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Cauliflower Mosaic Viral Promoter - A Recipe for
Disaster?
(Microbial Ecology in Health and Disease)
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Mae-Wan Ho, Angela Ryan, Biology Department
Open University, Walton Hall Milton Keynes, MK7 6AA, UK
and Joe Cummins Dept. of Plant Sciences,
University of Western Ontario, Ontario, Canada.
Abstract
Concerns have been raised over the spread of transgenic DNA by
horizontal gene transfer. One main factor determining the success of
horizontal gene transfer is its tendency to recombine. This paper examines
the safety implication of recent revelations on the recombination hotspot
of the cauliflower mosaic viral (CaMV) promoter, which is in practically
all current transgenic crops released commercially or undergoing field
trials.
As a precautionary measure, we strongly recommend that all transgenic
crops containing CaMV 35S or similar promoters which are recombinogenic
should be immediately withdrawn from commercial production or open field
trials. All products derived from such crops containing transgenic DNA
should also be immediately withdrawn from sale and from use for human
consumption or animal feed.
The release of transgenic crops into the environment has raised concerns
over the spread of transgenic DNA, not only by cross-pollination to
related species, but especially by horizontal gene transfer to unrelated
species (reviewed by Ho et al (1) and Traavik (2)). On account of the
persistence of DNA in all environments, and the ability of practically all
cells to take up 'naked' or free DNA, the success of horizontal gene
transfer may depend largely on the nature of the DNA itself. New
revelations concerning the CaMV recombination hotspot (3) have prompted us
to consider the safety implications of the CaMV promoter. That is all the
more urgent as CaMV promoter is in practically all transgenic crops
already released commercially or undergoing field trials.
Cauliflower Mosaic Virus (CaMV) is a pararetrovirus of crucifer plants.
The genome is an 8-kbp double-stranded circular DNA with three single
strand gaps. Two major RNA transcripts (19S and 35S) and six large open
reading frames are encoded by the DNA. Transcription occurs from a
nonintegrated, circular minichromosome in the nucleus of the plant cell,
and virion DNA is synthesised in the cytoplasm by reverse transcription of
the 35S RNA transcript (4, 5). Phylogenetically, CaMV belongs to a group
of caulimoviruses most closely related to the hepadnaviruses of animals,
which includes the human hepatitis B virus. The reverse transcriptase (RT)
of CaMV, however, is most similar to that of retrotransposons belonging to
the Gypsy group and also to that of retroviruses (6). This suggests that
CaMV evolved subsequent to the horizontal transfer of a retrotransposon to
the cruciferae, either as the result of capture of RTgene by a
pre-existing virus or by the transposable element acquiring additional
genes to become a virus.
The CaMV promoter is a sequence of about 350 basepairs upstream of the
35S transcript (-343 to +8, with Cap site at +1), about 250 basepairs of
which overlap with the 3' end of gene Vl, the last of the six large open
reading frames. There are three domains in the promoter, the core promoter
containing the TATA box (-46 to +8), and two other major domains with
enhancer functions. Region A (-90 to -46) is mainly required for
expression in roots, and region B (-343 to -90) for expression in leaves
(see (7, 8) and references therein). Subdomains of the B region (B1 to B5)
can be recognised based on differential interactions with various
transcription factors. However, the complete B region allows a more
general constitutive expression than expected from the combinations of
subdomains. This suggests that important sequence elements are at the
interfaces of the subdomains, or that the combination of subdomains is not
simply additive but results in qualitatively novel specificities.
The roles of the different 35S promoter domains in pathogenesis of CaMV
have only been studied fairly recently (4, 9). These studies show that the
loss of up to 40 amino acids from the 3' end of gene Vl (which overlaps
with the 35S promoter) had no effect on pathogenesis whereas further
truncation into a putative zinc finger region was fatal to the virus.
Removing the TATA box also abolished infectivity. However, upstream
deletions within the enhancer region between -207 and -56 were tolerated
even though complete removal of this fragment caused loss of infectivity.
Two separate enhancer domains for infectivity were identified, -207 to
-150 and -95 to -56, only one of which is necessary. The enhancer region
could even work in reverse orientation. Foreign gene sequences could be
inserted into deletion mutants, which may alter the infectious
characteristics of the virus.
Various hybrid or combination promoters have been constructed from the
CaMV 35S promoter which led to improved expression of transgenes: double
35S promoters (10), a hybrid containing the core 19S promoter from CaMV
and the 35S upstream enhancers (11), and combination of CaMV 35S with
mannopine synthase elements (12), or with Adh1- and ocs-promoter
elements for expression in monocotyledons (13). These results emphasise
the modularity and interchangeability of promoter elements (8), which have
important implications for the safety of transgenic plants. It means, in
effect, that recombination of the CaMV promoter elements with dormant,
endogenous viruses may create new infectious viruses in all species to
which the transgenic DNA is transferred.
Another factor which affects the safety of transgenic plants containing
CaMV promoters and related constructs is that although CaMV itself infects
only dicotyledons, its promoter is promiscuous; and functions efficiently
in monocotyledons (14), in conifer cell lines (15), green algae (16),
yeasts (17) and E. coli (18). The transfer of CaMV promoter to
these other species could also give rise to unpredictable effects on gene
expression, which may impact on the ecosystem as a whole.
