Hazards of CaMV Promoter

Joe Cummins - Dept. of Plant Sciences, University of Western Ontario, Ontario, Canada
Mae-Wan Ho and Angela Ryan, Department of Biological Sciences, Open University, Milton Keynes
(To appear in Nature Biotechnology April 2000)

This is a rebuttal to an article in Nature Biotechnology (Jan. 2000) attacking an earlier article, now published (Ho, M.W., Ryan, A., Cummins, J. (1999) The cauliflower mosaic viral promoter – a recipe for disaster? Microbial Ecology in Health and Disease 11, 194-197).

Keywords, CaMV 35S promoter, horizontal gene transfer, precautionary principle, hazards of GM crops

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In your account (Jan. 2000) (1) of our pre-publication manuscript, you quote the criticisms but ignore completely our full rebuttal, which was posted on the web last November. We shall outline the main points made in reply to the criticisms. The full details and references are available on our website (2).

Our manuscript (3) reviews and synthesizes the scientific literature on the 35S promoter of the cauliflower mosaic virus (CaMV) used to give constitutive over-expression of transgenes in practically all GM crops already commercialized or undergoing field trials. The promoter functions efficiently in all plants, as well as green algae, yeast and E. coli. It has a modular structure, with parts common to, and interchangeable with promoters of other plant and animal viruses. It also 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 – double-stranded DNA break-repair - requires little or no DNA sequence homologies. Finally, recombination between viral transgenes and infecting viruses has been demonstrated in the laboratory (4).

The findings suggest that transgenic constructs with the CaMV 35S promoter may be structurally unstable and prone to horizontal gene transfer and recombination. The potential hazards are mutagenesis, carcinogenesis, reactivation of dormant viruses and generation of new viruses. These considerations are especially relevant in the light of recent findings that certain transgenic potatoes - containing the CaMV 35S promoter - may be unsafe for young rats, and that a significant part of the effects may be due to "the construct or the genetic transformation (or both)" (5).

Our critics believe the CaMV 35S promoter is not harmful because people have been eating the virus in infected cabbages and cauliflower for many years. What we have been consuming is predominantly intact virus and not naked viral genomes. Naked viral genomes have been found to give full-blown infections in non-host species that are not susceptible to the intact virus (6). Moreover, the 35S promoter in the CaMV is a stable, integral part of the virus, and cannot be compared to the 35S promoter in artificial transgenic constructs. Artificial constructs are well-known to be structurally unstable (7). We know that the 35S promoter in the virus does not transfer into genomes because pararetroviruses, such as CaMV, do not integrate into host genomes to complete their lifecycle; and viral replication takes place in the cytoplasm (8). But that says nothing about the 35S promoter in transgenic constructs that are integrated into host genomes.

Proviral sequences are present in all genomes, and 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. The CaMV 35S promoter has been joined artificially to the cDNAs of a wide range of viral genomes, and infectious viruses produced in the laboratory (9). There is also evidence that proviral sequence in the genome can be reactivated (10).

The fact that plants are "loaded" with potentially mobile elements can only make things worse. Most, if not all of the elements will have been ‘tamed’ in the course of evolution and hence no longer mobile. But integration of transgenic constructs containing the 35S promoter may mobilize the elements. The elements may in turn provide helper-functions to destabilize the transgenic DNA, and may also serve as substrates for recombination to generate more exotic invasive elements.

In signing on to the International Biosafety Protocol in Montreal in January, more than 150 governments agreed to implement the precautionary principle. The available evidence clearly indicates that there are serious potential hazards associated with the use of the CaMV promoter. All GM crops and products containing the CaMV promoter should therefore be withdrawn both from commercial use and from field trials unless and until they can be shown to be safe.

Article first published 24/07/00

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References

1. Hodgson, J. (2000). Nature Biotechnology 18, 13.
2. Institute of Science in Society website: <www.i-sis.org.uk>
3. Ho, M.W., Ryan, A. and Cummins, J. (1999). Microbial Ecology in Health and Disease , in press, and available in electronic form www.scup.no/mehd/ho;.
4. Wintermantel, W. and Schoelz, J. (1996). Virology 223, 156-64
5. Ewen, S.W.B. and Pusztai, A. (1999). The Lancet 354, 1353-1354.
6. See for example, Rekvig, O.P., et al (1992). Scand. J. Immunol. 36, 487-95.
7. Structural instability of artificial vectors is a text-book topic. See Old, R.W. and Primrose, S.B. (1994). Principles of gene manipulation, 5th ed., Blackwell, Oxford.
8. Covey, S., et al (1990). Proc. Nat. Acad. Sci. USA 87, 1633-7.
9. Maiss, E., et al (1992). J. Gen. Virol. 73, 709-13; Meyer, M and Dessens, J. (1997). J. Gen. Viol. 78, 147-51.
10. Nowora, T. et al (1999). Virology 255, 214-20.