The use of Cauliflower Mosaic Virus (CaMV) in genetic engineering

35S Promoter (CaMV) in Calgene's Flavr Savr Tomato Creates Hazard

Joseph E. Cummins
Professor Emeritus (Genetics) Dept. of Plant Sciences University of Western Ontario

June 3, 1994

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 genes' 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 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 Vo.11, Retroviruses, Viroids and RNA Recombination pp. 23-42, 1988). CaMV is closely related to hepatitis B and is closely related to HIV (Doolittle et al. Quart.Rev.Biol. 64,2, 1989; Xiong and Eickbush, EMBO Joumal 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 analogous 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 analogous 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 tomatos, 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 recombinants 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: 4l5, 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 tomatos recombination between CaMV and hepatitis B viruses will take place creating a supervirus propagated in plants, insects and humans.

Plant biotechnology has grown out of recombinant DNA research that began in the early 1970's. The special nature of recombination has been debated since that time. In recent years, government regulators on the American and European continents, under pressure from well-funded lobby representing the biotechnology industry, have chosen to ignore the special nature of recombination. They have chosen instead to base regulations on existing frameworks for toxic chemicals and pathogenic organisms. Ignoring the special nature of recombination is likely to have costly, if not terminal, environmental consequences. A worst-case example includes the complete cloning of Human Immunodeficiency Virus (HIV) on an E. coli plasmid. When the plasmid is used to transform animal cells, intact HIV viruses are released from the cells. A careless (but legal) release of HIV bacteria to the environment would allow the plasmid to transfer to Salmonella as well as E. coli. Thus, numerous mammals and birds could contain HIV bacteria which could transform the animals, which would in turn produce HIV particles unable to target the animals T-cell receptors but easily transmitted to humans. When all the animals are HIV carriers, human survival would be marginal. The special concerns of recombination in plant biotechnology include the viruses and bacteria used in crop plant construction and gene flow between related crop plants and weeds in the field.

Currently most experts agree that virus diseases such as influenza gain strength for epidemics by alternating between animal hosts (pigs and ducks) and man. Epidemics begin when rare combinations appear in large closely associated populations such as in asia. CaMV can propagate in plant and insect hosts following recombination. It may not be outlandish to predict that CaMV may recombine with related Hepatitis B or for that matter HIV to create a most powerful disease. The salient feature being large number of people or animals consuming large numbers of virus genes incorporated into crop plants making up a major part of human and animal diet.

The use of CaMV promoter is seldom an issue in reviews of safety of gene tinkered crops. Few people have raised the important issue and more often than not their concerns are ignored by government officials "protecting" public safety. This omission may be a fatal one because it has potentially the most damaging impact, and the one perceived at the beginning of gene splicing.

Comment

CaMV promoter genes are used both in Monsanto's Roundup Ready Soy and in Ciba's Bt Maize. About 15 percent of the soy crop in USA is expected to be RR-soy.

The FlavrSavr tomato brand has been withdrawn because it turned out to have unexpected deficiencies that did not make it useful for commercial production.

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