(Editings including subheaders added by PSRAST)

The Killing Fields

Terminator Crops at Large

Mae-Wan Ho, Joe Cummins and Jeremy Bartlett

Abstract

'Terminator technology' renders harvested seeds sterile, for no other reason
than to enforce corporate patents on GM seeds. The first terminator patents
that came to the attention of the public were those jointly owned by US
Department of Agriculture and Delta and Pine Land Company, which Monsanto
had intended to acquire. As a result of universal condemnation and rejection
by farmers and non-Government organisations world wide, Monsanto had
announced it will not commercialise terminator crops, and assurances were
given that such crops do not yet exist. It was clear that research had
continued unabated, and other companies were actively developing terminator
crops.

Towards the end of last year, the US and UK Governments carried out 'public
consultations' in which terminator technology is being promoted as a means
of preventing the spread of GM genes. In the course of preparing our
submission to the UK Government, ISIS discovered that terminator crops have
been field-tested in Europe since the beginning of 1990, while 132 field
trials have been carried out in the United States starting in 1992, the vast
majority done without risk assessment.

The public consultations can only be seen as an exercise in smoothing the
path for commercial development of a technology condemned as contrary to
basic human rights, because it prevents farmers from saving, replanting and
exchanging seeds, practices going back thousands of years that are essential
to food security. To require containment of GM genes by terminator
technology is to admit that those genes are unsafe. It is an argument for
stopping GM crop development altogether, and is not an excuse for validating
a morally bankrupt technology.

Moreover, terminator technology uses genes and constructs that introduce
serious hazards over and above those of GM crops in general.

Key words: terminator technology, seed/pollen sterility, barnase,
recombinase, barstar

Field trials with terminator crops launched

Are the regulators knowingly deceiving us or have they been fooled?
Last December, one of us (MWH) was acting as expert witness in defence of
citizens who have taken civil disobedience action against GM crops. Among
the crops in question were GM oilseed rape varieties used to produce F1
hybrids, as described in the application for release from AgrEvo UK (now
Aventis) [1]. At the time, we were just preparing our submission to the
consultation document, "Guidance on Best Practice in the Design of GM Crops"
put out by the UK Government's Advisory Committee for Release to the
Environment (ACRE). One of the main 'enabling technologies' for 'best
practice' - to prevent gene flow - is precisely the seed/pollen sterility
system mentioned in AgrEvo's application.

It soon dawned on us that the GM oilseed rape lines undergoing field trials
in the UK are engineered using 'terminator technology' - so named by critics
because it can render harvested seeds sterile - for no other reason than to
enforce corporate patents on GM seeds. Not only that, according to AgrEvo's
application, similar crops produced by the company Plant Genetic Systems
(PGS), a subsidiary of AgrEvo, have been undergoing field-trials in France
and Belgium since the beginning of 1990, and subsequently on larger scales,
also in Sweden and Canada before coming to the UK.

A search on the US database on field trials [2] revealed that similar male
sterile lines engineered with the 'terminator-gene', barnase (see below),
have been tested at least as early as 1992. Since then, there have been 132
field trials, the vast majority of them done without risk assessment, as the
first environmental assessment came up with 'FONSI' - Finding of No
Significant Impact [3] Crops modified for male sterility include rapeseed,
corn, tobacco, cotton. Brassica oleracea, potato, poplar, Cichorium
intybus, petunia and lettuce.

Separately, the other genetic component in terminator crops, the
site-specific recombinase (see below), has also been engineered into corn
and papaya, and there have been 14 field trials between 1994 and 1998. No
environmental impact assessment had been carried out at all, as it was
deemed unnecessary.

There are more than 150 US patents listing barnase or site-specific
recombination or both [4] the oldest, on site-specific recombinase, going
back to 1987 [5]. The first terminator patents that came to the attention of
the public were those jointly owned by US Department of Agriculture and
Delta and Pine Land Company, which Monsanto had intended to acquire. The
novelty in those patents is the proposal to combine the terminator-gene
system with the site-specific recombinase system, giving the company
complete control over the hybrids as well as proprietary chemicals that
control gene expression.

As a result of universal condemnation and rejection by farmers and
non-Government organisations world wide, Monsanto had announced it will not
commercialise terminator crops, to everyone's relief. Research and
development, however, have continued unabated, and the technology kept
surfacing in different forms [6]. But on the whole, everyone has been duped
into thinking that such crops only exist in theory, when they have been out
there in one form or another for more than 10 years.

