Mae-Wan Ho and Angela Ryan Institute of Science in
Society and Biology Department, Open University, Walton Hall, Milton Keynes,
MK7 6AA, UK Joe Cummins Department of Plant Sciences, University of Western Ontario,
(We are ignoring the comments of P. Christou, as they bear little
relationship to the actual article that we submitted, and was published in
your Journal. Our remarks are directed to the critiques from Hull, R.,
Covey, S.N. and Dale, P. of the John Innes Centre, and from Oliver
Rautenberg of Biolinx.)
We reviewed and synthesized existing findings to predict potential
As Rautenberg (1) rightly points out, our paper (2) was not drawn from
research work that we have done ourselves, rather it was written to review
and synthesize the scientific literature on and around the CaMV 35S
promoter. This is a legitimate and important part of scientific activity,
as science does not consist of isolated facts which bear no relationship
to one another. It is precisely the web of mutual interrelationships of
the findings that constitute science. More importantly, this maps out the
universe of possibilities both for further research and for predicting
potential hazards in risk assessment. Our critics disagree with the
implications we draw from the scientific findings, and especially with our
conclusions and recommendation.
To recapitulate, we pointed out that the CaMV 35S promoter is
promiscuous in function, and works efficiently in all plants, as well as
green algae, yeast and E. coli. It has a modular structure, with
parts common to, and interchangeable with promoters of other plant and
animal viruses. It also has a recombination hotspot, flanked by multiple
motifs involved in recombination, and is similar to other recombination
hotspots including the borders of the Agrobacterium T DNA vector
most frequently used in making transgenic plants. The suspected mechanism
of recombination double-stranded DNA break-repair - requires little
or no DNA sequence homologies. Finally, recombination between viral
transgenes and infecting viruses has been demonstrated in the laboratory.
Transgenic constructs are already well-known to be unstable, and the
existence of a recombination hotspot will exacerbate the problem.
Consequently, transgenic constructs containing the CaMV promoter may be
more prone to horizontal gene transfer and recombination than
nontransgenic DNA. The
potential hazards include genome rearrangement, insertion mutagenesis,
insertion carcinogenesis, the reactivation of dormant viruses and
generation of new viruses (reviewed in refs. 3 and 4). These
considerations are especially relevant in the light of recent findings
that certain transgenic potatoes - containing the CaMV 35S promoter and
transformed with Agrobacterium T-DNA - may be unsafe for young
rats, and that a significant part of the effects may be due to "the
construct or the genetic transformation (or both)" (5). Consequently,
we called for all transgenic crops and products containing the CaMV
promoter to be withdrawn and banned, which is in accordance with the
precautionary principle as well as sound science.
Objections raised by our critics
Our critics do not disagree with our description of the findings but
with the implications we draw, and with our conclusion. They raised a
number of objections, which are listed as follows.
Cauliflower mosaic virus infects a
wide range of crucifers eaten by human beings, and no ill effects have
been found either through recombination to cause over-expression of
normal genes or by producing new viruses (1,6).
CaMV and other pararetroviruses, as well as the numerous related
retrotransposons present in plant genomes have never been found to
generate new viruses by recombination "inspite of intensive
research on these virus groups" (6).
CaMV promoter is not unique. Many plant promoters are expected to
share similar features. Recombination is a normal feature of
conventional plant breeding and of all populations. Therefore, no new
viruses could be generated by CaMV promoter in transgenic plants.
The recombination events described by Kohli et al (7) which
we refer to, gives evidence for recombination events before the
transgene is integrated and is therefore not relevant to its stability
in the transgenic plants and may be due to their particular construct
consisting of the CaMV 35S promoter being associated in all three linked
gene-expression cassettes (6).
Relationship between CaMV and hepadnaviruses such as the human
hepatitis B virus is not sufficient for the CaMV promoter to recombine
with it. They have significantly different replication cycles and there
is no sequence similarity between the hotspot of the CaMV 35S promoter
and the hepadnavirus sequences important in replication (6).
For CaMV promoter to recombine with human or other animal or plant
viruses, or to cause over-expression of host genes, the entire promoter
would have to be excised and reinserted precisely at the new site or its
3 end linked precisely with the host gene or the viral gene. This
is not likely, as there is only one recombination hotspot associated
with the CaMV 35S promoter (6). Furthermore, the intact promoter will
not survive digestion in the animals gut.
