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CaMV Promoter is A Recombination Hotspot - No Transgenic
Plant Containing CaMV Promoter Should be Released
Angela Ryan
Lay summary
A recent study of transgenic rice carried out at the John Innes
Institute [1] supports previous evidence that there is a 'recombination
hotspot' in the CaMV 35S promoter. A recombination hotspot is a site prone
to recombination, ie, breaking and joining with other DNA. Furthermore,
some of the recombination events are 'illegitimate' or nonhomologous, and
do not require substantial similarity in nucleic acid base sequence.
Implications
The results show that the CaMV promoter is very likely to recombine with
other DNA in the host genome, including dormant viral DNA, as well as with
other viruses in the host cell. Transgenic lines containing CaMV
promoters, which includes practically all that have been released, are
therefore prone to instability due to rearrangements, and also have the
potential to create new viruses or other invasive genetic elements.
Such elements cannot be contained or controlled once they have entered
the wider environment. It is now indisputable that recombination events
will take place at the CaMV promoter in the current generation of
transgenic plants. The continued release of such transgenic plants is
unwarranted especially in the light of the new findings.
Technical details
Twelve representative transgenic rice lines were analyzed, carrying a
range of transforming plasmid rearrangements, which predominantly
reflected micro-homology mediated illigitimate recombination involving
short complementary patches at the recombining ends. Direct end-ligation
(ie, joining of ends), in the absense of homology between recombining
molecules, was also observed but occurred less frequently. Filler DNA was
found at some of the junctions and short purine rich tracts were also
found either at the junction or in the immediate flanking regions.
Furthermore, putative DNA topoisomerase I binding sites were found in
clusters around the junction.
Links between DNA double strand break repair (DSBR), illegitimate
recombination and plasmid DNA integration have previously been established
and involve sequences with either microhomology or no homology. This study
reveals that there are similarities between recombination junctions
generated by various transformation methods and this strongly suggests
that the underlying mechanisms controlling plasmid rearrangement and
transgene integration in plants are likely to be the same.
Intergration of foreign DNA has been studied in detail in animal genomes
and it appears that large amounts of DNA ends up stimulating the
production of DNA ligase, which in turn promotes illegitimate
recombination. A wound response is elicited in both Agrobacterium-mediated
DNA delivery and direct physical DNA transfer into plant cells. This
involves the activation of nucleases and DNA repair enzymes which maintain
the integrity of the host genome. When unorthodox substrates are present,
illegitimate recombinations can lead to large scale genome rearrangements
and the integration of exogenous DNA. Any exogenous DNA entering the cell
is therefore exposed to breakdown and repair enzymes, resulting in some
rearrangement and/or incorporation of it into the recipient genome. DSBR
is the predominant mechanism of illegitimate recombination in higher
eukaryotes, probably due to the large genome size preventing homology
searching and also the higher order chromatin structure holding broken DNA
ends in close proximity.
Although different regions of transforming plasmid were involved in
plasmid-plasmid recombination, a 19 bp palindromic sequence, including the
TATA box of the CaMV 35S promoter acted as a recombination hotspot, ie, a
hotspot for breaking and joining up with other DNA. Furthermore, the
palindrome and surrounding DNA sequence were found to possess a number of
characteristics common to known recombination hotspots. The purine-rich
half of the palindrominc sequence was specifically involved at the
recombination junctions. AT-rich sequences cause isotropic DNA bending and
influence DNA melting and have been shown to contain S/MAR motifs
(Sawasaki et al 1998) which intrinsically harbor curved DNA. There is a
short tract of alternating purine-pyrimidine residues situated 50 bp
upstream. Such sequences are known to adopt a Z-DNA conformation which in
turn is known to influence transcription and recombination . These
sequences are also known to bind DNA topoisomerase II which is involved in
the resolution of recombination intermediates. In addition, the 3' end of
the CaMV promoter was found to have structure and sequence similarity to
the petunia transformation booster sequence which is shown to increase
plant transformation efficiency, most likely by stimulating recombination.
Other similar structures were found in recombinogenic regions of SV40 DNA
and HeLa cells. Furthermore the 25 bp border repeats of T-DNA shows a
remarkable similarity to the recombination hotspot of the CaMV promoter:
There is an 11 bp palindromic sequence involving a TATA box-like structure
in the right border and the left border has a short purine-rich sequence
in the center. This study predicts that these two regions of T-DNA could
be involved in rearrangements and indeed certain crossover events have
been previously documented.
The recombination hotspot described in the CaMV 35S promoter is found
within the highly recombinogenic region of the full-length CaMV RNA and
this study shows that recombination events can occur in this region even
in the absense of viral enzymes and other cis-acting elements. It was
shown that in CaMV RNA the recombination events were clustered around the
35S RNA transcription initiation site. This site is believed to be
involved in recombination during reverse transcriptase-mediated virus
replication. A template switch at the 5' end of the RNA is induced by the
19 S RNA terminal repeat. However, in this study concerning the 423 bp
fragment of the CaMV promoter, recombinogenic activity was maintained in
the absense of reverse transcriptase and the remainder of the virus
genome. These results prove that the plant cellular machinery alone is
sufficient to recognise and act on these viral sequences.
In one of the transgenic rice lines the junction included the insertion
of a 23 bp fragment of filler DNA and the presense of direct repeats
(5'TCCGG 3') flanking the insert, suggesting one of two possible
mechanisms. The synthesis of untemplated nucleotides by illegitimate
recombination between the two ends representing short tails of imperfect
complementarity. Alternatively, the insertion may represent a
transposition event whereby the presense of staggered breaks in a target
DNA molecule may have acted as a substrate for the transposase or
integrase encoded by an endogenous plant transposable element. Insertions
ranging from 2 bp to 1.2 kb were found in another study in nearly 30% of
the plasmid junctions analyzed. This so called filler DNA was sometimes
genomic in origin, sometimes it appeared to have been derived from the
transforming plasmid and in other cases the origin was unknown. The entire
insertion could itself be defined as filler DNA or captured DNA and the
possible involvement of transposase in the generation of plasmid-plasmid
junctions exemplifies a discrete form of illegitimate recombination
characterised by the use of incorrect substrates by various DNA processing
enzymes. Such rearrangements have been seen frequently with transposases
and integrases, and with the enzymes that catalyze site-specific
recombination (e.g. Cre recombinase, l integrase and Hin invertase).
Reference
1. Kohli, A. 1999. Molecular characterization of transforming plasmid
rearrangement in transgenic rice reveals a recombination hotsport in the
CaMV promoter and confirms the predominace of microhomology mediated
recombination. The Plant Journal 17(6), pp 591-601.
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