15.07.99 19:12

IMPORTANT! - Viral danger from GM crops confirmed - NEW STUDY


Thanks to NGIN for forwarding the 2 highly significant items below.

It is interesting that the latest study (see item 2 below) highlighting the viral recombination risks of GM crops should involve transgenic rice (although it has implications for almost all GM crops), and that the work was carried out at the UK's John Innes Centre in Norwich.

No mention of this risk was provided earlier today when the John Innes
Centre was actively publicising in the media other research it has carried
out which it claims may help increase rice yields.

It would appear that the Centre is being highly selective when deciding
which of its own studies it will chose to draw the media's attention to.

The study publicised today by the John Innes Centre does not demonstrate higher yields from transgenic rice. It is only able to provide speculation that this could be possible in the future if the approach can be successfully developed - which is by no means certain. Nonetheless, this is the study which has been profiled in public.

Meanwhile the same institute has simultaneously established GM viral
recombination as an existing scientific fact and threat. This is the study
which the biotechnology community choses to keep to itself.

By contrast the speculative yield study is likely to have been given a high public profile by the Institute in an attempt to divert attention from recent reports that commercial GM crops which have reached the market often produce lower yields than non-modified varieties (see
http://www.netlink.de/gen/Zeitung/1999/990708.htm and
www.btinternet.com/~nlpwessex/Documents/gmlemmings.htm ).

In circumstances such as these it is little wonder that a BBC TV Newsnight report earlier in the week confirmed that the mood of US farmers towards the biotech industry "is changing from a warm welcome to simmering resentment."
(see http://news.bbc.co.uk/hi/english/sci/tech/newsid_394000/394301.stm ).

NATURAL LAW PARTY WESSEX
nlpwessex@bigfoot.com
http://www.btinternet.com/~nlpwessex
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1. Dr. Mae-Wan Ho's letter re: cauliflower mosaic virus
2. CaMV Promoter is A Recombination Hotspot - No Transgenic Plant Containing CaMV Promoter Should be Released http://www.i-sis.dircon.co.uk
Prepared by Angela Ryan

===============================
Date Posted: 07/15/1999
Posted by: M.W.Ho@open.ac.uk
================================


Dr.Penny Maplestone
The British Society of Plant Breeders Limited
Woolpack Chambers, Market Street
ELY, Cambridge CB7 4ND

Dear Dr. Maplestone,

Thank you for your enquiry about the cauliflower mosaic virus (CaMV) and for
the opportunity to clear up a major misconception. I most certainly did not
say, "cauliflower mosaic virus gives you cancer". I was talking
specifically about the cauliflower mosaic viral promoter that is in
virtually all transgenic plants currently on the market or being
field-tested. This is a piece of the virus' genetic material which is being
used to drive the expression of many transgenes. Several factors make this
piece of viral genetic material hazardous.

First, the CaMV promoter is used in a 'naked' form - that is, without its
viral coat. It is now well-known that naked viral DNA is more infectious
than the intact virus, because the viral coat generally determines the host
specificity. For example, DNA from the human polyoma virus can give a full
blown infection when injected into rabbits while the intact virus is
harmless. So the while the intact virus will infect cauliflower and
cabbages, it will almost certainly not gain access to cells of human
beings. The naked viral promoter, however, may well be taken up by
mammalian cells including our own.

Now, foreign DNA taken up into cells are usually degraded, unless they have
a propensity to integrate into the cell's genome. It so happens that the
CaMV promoter is known to have a recombination hotspot, which means it is
especially prone to break and join with other DNA at that point. This
enhances the likelihood of the promoter (and other genes linked with it)
being integrated into the cell's genome.

Integration of foreign DNA into the genome of mammalian cells is well-known
to have harmful effects such as inactivation or activation of host genes
that could lead to cancer.

Another potential hazard of having the cauliflower viral promoter in the
genome is that it could reactivate dormant viruses, which are in the genomes
of all higher organisms including plants and animals, or it could generate
new viruses by recombination. The CaMV is known to be closely related to
human hepatitis B virus and also to retroviruses including HIV and others
that cause cancer.

My research assistant, Angela Ryan and I were among the scientists invited
to Michael Meacher's office to discuss the specific hazards of GM crops at
the end of last March. There, the question of the safety of the CaMV
promoter was specifically raised. The details of that meeting and a briefing
paper I wrote for Michael Meacher afterwards can be found in our Institute
of Science in Society website:
http://www.i-sis.dircon.co.uk

The latest article on the recombination hotspot of CaMV is Kohli et al
(1999). The Plant Journal 17(6), 591-601. You can find a summary of that
paper on our website.

Yours sincerely,


Dr. Mae-Wan Ho
Institute of Science in Society
and Biology Department, |
Open University.
Walton Hall, Milton Keynes
MK7 6AA, UK
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CaMV Promoter is A Recombination Hotspot - No Transgenic Plant
Containing CaMV Promoter Should be Released

Prepared by Angela Ryan
Molecular Biologist
Open University

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.

Overview