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Dangers Inherent in the Process Itself
The Use of Cauliflower Mosaic Virus

35S Promoter (CaMV) in Calgene's Flavr Savr Tomato Creates Hazard
Joseph E Cummins Associate Professor (Genetics) Dept of Plant Sciences University of Western Ontario
3 June 1994
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The majority of crop plant constructions for herbicide or disease resistance employ a Promoter from cauliflower mosaic virus (CaMV). Regardless of the gene transferred, all transfers require a promoter, which is like a motor driving production of the gene's message. Without a promoter, the gene is inactive, but replicated, CaMV is used because it is a powerful motor which drives replication of the retrovirus and is active in both angiosperms and gymnosperms. The CAMV pararetrovirus replication cycle involves production of vegetative virus containing RNA which is reverse transcribed to make DNA similar to HIV, Human Leukemia Virus and Human hepatitis B. (Bonneville et al RNA Genetics Vol 11, Retroviruses, Viroids and RNA Recombination pp 23-42, 1988). CaMV is closesly related to hepatitis B and is closely related to HIV (Doolittle etal Quart Rev Biol 64, 2, 1989; Xiong and Eickbush, EMBO Journal 9, 3353, 1990). The CaMV promoter is preferred above other potential promoters because it is a more powerful promoter than others and is not greatly influenced by environmental conditions or tissue types. CaMV has two promoters 19S and 35S, of these two the 35S promoter is most frequently used in biotechnology because it is most powerful. The 35S promoter is a DNA (or RNA) sequence about 400 base pairs in length. The use of the CaMV promoter in plants is analagous to the use of retrovirus LTR promoters in retrovirus vectors used in human gene therapy. The majority of human gene therapy trials employ LTR promoters to provide motors to activate genes.

Antisense genes are genes constructed to have a complementary sequence to a target gene, thus producing a product that combines with a gene message to inactivate it. Antisense is analagous to an antibody which combines with an antigen like a key fitting a lock. Antisense is being used to treat human cancer and HIV infection. Antisense is used to prevent spoilage in tomatoes, either by targeting an enzyme degrading cell walls (polygalacturonase), or production of ethylene a hormone promoting ripening (P Oeller et al Genetic Engineering 49, 1989; R Fray and D Grierson, Trends Genetics 9, 438, 1993). Most frequently antisense targets production of a chemical metabolite producing ethylene. The antisense gene also influenced polyamines spermine and spermidine production through S-adenosylmethionine. The implication is that the plant antisense gene product should be tested in animals to ensure that critical functions including gene replication, sperm activity and gene imprinting are not disrupted.

The perceived hazards of CaMV in crop plants include the consequences of recombination and pseudo recombination. Recombination is the exchanges of parts of genes or blocks of genes between chromosomes. Pseudorecombination is a situation in which gene components of one virus are exchanged with the protein coats of another. Frequently viruses may incorporate cellular genes by recombination or pseudorecombination, it has been noted that such recombinations have selective advantages (Lai, Micro. Rev. 56, 61, 1992).

It has been shown that the CaMV genes incorporated into the plant (canola) chromosome recombine with infecting virus to produce more virulent new virus diseases. The designers of the experiment questioned the safety of transgenic plants containing viral genes (S Gal et al, Virology 187:525, 1992). Recombination between CaMV viruses involves the promoter (Vaden and Melcher, Virology 177: 717, 1992) and may take place either between DNA and DNA or RNA and RNA and frequently creates more severe infections than either parent (Mol Plant-Microbe Interactions 5, 48, 1992). Recently related experiments suggest altered plants may breed deadlier diseases (A Green and R Allison, Sciences 263: 1423, 1994). DNA copies of RNA Viruses are frequently propagated using the CaMV 35S promoter to drive RNA virus production (J. Boyer and A Haenni, Virology 198: 4165, 1994 and J Desuns and G Lomonossoff, J Gen Vir 74:889, 1993). In conclusion CaMV promoters recombine with the infecting viruses to produce virulent new diseases. CaMV viruses and promoter may incorporate genes from the host creating virulent new diseases. CaMV can recombine with insect viruses and propagated in insect cells (D Zuidema et al J gen Vir 71: 312, 1990). Thus it is likely that as large numbers of humans consume CaMV modified tomatoes recombination between CaMV and hepatitis B viruses will take place creating a supervirus propagated in plants, insects and humans.