Recombination involving the CaMV 35S promoter, if it occurs, is a
rare event, as transgenic sequences are present in very low
concentrations, ie, one or at most several copies per cell. Furthermore,
rare recombinants will be selected out when seeds are bulked up at an
early stage (6).
Potentially carcinogenic compounds already exist in abundance in
natural food plants (6). Therefore it is unnecessary to be concerned
about generating new toxins or carcinogens in transgenic plants.
We shall try to deal with all of these objections, in the spirit of "enhancing
the debate" (6)
The intact virus differs significantly from the naked viral genomes
The intact, encapsidated CaMV, consisting of the CaMV genome wrapped in
its protein coat, is not infectious for human beings nor for other
non-susceptible animals and plants, as is well-known; for it is the coat
that determines host susceptibility in the first instance. So eating the
intact virus (objection 1 above) is of little consequence. However, the
naked or free viral genomes may be more infectious and have a wider
host-range than the intact virus. Human T-cell leukemia viral genomes
formed complete viruses when injected into the bloodstream of rabbits (8).
Similarly, the genomes of the human polyomavirus BK (BKV) gave a
full-blown infection when injected into rabbits, despite the fact that the
intact BKV is not infectious (9). Recent developments in gene therapy and
nucleic acid vaccines leave little doubt that naked or free nucleic acids
can readily gain access to all cells of model animals and human beings
(4). The fate of such nucleic acids, once internalized, depends on the
particular constructs. For example, it is recently found that integrated
viral sequences in genomes of dead cells are much more readily transferred
horizontally to the genomes of live cells that have taken up the
fragmented DNA (10). It is also found that non-integrated viral sequences
replicated as episomes are rarely, if ever, transferred.
There is a world of difference between viral genomes containing the CaMV
35S promoter as an integral part of the virus - adapted to the virus and
to the host over millions of years of evolution - and the CaMV 35S
promoter taken out of context, joined up with a strange gene and inserted
into a strange genome. So, eating naked viral genomes (objection 1) may
have little effect, but that says nothing about the transgenic DNA
containing the CaMV 35S promoter.
The CaMV 35S promoter in the viral genome differs significantly from
the CaMV 35S promoter in transgenic DNA
As Hull et al (6) emphasize, there are many constraints on
natural recombination, and natural recombination between viruses is very
rare (objection 7), possibly because each viral genome has a natural
integrity and stability resulting from millions of years of evolution. The
35S promoter in the virus does not transfer into genomes because
pararetroviruses like CaMV, do not need to integrate into host genomes to
complete their lifecycle; and the virus replicates in the cytoplasm away
from the host genome. Nevertheless, some pararetroviral sequences have
been found integrated into plant genomes (11).
It is also clear that recombination between viral transgenes and
infecting viruses can occur. A number of studies have demonstrated
that plant viruses can acquire a variety of viral genes from transgenic
plants. It indicates that the viral transgene, isolated from the virus and
integrated into the host genome, cannot be equated with the same gene in
the virus itself. This is relevant to objections 2, 3, 4 5 and 7 raised by
Defective red clover necrotic mosaic virus lacking the cell-to-cell
movement gene, and hence not infectious, recombined with a copy of that
gene in transgenic Nicotiana benthamiana plants to regenerate
infectious viruses (12). Transgenic Brassica napus containing CaMV
gene VI, a translational activator, recombined with the complementary part
of the virus missing that gene (13), and gave infectious virus in 100% of
the transgenic plants. The same experiment carried out in Nicotiana
bigelovii (14) gave infectious recombinants that expanded the host
range of the virus. N. benthamiana plants expressing a segment of
the cowpea chlorotic mottle virus (CCMV) coat-protein gene recombined with
defective virus missing that gene (15). A later report stated that
recombination between transgenes and infecting virus in CCMV was much more
frequent than recombination between co-infecting viruses (16), despite the
fact that transgenic sequences occur at very low concentrations compared
with co-infecting viruses (objection 7).
