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The CaMV promoter story

The Biosafety Protocol concluded in Montreal reaffirms the precautionary principle but the problem is one of ensuring that the principle is implemented, as illustrated by the case of the CaMV promoter. The CaMV promoter is a gene-switch from the cauliflower mosaic virus which is incorporated into practically all current GM crops. Recent scientific findings reveal it may be highly unsafe. But many of the scientists themselves are refusing to read the implications of the findings or to draw the right conclusions in accordance with the precautionary principle.

By Mae-Wan Ho


The CaMV promoter - a recipe for disaster?

THIS was the title of a scientific paper co-authored by myself and my colleagues, Angela Ryan from the Open University UK and Prof. Joe Cummins from the University of Western Ontario, Canada, and submitted to the journal Microbial Ecology in Health and Disease last October. The journal-s Editor, Prof. Tore Midtvedt, a distinguished medical microbial ecologist working in the Karolinska Institute of Sweden, promptly posted our paper on the journal's website before publication and put out a press release. Within two days, someone managed to solicit at least nine critiques, including one from Monsanto, which were posted on a website funded by the biotech industry and widely circulated on the Internet. The critiques varied in tone from the moderately polite to the ill-mannered.

We wrote a detailed rebuttal, which was likewise circulated and posted to the same website. In January, Nature Biotechnology published a distorted, one-sided and offensive account of our paper, concentrating on the criticisms and ignoring our rebuttal completely.

Our paper reviews and synthesises existing scientific findings on the cauliflower mosaic viral (CaMV) promoter that is in practically all GM crops already commercialised or undergoing field trials. The findings suggest to us that artificial gene-constructs containing the CaMV promoter may be especially prone to breaking and joining up with other genetic material, thereby increasing the chance that it can be transferred horizontally to unrelated species. The potential hazards are harmful mutations, cancers, reactivation of dormant viruses and generation of new viruses. These considerations are especially relevant in the light of recent findings by Arpad Pusztai and his collaborator Stanley Ewen (The Lancet 354, p.1353, 1999), that transgenic potatoes - containing the CaMV 35S promoter - may be unsafe for young rats, part of the effects being attributed to the construct or the genetic engineering process, and hence common to all GM crops.

More significant still, secret documents belonging to the US Food and Drug Administration have come to light as the result of a civil lawsuit against the agency's approval of GM foods mounted by a coalition of scientists and religious leaders. These documents reveal that the first GM crop to be commercialised, the Flavr Savr tomato, actually failed to pass the standard safety tests (see www.biointegrity.org). Since then, no comprehensive safety testing has been done on any GM foods. In line with the precautionary principle, we recommend the immediate withdrawal of all GM crops and products containing the CaMV promoter, until and unless they can be proven safe.

Prof. Joe Cummins was the first scientist to question the safety of the CaMV promoter back in 1994 in connection with the Flavr Savr tomato. He pointed out that the promoter could recombine with other viruses to generate new disease-causing viruses. His warning was ignored.

Like supporters of the biotech industry who insist there is no difference between genetic engineering and conventional breeding, our critics deny any difference between the CaMV promoter as a stable, integral part of the virus itself and the promoter in artificial gene constructs which are already known to be unstable. So much so that the structural stability of artificial vectors - made by joining bits of genetic material from viruses and other genetic parasites of many different organisms - is a textbook topic (see Old, R.W. and Primrose, S.B. (1994). Principles of Gene Manipulation 5th ed., Blackwell, Oxford).

What is a 'promoter'?

A 'promoter' is a stretch of genetic material that acts as a switch for turning genes on. Every gene needs its own promoter. But the promoter is not a simple switch like that for an electric light, which has only two positions, either fully on or fully off. Instead, the gene promoter has many different parts or modules that act as sensors, to enable it to respond, in ways we do not yet fully understand, to signals from other genes and from the environment. These signals tell it when and where to switch on, by how much and for how long. And under certain circumstances, the promoter may be silenced, so that it is off all the time.

The role of the promoter of a normal gene in an organism is to enable the gene to work appropriately in the complex regulatory circuits of the organism as a whole.

The promoter associated with each of the organism's own genes is adapted to its gene, while the totality of all the genes of the organism have been adapted to stay and work together for millions, if not hundreds of millions of years.

When genetic engineers transfer foreign genes into an organism to make a GMO, they also have to put a promoter in front of each of the genes. The promoter plus the gene it switches on make up a 'gene-expression cassette'. Several gene-expression cassettes are usually stacked, or linked in series; one or more of them will be genes that code for antibiotic resistance, which will enable those cells that have taken up the foreign genes to be selected with antibiotics. The stacked cassettes are then spliced, in turn, into an artificial gene carrier or 'vector'.

The vector itself is generally made by joining together parts of viruses and other infectious genetic parasites (plasmids and transposons) that cause diseases or spread antibiotic and drug resistance genes.

In the case of plants, the most widely used vector is the 'T-DNA' which is part of the tumour-inducing plasmid ('Ti plasmid') of Agrobacterium, a soil bacterium that infects plants and gives rise to plant tumours or galls.

The role of the vector is to smuggle genes into cells that would otherwise exclude them. More importantly, the vector can invade the cell's genome and so enable the gene-expression cassettes it carries to become incorporated into the genetic material of the cell.

