Mae Wan Ho
Institute of Science in Society (ISIS)

The story of CaMV promoter encapsulates and draws attention to the hazardous nature of the genetic engineering process itself as well as the foreign gene constructs created and released into the environment.

Contrary to our usual practice, we are reviewing this publication before it is actually in print because of the overwhelming number of attacks on it which have already appeared on the net, and because we have been asked to provide an accessible account of this somewhat technical article. Our official rebuttal has been circulated earlier. In case you have missed it please visit our ISIS website.

Prof. Joe Cummins of the University of Western Ontario was the first scientist to question the safety of the cauliflower mosaic viral (CaMV) promoter, which is in practically all GM crops currently grown commercially or undergoing field trials. His initial concern was that the promoter could recombine with other viruses to generate new disease-causing viruses. In our paper, we review some recent findings which give further grounds for concern, and have recommended the immediate withdrawal of all crops and products containing the CaMV promoter.

Ref.: Ho, M.W., Ryan, A. and Cummins, J. (1999). The cauliflower mosaic viral promoter - a recipe for disaster? Microbial Ecology in Health and Disease (in press).

To begin with, a 'promoter' is a stretch of genetic material that acts as a switch for turning genes on. Every gene needs a promoter in order to work, or to become expressed. But the promoter is not a simple switch like that for an electric light, for example, which has only two positions, either fully on or fully off. Instead, the promoter has many different modules that act as sensors and to enable it to respond, in ways we do not yet fully understand, to different signals from other genes and from the environment, which 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.

All in all, the role of the promoter of a normal gene in an organism is to enable the gene to work appropriately in the extremely 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. The genome of each organism is organised in a certain way which is more or less constant across the species so individuals within a species can freely interbreed. Each species protects its integrity and remains genetically stable because there are biological barriers that prevent distant species from interbreeding or otherwise exchanging genetic material. Foreign DNA are generally broken down or inactivated. Genetic engineering attempts to break down these biological barriers so genes can be arbitrarily transferred between species that would never interbreed in nature. In order to do so, special tricks are needed.

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 transferred, otherwise it would not work. The promoter plus the gene it switches on constitutes a 'gene-expression cassette'. Many of the genes are from bacteria and viruses, and the most commonly used promoter is from the cauliflower mosaic virus. 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 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 give rise to plant tumours or galls. The role of the vector is to smuggle genes into cells that would otherwise exclude them. And more importantly, the vector can jump into the cell's genome and so enable the gene-expression cassettes it carries to become incorporated into the genetic material of the cell. The genetic engineer cannot control where and in what form the vector jumps into the genetic material of the cell, however. And this is where the first unpredictable effects can arise. Each transgenic line is unique, and gives rise to different unintended effects, and in the case of food, can include unexpected toxins and allergens.

The foreign genetic material transferred to make a transgenic organism - referred to as the 'transgenic DNA' or the 'construct' - is quite complicated. It consists of new genes and new combinations of genes - from diverse species and their genetic parasites - which have never existed in nature. Such chimaeric constructs are already known to be structurally unstable, that is, they are prone to make and break and rearrange. It is to be expected that such structural instability can only increase when the construct is introduced, by a totally hit or miss process, into a new genome. Transgenic instability is a well-known problem for the industry. Transgenic lines often do not breed true (see Srivastava et al, 1999, in item #3 below).

Why use a promoter from a virus such as the CaMV? A virus is a genetic parasite that has the capability to infect the cell and hi-jack the cell to make many copies of itself in a short period of time. Its promoter is therefore very aggressive and hence popular with genetic engineers, as it effectively makes the gene placed next to it turn on full blast, at perhaps a thousand times the volume of any of the organism's own gene. Having it in the genome is rather like having the loudest phrase of a heavy-metal piece played with the most powerful amplifier simultaneously over and over again throughout a live performance of a Mozart concerto. What the CaMV promoter actually does is to place the foreign gene outside the normal regulatory circuits of the host organism, subjecting the host organism effectively to a permanent metabolic stress. This will multiply the unintended, unpredictable effects, which are legion in transgenic organisms. It may also be another reason why transgenic lines are notoriously unstable (Finnegan, J. & McElroy, D. 1994, Bio/Technology 12, 883). The organism generally reacts to the presence of foreign genetic material by breaking it down or inactivating it. Even after the genetic material is incorporated into the genome, it can silence the foreign genes so that they are no longer expressed (see Item #3 below).

The key recent finding, which provoked our review, was the report (Kohli et al, (1999) The Plant Journal 17, 591) 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. Around the hotspot are several short stretches or modules for binding various enzymes, all of which are also involved in recombination, ie, breaking and joining DNA. Furthermore, the CaMV promoter recombination hotspot bears a strong resemblance to the borders of the T-DNA vector carrying the transgenes, which are also known to be prone to recombination. It is that which enables the vector to invade the cell's genome in the first place.

The aim of our original paper, restated explicitly in our official rebuttal, was to review the relevant findings and, in particular, to point out the implications, which the researchers themselves are unwilling or unable to draw. The findings that transgenic DNA has the tendency to break and join in several places imply that parts or all of it may be more likely than the plant's own DNA to jump out of the genome and successfully transfer horizontally to unrelated species. Horizontal gene transfer, in this context, means the transfer of the genetic material directly by infection to the genetic material of unrelated species, in principle to all species interacting with the GMO: bacteria, fungi, earthworms, nematodes, protozoa, insects, small mammals and human beings. This process is uncontrollable and cannot be recalled. The damages done are hence irreversible. Transgenic DNA has been designed to be invasive and to overcome species barriers; once released, it will invade different organisms, especially bacteria which are in all environments, where it will multiply, mutate and recombine.

There are additional findings which suggest an increased potential for the horizontal spread of transgenic DNA. For example, enzymes that insert the transgenic DNA into the genome can also help them to jump out again; DNA released from both dead or live cells can survive without being degraded in all environments, including the mouth and gut of mammals; DNA can be readily taken up into cells; and all cells can take up naked or free DNA. The instability of transgenic DNA may also be enhanced as the result of the metabolic stress inflicted on the organism by the CaMVpromoter that gives continuous over-expression of transgenes.

The major consequences of the horizontal transfer of transgenic DNA 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 that we now know to be present in all genomes, plants and animals included. Recombination with defective, dormant animal viral promoters may also occur, as we know that there are modules within the promoter that are interchangeable between plant and animal promoters. Recombination of CaMV promoter modules with defective promoters of animal viruses may result in recombinant promoters that are active in animal cells, causing over-expression of one or the other of dozens of cellular genes which are now believed to be associated with cancer.

There is sufficient scientific evidence to support well-founded suspicion of serious, irreversible harm to justify the immediate withdrawal of all GM crops and products containing the CaMV promoter from environmental release.

(M.W.H.)