Jean-Benoît Morel and Mark Tepfer
Over the past year, the world of transgenic plants has been shaken by a series of controversies concerning questions of biosafety. The general public has often gone directly from a state of a complete lack of information concerning the question posed to that of a full-blown polemic, with all that the latter implies in terms of the absence of objective examination of the current state of knowledge. Quite recently, we have learned of a new potential controversy concerning transgenic plants. Since discussion of these newly raised questions has not reached an over-heated state, we present here a scientific evaluation of the questions raised, which we have attempted to treat in the most unbiased fashion possible.
The subject of the current controversy is the use of the 35S promoter, which has been introduced into a great number of transgenic plants. Transgenes composed of the 35S promoter and a region coding for the protein of interest allow high levels of accumulation of the gene products (RNA and protein) in almost all tissues of the transformed plant. It is precisely this dependably high level of expression that has been the reason for the widespread use of this promoter. Recently, Ho et al. (1) have proposed that there are dangers associated with the use of the 35S promoter, which is from the cauliflower mosaic virus (CaMV), since it might enhance recombination between DNA molecules.
Definition of recombination: covalent joining of of DNA or RNA segments that are normally not adjacent. It can occur either by cleavage and ligation (cut and paste), or in the case of RNA recombination, but a switch in the template used by the RNA polymerase during RNA synthesis.
The starting point of the questions concerning potential risks associated with the 35S promoter is a recent article by Kohli et al. (2), describing a hot spot of recombination in this promoter, which was observed in rice transformation experiments. Ho et al. proposed that this hot spot would destabilize the transgenic DNA, and thus lead to a greater mobility of the 35S promoter, which when introduced into a transgenic plant would be able to hop, and thus insert itself in various sites of the genome. From this initial hypothesis, Ho et al. develop a series of potential consequences of the hypothetical enhanced mobility of the 35S promoter.
The key hypothesis in the above scenarios is that a hot spot for recombination in the 35S promoter would cause its mobility in a plant or animal genome. We will thus examine with particular care whether this hot spot does in fact exist, and under what conditions. The 35S promoter is from a virus, CaMV, that infects numerous crucifers. During its replication, this virus goes through successive DNA and RNA phases, as do the human immunodeficiency virus (HIV) and hepatitis B virus, as well as certain related non-pathogenic molecules, called retrotransposons. Contrary to what is observed with HIV, the CaMV DNA does not integrate into the host's genomic DNA. The 35S promoter is the region of CaMV DNA that controls the synthesis of the RNA form of the genome, called 35S RNA, from which the viral DNA is synthesized in turn by the viral reverse transcriptase, thus closing the viral replication cycle.
The results of Kohli et al. do indeed show that there is a hot spot for recombination in the 35S promoter (2). However, since CaMV has been a model system for recombination studies, it is important to consider the meaning of these results in the context of the body of previous studies. Kohli et al. studied rice plants that had been transformed by biolistics (bombardment with microscopic DNA-coated metal beads) with three genes, all of which were under the control of the 35S promoter. They observed that multiple copies of the DNA had been inserted next to each other in the rice genome (although they did not indicate how frequently they observed multiple insertions). They then localized precisely the junctions between the different copies of the inserted foreign DNA, which do indeed constitute sites of DNA recombination. They have found that a precise site of the 35S promoter was involved in four of eleven recombination sites examined, which allows them to conclude that this site does constitute a hot spot for recombination, at least compared to the rest of the 35S promoter.
Nonetheless, if we examine their results closely, it becomes evident that we should impose certain limits on how they are interpreted. One point is that the 35S promoter is present in three copies in the foreign DNA, while the rest of the region studied is present only in a single copy. Logically, one would expect this to favor recombination between copies, and indeed the 35S hot spot was only used in recombination events occurred between copies of the 35S promoter. In addition, their analysis of the structure of the DNA transferred clearly shows that other numerous recombination events occurred during integration of the foreign DNA into the rice genome. The authors themselves propose that other hot spots for recombination exist in the foreign DNA. This raises the question of the importance of the hot spot in the 35S promoter relative to other sequences that favor recombination.