The artificial transgenic constructs are very complicated and unstable, and are designed for invading the genome - the totality of the cell's own genetic material which is organised in specific ways.

However, the genetic engineer cannot control where and in what form the vector jumps into the genome of the cell. And this is where the first unpredictable effects arise. Each transgenic line or GMO is unique, and gives rise to different unintended effects. In the case of food, this can mean unexpected toxins and allergens.

Why use a promoter from the CaMV?

Like all viruses, CaMV is a genetic parasite that has the ability to infect cells and hijack the cell to make many copies of itself in a short period of time. Its promoter is therefore very aggressive and, in its isolated form, is active in all plants, monocots, dicots, algae, and the E. coli bacteria that live in the gut of all mammals. Hence, the CaMV promoter is very popular with genetic engineers. It can make the gene placed next to it turn on full blast in any plant genome, at perhaps a thousand times the volume of any of the organism's own genes. Having it in the genome is rather like having the loudest phrase of a heavy-metal piece, played with the most powerful amplifier, over and over again, throughout a live performance of a Mozart concerto.

What the CaMV promoter does is to place the foreign gene outside the normal regulatory circuits of the host organism, subjecting the host organism to unremitting metabolic stress. This will multiply the unintended, unpredictable effects in the GMO. It may also be another reason why GMOs are notoriously unstable (Finnegan, J. and McElroy, D. (1994). Bio/Technology 12, 883).

Another characteristic of the CaMV promoter is that it is modular in structure, with parts that are common to and interchangeable with promoters of other plant and animal viruses.

A key recent finding is that the CaMV promoter contains a 'recombination hotspot' - a site where the DNA tends to break and join up with other DNA, thus changing the combination and arrangement of genes (Kohli et al (1999). The Plant Journal 17, 591, 1999). Furthermore, the CaMV promoter recombination hotspot strongly resembles the borders of the T-DNA vector carrying the foreign gene constructs, which are also known to be prone to recombination. Actual recombination has been demonstrated in the laboratory between viral genes engineered into plants and infecting viruses, resulting in new viruses.

Structurally unstable

Artificial constructs of all kinds are already known to be structurally unstable, and recombination hotspots are expected to further exacerbate the instability. This implies that parts, or all of the foreign gene-constructs may be more likely to jump out of the genome and successfully invade the genomes of unrelated species, in principle, all species interacting with the GM plant: bacteria, fungi, earthworms, nematodes, protozoa, insects, small mammals and human beings. This process is uncontrollable and cannot be recalled. The gene-constructs have been designed to be invasive and to overcome species barriers; once released, they will invade different organisms especially bacteria which are in all environments, where they will multiply, mutate and recombine.

The major consequences of the horizontal transfer of gene-constructs are the spread of antibiotic resistance marker genes among bacteria and the generation of new bacteria and new viruses that cause diseases from the many bacterial and viral genes used.

The generation of new viruses could occur by recombination with live or dormant viruses, which we now know to be present in all genomes, plants and animals included. Recombination between viral genes in GM plants and infecting viruses has been demonstrated (Wintermantel, W.M. and Schoelz, J.E. (1996). Virology 223, 156-64.) Recombination with defective, dormant animal viral promoters may also occur. Recombination of CaMV promoter modules with defective promoters of animal viruses may result in recombinant promoters that are active in animal cells. This may reactivate the virus, generate new viruses or give functional viral promoters causing over-expression of one or another of dozens of cellular genes now believed to be associated with cancer.

What our critics say, and why they may be mistaken

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.

First, what we have been eating is mainly intact virus and not naked viral genomes. One of the most surprising recent discoveries is that the naked viral genome - the genetic material taken out of the viral protein coat - is found to give full-blown infections in non-host species that are not susceptible to the intact virus (see Rekvig, O.P. et al (1992). Scand. J. Immunol. 36, 487-95).

Moreover, as said earlier, the 35S promoter in the CaMV is a stable, integral part of the virus, and cannot be compared to the 35S promoter in artificial gene-constructs. 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 replication of the virus takes place in the cytoplasm away from the genetic material of the cell (Covey, S., et al (1990). Proc. Nat. Acad. Sci. USA 87, 1633-7). But that says nothing about the 35S promoter in the gene-constructs that are integrated into host genomes.

As proviral sequences (non-active genomes of viruses) 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 artificial 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 (Meyer, M. and Dessens, J. (1997). J. Gen. Virol.78, 147-51). There is also evidence that proviral sequence in the genome can be reactivated (Nowora, T. et al (1999). Virology 255, 214-20).

Worse

Our critics point out that plants are 'loaded' with potentially mobile elements, and therefore adding another cannot be harmful. However, this 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. The integration of transgenic constructs containing the 35S promoter may mobilise the elements. The elements may in turn provide helper-functions to destabilise 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, the US, British, French and more than 150 other governments agreed to implement the precautionary principle,which is also sound science. 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.

Acknowledgment

I am very grateful to Joe Cummins and Angela Ryan for helpful discussions in preparing this account.

Dr Mae-Wan Ho, of the Institute of Science in Society, is a Reader in Biology at the Open University, UK, and a Fellow of the US National Genetics Foundation.

 


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