It has been known for some time that recombination can occur between
different CaMV viral strains in plants (19), between different homologous
parts of an integrated CaMV viral sequence in transgenic plants (20)and
between an integrated transgene and an infecting virus (21). Analysis of
the junctions of recombination suggests that one of the recombination
hotspots was at the 3'end of the 35S promoter, and was thought to be due
primarily to template switching during reverse transcription. This kind of
recombination depends on sequence homology between the recombining
partners as well as the action of virally encoded reverse transcriptase,
and is expected to have little impact on nonhomologous DNA belonging to
other organisms. However, it was suspected that recombination may also
occur between double-stranded DNA, as recombination junctions were found
away from the initiation site of DNA synthesis (where the 35S promoter is
located).
Double-stranded DNA break repair (DSBR) is recognized to be involved in
the illegitimate recombination which enables plasmid DNA to integrate into
plant genomes following plant transformation (22-23); and transgene
rearrangements have been identified in both Agrobacterium-mediated
transformation (24) and particle bombardment (25). Illegitimate
recombination was also observed between a resident transgene in a
transgenic tobacco plant and a newly delivered transgene (26).
Illegitimate recombination involves sequences with either microhomology or
no homology between the junctions, often resulting in filler DNA and
deletions of nucleotides from one or both of the recombining ends (27).
Kohli et al (3) analysed 12 multicopy transgenic rice lines
transformed with a co-integrate plasmid by means of particle bombardment
in order to investigate the fate of exogenous transforming DNA. They not
only discovered the same kind of illegitimate recombination between
plasmids, but also that many of the illegitimate recombinations were
located to the CaMV 35S promoter hotspot previously identified (19, 20).
Furthermore, recombination occurred at high frequency without the virally
encoded reverse transcriptase or other enzymes, suggesting that plant
factors can direct recombination events by recognising and using these
highly recombinogenic viral sequences.
The hotspot located by Kohli et al (3) was an imperfect
palindrome of 19bp at the 3’ end of the CaMV 35S promoter containing
the TATA box. The palindrome and surrounding DNA sequences were found to
have a number of characteristics common to known recombination hotspots.
One half of the 19 bp palindrome was purine rich, and it is known that
recombinase proteins bind to such regions. Topoisomerase I cleavage
sites, the trinucleotide AAG, are also found clustered around the
recombining junctions in the hotspot, which is either part of the junction
or present within 3 bp of it in 8 out of the 11 junctions analysed by
Kohli et al (3). A 32 bp region with 90% AT content was found in
the 35S promoter 28 bp upstream from the palindrome. AT-rich regions cause
isotropic DNA bending and influence DNA melting. They contain matrix
attachment region (MAR) motifs, which harbour intrinsically curved DNA,
and have been found in the vicinity of other recombination hotspots. The
19 bp palindrome itself contains a short tract of alternating
purine-pyrimidine (AT) residues situated 50 bp upstream from another
alternating purine-pyrimidine sequence in the transgene. Such
residues are known to adopt Z DNA conformation and have been shown to
influence transcription and recombination, and are also binding sites for
topoisomerase II, which is specifically involved in the resolution of
recombination intermediates.
The structure and sequence-specific properties of the 3’ end of
the CaMV 35S promoter are similar to the petunia transformation
booster-sequence which increased plant transformation efficiency, most
probably by stimulating recombination (28). Similar structures and
sequence-specific characteristics were identified for recombinogenic
regions of SV40 DNA in Hela cells (29). The 25bp border repeats of the
Agrobacterium T-DNA, the most commonly used vector for plant
transformation, also show remarkable similarities to the recombination
hotspot of the CaMV 35S promoter. There is an 11 bp imperfect plaindrome
sequence with a TATAbox-like structure in the right border whereas the
left border has a short purine-rich sequence in the centre. Kohli et
al (3) predicts that these two regions of T-DNA could be involved in
rearrangements which are often seen in T-DNA mediated plant
transformations.
It is clear that the CaMV 35S promoter is well-endowed with motifs
involved in recombination. An additional factor which may increase the
instability of the plasmid is the junction between CaMV 35S promoter and
foreign DNA. All these considerations make it highly likely that the CaMV
35S promoter will take part in horizontal gene transfer and recombination,
and also cause largescale genomic rearrangements in the process.
Horizontal transfer of the CaMV promoter not only contributes to the
known instability of transgenic lines (30), but has the potential to
reactivate dormant viruses or creating new viruses in all species to which
it is transferred, particularly in view of the modularity and
interchangeability of promoter elements (8). In this regard, the close
relationship of CaMV to hepadnaviruses such as the human hepatitis B is
especially relevant. In addition, because the CaMV promoter is promiscuous
in function (see above), it has the possibility of promoting inappropriate
over-expression of genes in all species to which it happens to be
transferred. One consequence of such inappropriate over-expression of
genes may be cancer.
Our considerations should be seen in the light of the results of the
first systematic safety testing of transgenic food backed up by
histological studies, which was carried out by Pusztai and his
collaborators. Ewen and Pusztai (31) conclude that a significant part of
the toxic effects of transgenic potatoes with snowdrop lectin was due to
the "construct or the genetic transformation (or both)". They
further state, "The possibility that a plant vector in common use in
some GM plants can affect the mucosa of the gastrointestinal tract and
exert powerful biological effects may also apply to GM plants containing
similar constructs…" The plant vector in common use is the T-DNA
of Agrobacterium, and the construct in question is the CaMV 35S
promoter, both of which are in the transgenic potatoes tested by Ewen and
Pusztai (31).
As a precautionary measure, we strongly recommend that all transgenic
crops containing CaMV 35S or similar promoters which are recombinogenic
should be immediately withdrawn from commercial production or open field
trials. All products derived from such crops containing transgenic DNA
should also be immediately withdrawn from sale and from use for human
consumption or animal feed
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Corresponding author: Phone 44-1908-65-3113, Fax:
44-1908-654167, E-mail m.w.ho@i-sis.org.uk
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