The same plants transformed with three
different constructs containing the coat protein coding sequence of
African cassava mosaic virus (ACMV) recombined with a deletion mutant of
the coat protein to regenerate the wild-type virus that produced severe
symptoms of infection in the transgenic plants (17).
As all the experiments involved recombination between defective virus
and transgene, it was thought that under natural conditions, when viruses
are not defective, no recombinant viruses would be generated (18).
However, recombination between wild-type CaMV and transgene VI was
demonstrated in N. bigelovii (19). At least one of the recombinant
viruses was more virulent than the wild type.
Green and Allison (20) found that trimming the 3 end of the CCMV
transgene containing the untranslated region (UTR) reduced recombination
to zero, as compared with 3% in the controls. As ribonucleotide sequences
within the 3 UTR are involved in initiating viral replication, the
presence of this sequence may encourage the participation of the transgene
in RNA recombination. This suggests that most, if not all of the
recombinations may involve template-switching between homologous sequences
during viral replication. Recent findings also indicate that the viral
RNA-dependent RNA polymerase of several potyviruses and tomato aspermy
virus have the ability to recognize heterologous 3 UTR (from lettuce
mosaic virus and cucumber mosaic virus) included in transgene mRNAs, and
to use them as transcription promoters (21). These findings have important
implications for the safety of viral resistant transgenic plants in
It has been noted in experiments involving CaMV (19), that the frequency
of recombination is much higher than for other viruses. While recombinant
CCMV was recovered from 3% of transgenic N. benthamiana containing
CCMV sequences, recombinant CaMV was recovered from 36% of transgenic N.
bigelovii. It was suspected that double-stranded DNA breaks may be
involved in the case of CaMV recombination. This may be due to the fact
that the transgenic DNA included the CaMV 35S promoter.
Structural instability of transgenic DNA versus natural host DNA
Our critics believe that the recombination hotspot of CaMV 35S promoter
in transgenic DNA is not unique, as all promoter contain recombination
hotspots and recombination in genomes is a normal event (objection 3).
It is well-known, however, that pieces of DNA taken out of context and
recombined in novel configurations are likely to be unstable, so much so
that structural instability of artificial vectors for genetic engineering
- made by joining pieces of DNA from different viruses, plasmids,
transposons and other sources - is a topic discussed in a text-book on
genetic engineering (22). This instability also renders the artificial
vectors prone to recombination and rearrangement.
The CaMV 35S promoter in the transgenic plant is part of the transgenic
DNA introduced, which is a highly mosaic construct. The CaMV promoter is
joined to a gene it has never been linked to before, to form an expression-cassette.
Several expression-cassettes are often stacked in series, and spliced in
turn into an artificial vector, most often T DNA, which is also known to
be flanked by recombination hotspots (see ref. 7). Such a structure
typical of transgenic DNA is recognized to be unstable and to have a
propensity for rearrangements and for horizontal gene transfer (23, 24).
This is stated explicitly in a recent scientific report commissioned by
the UK Health and Safety Executive (25),
"The location of released genes on mobile
genetic elements or in close association within IS sequences,
transposons, gene cassettes or hot-spots for recombination . can all
increase the probability of horizontal gene transfer." p. 70.
CaMV promoter in transgenic DNA differs significantly from the plants
own promoters, integrated viruses and other possible recombination hotspots
The CaMV promoter in transgenic DNA is also quite different from the
promoters of the plants own genes (objection 3). Structurally, the
plants own promoters will be expected to be much more stable than
the CaMV promoter in transgenic DNA for the following reasons. First, the
plants genes and their promoters exist in an organized genome, where
recombination is predominantly between homologous alleles, so each
promoter will remain associated with alleles of the same gene after
recombination. Second, each host gene and its promoter have been adapted
to each other, structurally and functionally, in the context of the whole
genome, for hundreds of millions of years, and therefore expected to be
much more stable than the transgenic DNA containing CaMV promoter.
Integrated, inactive viruses and retrotransposons, similarly, have been tamed
by the plant, probably for millions of years and hence, again, more likely
to be stable than the new intruder (objection 3). As Hull et al
(6) state, most, if not all of the retrotransposons are no longer mobile.