It is also important to note that during the biolistic transformation process, there is an initial phase in which massive quantities of foreign DNA are introduced into the bombarded cell, of which only a small part is integrated into the DNA of the target cell. It is well known that large quantities of broken DNA stimulate the cell's DNA repair machinery, and also favor recombination. A relatively old study has shown that even the structure of the DNA integrated is influenced by the amount of DNA initially introduced into the cell, since this is determined by the degree of induction of the DNA repair system (3). It is also important to note that when plants are transformed with a more natural transformation vector, Agrobacterium, the 35S promoter does not behave as a hot spot for recombination; this observation, made in numerous labs over the last 20 years, in itself suggests that the hot spot observed by Kohli et al. is an artefact of the transfomation technique used. The recombination events described by Kohli et al., which in all probability occurred during the process of integration of the foreign DNA, took place under conditions of introduction of massive amounts of broken DNA, which do not at all correspond to those occurring in a stably-transformed transgenic plant that has been regenerated from the initially transformed cell, in which one would expect to find few or no fragments of broken DNA.
Studies of recombination in CaMV carried out ten years ago make it possible to put the results of Kohli et al. in the perspective of more normal physiological conditions. In these experiments, nontransgenic plants were infected with two slightly different CaMV strains, and the recombination events between the two CaMV genomes were mapped. If the hot spot localized by Kohli et al. is truly significant, then we should also find it as a hot spot for recombination between CaMV genomes. The results of Vaden and Melcher (4) are particularly pertinent in this regard. These authors localized 22 sites of recombination between CaMV genomes, among which only two are located in the area of the 35S promoter. Unfortunately, there are too few differences in the sequences of the two CaMV strains to determine if these two recombination sites are localized at the hot spot of Kohli et al., or whether they are at a nearby site, which is well known to be a recombination site that is used during normal replication of the viral genome by the virus's reverse transcriptase (5). In any case, compared to the rest of the CaMV genome, the 35S promoter could not be considered to be a hot spot of recombination, and should not have any particular mobility in the genome of a transformed plant. Finally, we are not aware of any reports of instability in the numerous transgenic plants whose genome has been transformed with multiple genes bearing the 35S promoter.
As we have mentioned above, insertion in the host genome is a normal feature of the retrovirus replication cycle. Retroviruses infecting plants are not known, and the only related viruses, such as CaMV, are not known to normally have an integrative phase. Nonetheless, it is important to note that viral sequences have recently been found inserted in the genome of petunia (6), banana (7, 8) and tobacco (9, 10). In the tobacco genome one finds numerous copies of the genome of two viruses, and in both cases the copies that have been sequenced are incomplete and have numerous mutations. Considering this, the accidental insertion of a promoter adjacent to these viral sequences, even if it did allow their transcription, would not be able to generate a functional viral genome. In contrast, in the case of banana, functional copies of the inserted viral genome are likely to exist, since under certain conditions, independent virus can be generated from the inserted sequences. In petunia, the integrated viral sequence have not been characterized, but they can be presumed to be functional, since, here too virus can be generated spontaneously from the integrated sequences. However, considering the alternation of RNA and DNA molecules in the replication cycle of these viruses, insertion of a 35S promoter would not itself suffice to generate an infectious virus, since several essential elements (including the 35S promoter itself!) would be absent from the RNA transcribed. In addition, in putting these results in perspective it is important to recall that plant genomes contain enormous numbers of naturally mobile elements, called transposons, which have strong promoters, and that activation of dormant genes or viruses is much more likely to occur by insertion of a transposon than by the purely hypothetical insertion of foreign DNA that is normally not mobile. This same reasoning applies to the hypothesis of activation of a gene coding for a toxin.
Among the potential risks that Ho et al. attribute to the 35S promoter, the most frightening is the proposition that it could activate dormant oncogenes in animals, including humans, that consume transgenic plants that include the 35S promoter. Here the mechanism would be similar to that proposed above: we consume DNA containing the 35S promoter, this DNA could insert adjacent to a dormant oncogene, the promoter could transcribe the gene, a cancer could be induced. In this hypothetical chain of events, there is at least one link that is relatively plausible. It has long been known that the 35S promoter is functional, not only in plants, but also in yeasts and certain bacteria. Its activity has also been shown in the oocytes of Xenopus, an amphibian (11). How far this can be generalized is at this time unknown, since to the best of our knowledge the activity of the 35S promoter has never been studied in mammalian cells, or in vertebrates other than Xenopus.
