the precautionary principle" by Mae-Wan Ho, ISIS News 5, 2000 <www.i-sis.org.uk>

8. See my latest attempt to put evidence before ACRE and ACNFP. Ho MW. Recent Evidence Confirms Risks of Horizontal Gene Transfer ISIS' Written Contribution to ACNFP/Food Standards Agency Open Meeting 13 November 2002 <www.i-sis.org.uk>

9. Hohlweb U. and Doerfler W. On the fate of plant or other foreign genes upon the uptake in food or after intramuscular injection in mice. Mole. Genet Genomics 2001, 265, 225-33.

10. "Suppression & denial" in Horizontal gene transfer special series, by Mae-Wan Ho, SiS16, 2002.

11. "Stacking the odds" in Horizontal gene transfer special series, by Mae-Wan Ho, SiS16, 2002.

12. Ho MW, Ryan A and Cummins J. Cauliflower mosaic viral promoter - a recipe for Disaster? Microbial Ecology in Health and Disease 1999 11, 194-7.

13. Ho MW, Ryan A and Cummins J. Hazards of transgenic plants with the cauliflower mosaic viral promoter. Microbial Ecology in Health and Disease 2000, 12, 6-11.

14. Ho MW, Ryan A and Cummins J. CaMV35S promoter fragmentation hotspot confirmed and it is active in animals. Microbial Ecology in Health and Disease 2000, 12, 189.

15. "GM maize approved on bad science in the UK" by Mae-Wan Ho, SiS 15, 2002.

16. Kohli A, Griffiths S, Palacios N, Twyman R, Vain P, Laurie D and Christou P. Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hot spot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant.J. 1999, 17,591-601.

17. Kumpatla SP and Hall TC. Organizational complexity of a rice transgenic locus susceptible to methylation -based silencing. IUBMB Life 1999, 48, 459-67.

18. Christou P, Kohli A, Stofer E, et al. Transgenic plants: a tool for fundamental genomics research. John Innes Centre & Sainsbury Laboratory Annual Report 1999/2000, p.29.

19. See "Who's afraid of horizontal gene transfer" and other articles in GM maize wars series, SiS 15, 2002

20. Roger Hull's letter to editor, Sunday Herald, 19 Dec 2002.

21. See Ho, MW, Ryan A, Cummins J and Traavik T. Slipping Through the Regulatory Net. 'Naked' and 'Free' Nucleic Acids, Third World Network Biotechnology and Biosafety Series 5, TWN, Penang, 2001.

22. "Gene therapy a suspect in leukemia-like disease", by Eliot Marshall, Science Oct 4 2002, 34-35.

23. "Predicted hazard of gene therapy a reality" by Mae-Wan Ho, ISIS Report, October 2002 <www.i-sis.org.uk>; "Gene therapy's first cancer victim" by Mae-Wan Ho SiS 17, 2003.

24. "Gene therapy trials halted" by Andrew Pollack, 15 January 2003, New York Times.
25. Hull R, Harper G and Lockhart B. Viral sequences integrated into plant genomes. Trends in Plant Science 2000, 5, 362-5.

26. Haas M, Bureau M, Feldreich A., Yot P and Keller M. Cauliflower mosaic virus: still in the news. Molecular Plant Pathology 2002, 3, 419-29.

27. See Transgenic Instability, ISIS reprints, ISIS members' website <www.i-sis.org.uk>, also available in hardcopy, enquire <mailto:sam@I-sis.org.uk>

28. Harwood, WA, Hardon J, Ross SM, Fish L, Smith J and Snape JW. Analysis of transgenic barley in a small scale field trial. John Innes Centre & Sainsbury Laboratory Annual Report 1999/2000, p.28.

29. Windels P, Taverniers I, Depicker A, Van Bockstaele E and De Loose M (2001). Characterisation of the Roundup Ready soybean insert. Eur Food Res Technol DOI 10.1007/ s002170100336, © Springer-Verlag.

30. "Scrambled genome of Roundup Ready soya" by Mae-Wan Ho, ISIS News 9/10, July 2001, ISSN: 1474-1547 (print), ISSN: 1474-1814 (online) <www.i-sis.org.uk>