Third, the mere integration of transgenic DNA into a host chromosome
creates regions of non-homology, which will be expected to further disrupt
chromosomal structure due to unequal cross-over in mitotic and meiotic
Instability of transgene expression as the result of gene-silencing
is now well-recognized and actively researched (see ref. 26 for the latest
mechanism discovered). Structural instability involving secondary mobility
or rearrangement of integrated transgenes is also a common cause of
breakdown of transgenic lines (discussed in ref. 27).
Structural instability of transgenes is generally underestimated, as
cells which lose transgenes are automatically eliminated during the
selection process necessary for producing transgenic lines. However,
instability may arise even in later generations of plant propagation
(objection 7). We are aware of no published data documenting the
structural stability of transgenic lines in successive generations, even
though phenotypic instability has been documented, for example, in
transgenic bt-cotton commercially grown in Southern United States in 1996
(28), and Roundup cotton in 1997 (29). Physiological stress due to
extremes of temperature, or drought, which can mobilize transposons, may
increase transgene instability. The constitutive over-expression of
transgenes linked to the CaMV 35A promoter, similarly is a metabolic
stress-factor that may increase transgene instability (30).
Hull et al (6) suppose that as there is only one recombination
hotspot in the CaMV 35 promoter, it is not likely to undergo horizontal
gene transfer (objection 6). Of course, even one double-stranded break can
give rise to genetic rearrangement, and result in the CaMV 35S promoter
being associated with host gene or proviral sequences. But more to the
point, the transgenic construct typically contains multiple recombination
hotspots. For example, most transgenic plants created with the Agrobacterium
T-DNA vector will have transgenic DNA flanked by the left and right
borders, both recombination hotspots. In addition, gene expression
cassettes include terminators that are also recombination hotspots (as
discussed in ref. 7). So all or part of the transgenic DNA may be prone to
It is quite likely that stacking CaMV promoters in three successive
expression cassettes, as Kohli et al (7) have done, will increase
structural instability further (objection 4). The recombinations and
rearrangements they have observed in the different transgenic lines,
however, may have occurred both before and after the primary
transformation events, during propagation and selection in cell and plant
culture. This is something that must be addressed by empirical
Functional consequences of the modular construction of all promoters,
including the CaMV 35S promoter
The modular construction of promoters has two important consequences,
the first of which is related to the function of promoters. Different
modules or elements in the promoter respond to signals from a battery of
other genes coding for transcription factors, which determine the tissue
specificity, timing and amplitude of gene expression. Thus, promoters
allow genes to talk to one another, forming complex intercommunication
networks that enable the tens of thousands of genes in an organism to
function as a coherent whole, and as appropriate to the environment. Some
of the elements in the promoter also enable it to respond to recombinases
and other enzymes that result in recombination, rearrangement, mobility
and mutation. This kind of natural genetic engineering (31) is
quite precise, as it is regulated by the organism as a whole, in ways that
we still do not understand (32). The structure of chromatin itself is now
known to contribute to this regulation. Histones are modified in
accordance to an as yet unknown histone code
which modulates gene function through chromatin structure in all
eukaryotic genomes (33).
One can see why random insertion of foreign DNA into this incredibly
complex, mutually entangled and subtle regulatory system will give a range
of unexpected, unintended effects, especially when the foreign DNA
includes the strong constitutive CaMV 35S promoter. The integration of
transgenic DNA into genomes is known to have many unexpected effects,
including mutations, cancers (in the case of mammalian cells) and changes
in DNA methylation, a chemical modification of DNA which can affect
activities of host genes. The effects are known to extend far away from
the site of insertion (34). Hull et al (6) are mistaken to suppose
that the CaMV promoter has to be placed exactly next to a gene in order to
make it over-express (objection 6). In a recent experiment in insertion
mutagenesis using a synthetic mini-transposon, researchers discovered an
event resulting in the over-expression of a host gene which is 164
basepairs away from the site of insertion (35).