Nonetheless, examination of the significance of the first step in the hypothetical chain of events (we consume DNA containing the 35S promoter) makes it possible to invalidate this hypothetical chain of events. The essential point is that in fact we already consume large quantities of DNA containing the 35S promoter in non-transgenic plants. As we mentioned near the beginning of this article, CaMV infects various members of the cabbage family (including radish, cabbage, broccoli, cauliflower, watercress, etc.).
Several scientists have estimated that on the order of 10% of cabbages are infected with CaMV (12). In an epidemiological study of cauliflower, it was shown that on the order of 50% of the plants were infected with CaMV (13), and each infected cell contains tens of thousands of viral genome copies, including both "naked" DNA and DNA in viral particles (14). Thus, if comsumption of DNA containing the 35S promoter is indeed dangerous, then we have much more to fear from comsuming CaMV-infected plants than from transgenic ones.
It is always useful to periodically re-examine potential risks in the light of scientific knowledge, and in particular in the light of new information. But, finally, what should we conclude after a careful examination of the questions raised concerning the 35S promoter?
· The results of Kohli et al. do show that in an experiment of biolistic gene transfer there is a hot point of recombination in the 35S promoter. In fact what they have shown is that at the step of integration of the foreign DNA, the precise site in question does seem to be favored relative to the rest of the 35S promoter. This does not however show that the 35S promoter is a hot spot relative to other DNA regions, since Kohli et al. also show that there are numerous other uncharacterized recombination sites elsewhere in the DNA that was integrated in their transgenic plants. In addition, previous work shows that the site described by Kohli et al. is not a hot spot relative to the rest of the CaMV genome. Thus, nothing in the current state of knowledge leads us to conclude that the DNA of the 35S promoter would have any particular mobility in the genome into which is has been introduced. The 35S promoter would not be expected to have anything like the degree of mobility of the transposons which are extremely numerous in plant genomes, and which possess biochemical tools whose role is to favor their mobility in the host genome.
· In genetic transformation experiments, the DNA is integrated at random, and thus can modify the expression of genes adjacent to the site of insertion. In the absence of any evidence for DNA mobility after integration, any such effects would be detectable in the regenerated plants, and would not be expected to change appreciably thereafter. In any case, the activation of inserted viral sequences, or that of genes encoding a toxin would be much more likely to occur due to the natural phenomenon of transposon mobility than due to mobility of the DNA inserted stably in the genome of a transgenic plant.
· As we have mentioned, the 35S promoter is active in cells of organisms other than plants, including a vertebrate, Xenopus. One can indeed wonder about its activity in mammalian cells, and to the best of our knowledge this has not been studied. However, we consume DNA containing the 35S promoter each time we eat CaMV-infected cabbage or other crucifer plants. In addition, we consume enormous quantities of foreign DNA promoters that are perfectly adapted to be active in mammalian cells each time we eat animal-derived foods. To the best of our knowledge, consumption of promoters in animal DNA has not yet been shown to be deleterious, much less those in plant DNA.
A general point that is underlined by this evaluation of potential risk associated with the 35S promoter is the necessity to use the situation in equivalent non-transgenic organisms as a baseline for comparison to the situation including transgenics. The risks that are essential to scrutinize with care are the new ones, the ones that are additional relative to the pre-existing non-transgenic situation. For the questions examined here, the very hypothetical potential risks proposed do not present any hazards that are new, since the DNA in question is abundant in non-transgenic plants. Considering the complexity of the debate concerning GMOs, which is not only scientific (risk assessment), but also touches on important questions of an economic, sociological or political nature, it is essential to take care to make the distinction between scientific questions raised for reasons that are primarily political, and the truly scientific ones that merit devoting considerable effort of analysis and reflection.
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Last Updated on 6/14/00
By Karen Lutz Benbrook