Thus, the possibility of new toxins and allergens arising cannot be
easily dismissed on account of both position effects due to random
insertion of transgenic DNA and pleiotropic effects due to functional
interactions with host genes. The suggestion that potentially carcinogenic
compounds occur in abundance in natural plants (objection 8) is
irrelevant. These foods have been eaten for tens of thousands of years,
and compounds which are carcinogens in isolation may have very different
effects when eaten in combination with all the other constituents of the
The important point is that transgenic constructs do contain new genes
and new combinations of genes that have never existed in nature, not in
billions of years of evolution. That, at least, is one reason on which
patent claims are made. Furthermore, over-expression of host genes that
become associated with the CaMV 35S promoter as the result of horizontal
gene transfer or genomic rearrangement may also increase the concentration
of otherwise safe metabolites to toxic or carcinogenic levels.
Structural consequences of the modular structure of promoters
Hull et al (6) may also be mistaken to think that the entire
CaMV 35S promoter has to be transferred before it can either lead to
over-expressing of host genes or to reactivating or generating new viruses
(objection 6). On account of the modular construction of all promoters, it
is already clear that many elements are common to many promoters, so much
so that gene therapists are now making synthetic super-promoters by random
recombination of different elements (36). As reviewed in our earlier paper
(2), the CaMV 35S promoter is promiscuous in function, and is active in
combination in whole or in part with other promoters. Therefore, the
transfer of parts of the CaMV 35S containing enhancer or other elements
into genomes may be sufficient to cause over-expression of genes or to
reactivate dormant viruses or generate new viruses.
As pointed out by Hull et al (6), proviral sequences (37) and
related retrotransposons are now found to be present in all genomes,
including those of higher plants (38). The fact that the CaMV promoter is
different in sequence from other promoters does not prevent it from
substituting for a range of viruses. The CaMV 35S promoter has been joined
artificially to the cDNAs of a wide range of viral sequences, and
infectious viruses produced in the laboratory (39, 40). There is also
evidence that proviral sequence in the banana genome can be
reactivated, especially in tissue culture, as demonstrated by a group of
researchers that include one of our critics, Roger Hull (41). He had
earlier warned that viral coat proteins in transgenic plants not only can
offer disguise to related viruses to move around the plant and infect it,
but also that the protein may wrap up retrotransposons in plants and allow
them to be transmitted horizontally to other species (42).
The fact that plants are "loaded" with potentially mobile
elements, such as retrotransposons, can only make things worse. Most, if
not all, of the elements are no longer mobile. But integration of
transgenic constructs containing the 35S promoter may mobilize the
elements. The elements may in turn provide helper-functions to destabilize
the transgenic DNA, and may also serve as substrates for recombination to
generate more exotic invasive elements. It is already known, from
experiments in gene therapy, that retroviral and other sequences can
integrate into mammalian genomes in the absence of viral integrase (43).
Although CaMV 35S promoter and promoters of animal viruses do not have the
same base sequence, they have at least one element (the TATA-box) in
common, if not more. It is therefore possible therefore, for host
protooncogenes and proviral sequences to become activated and reactivated
(objections 5 and 6). Also, completely new cross-species viruses may arise
from recombination between elements and motifs of the CaMV 35S promoter
and those of animal viruses, dormant or otherwise (objection 5).
New research in our critics own research institute are revealing
how plants naturally resist viral infections by making small antisense RNA
of 25 nucleotides against viral genes. Exactly the same mechanism is
directed against transgenes to silence them (26). The authors remark that
the gene-silencing "may represent a natural antiviral defence
mechanism and transgenes might be targeted because they, or their RNA are
perceived as viruses." So much for the claim that genetic engineering
is just like conventional plant breeding.
In signing on to the International Biosafety Protocol in Montreal in
January, more than 130 governments agreed to implement the precautionary
principle. The available evidence clearly indicates that there are serious
potential hazards associated with the use of the CaMV 35S promoter. The
hallmark of science is that it is always provisional and uncertain.
Molecular genetics is a new discipline and our ignorance regarding gene
regulation and the ecological impacts of horizontal gene transfer is
profound. The social responsibility of science and the proper use of
scientific evidence are therefore to set precaution. We appeal to our
critics as fellow scientists to join us in calling for the withdrawal of
all GM crops and products containing the CaMV 35S promoter, both from
commercial use and from field trials, unless and until they can be shown
to be safe. Meanwhile, much more good quality basic research - such at
that carried out in the John Innes Institute should continue under
strictly contained conditions.
We thank Mark Griffiths for drawing our attention to some important
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