ACRE's consultation can only be seen as an exercise in smoothing the path
for commercial development of a technology condemned as contrary to basic
human rights, because it prevents farmers from saving, replanting and
exchanging seeds, practices going back thousands of years that are essential
to food security.

It is no coincidence that simultaneous consultation is going on in the
United States on the USDA-Delta and Pine terminator patents. The USDA is
indeed considering commercial development of the technology, and also
recommends it for preventing GM gene flow. Surely, to require containment of
GM genes is to admit that they are unsafe, which is an argument for stopping
GM crop development altogether. It is not an excuse for validating a morally
 bankrupt technology.

Hazards with the Terminator technology

What the regulators and the public are not yet aware of is that the
technology introduces serious hazards over and above those of GM crops in
general [7]. The terminator-gene barnase is a universal poison that breaks
down RNA, an intermediate in the expression of all genes. The recombinase,
in theory, breaks and rejoins DNA at specific sites, but is far from
accurate, so it has the potential to break and rejoin DNA inappropriately,
thereby scrambling the genome in unpredictable, lethal ways.

After a report in the Scottish press, a spokesperson from the UK Department
of the Environment, Transport and the Regions (DETR) denied that the enzyme
barnase was in the crops undergoing field trials. The DETR spokesman was
reported to have said that it was the barnase gene and not the enzyme which
was present in "a few oil seed rape crops currently being trialled." and
that "where the enzyme would be poisonous, the gene was not harmful."[8]
Obviously, he did not know that the barnase gene had to be expressed to make
the barnase enzyme in order to have male sterility. Furthermore, the barnase
gene could spread, either by crossing with related species, or by the GM DNA
being taken up and integrated into the genome of unrelated species, and it
may become expressed in other cells and tissues, with potentially fatal
consequences.

On seeing our press releases,[9] Dr. Ian Woiwod of Rothampstead, a scientist
involved in overseeing the UK field trials, indicated that he had no
knowledge of such crops in the field trials [10]. Indeed, in a
correspondence describing the trials published in Nature in 1999 [11], there
was no mention of the male sterile spring and winter oilseed rape. Have our
regulators been kept in the dark? During a workshop at the first meeting of
the Intergovernmental Committee on the Cartagena Protocol on Biosafety held
in Montpellier last December [12], the UK Government delegate from the DETR
actually thanked MWH for providing the information on terminator crops.

How seed/pollen sterility is engineered into crops

There are two key components to terminator technology, which is being widely
used, not only in plants but in animals as well, as revealed by the 150 plus
patents filed in the US alone (see above). The first component is
'site-specific recombination', carried out by a recombinase, an enzyme that
recognises specific 'sites', or short DNA sequences, labelled 's' in the
diagram below. Any stretch of DNA sequence flanked by two such sites will be
spliced out by the recombinase.

...s-any DNA sequence-s...

The other key element is literally the 'terminator'. It is barnase, an
enzyme that breaks down RNA. RNA is an intermediate in the expression of all
genes, and that is why barnase is lethal to all cells in which it is
expressed, unless its specific inhibitor, barstar, is also present. Both
barnase and barstar are produced by a soil bacterium, Bacillus
amyloliquefaciens. Inside the bacterial cell, barstar binds to barnase in a
one-to-one complex, disarming the latter so it can do no harm. However, when
barnase is secreted outside, it is no longer bound to barstar and is thus
harmful to other cells.

To engineer pollen sterility, the barnase gene is placed under the control
of a promoter that allows the gene to be expressed only during anther
development, ie, in the male part of the flower. The barnase with its
anther-specific promoter is stitched next to the transgene of interest, say,
a gene coding for herbicide tolerance, also with its own promoter.
Theoretically, there will be no fertile pollen from this transgenic crop. In
the case of crops that are normally self-fertilised, there will be no seeds
set. In out-crossing plants, the only fertile seeds set will be those
fertilised by non-GM varieties nearby, which will not be herbicide tolerant;
so farmers who want the herbicide tolerant trait will have to buy fresh
seeds from the company every season. The problem is that such a male-sterile
line by itself cannot be propagated, it does not breed true.

To propagate the line, the company makes use of site-specific recombination.
For example, the promoter of the barnase could normally be blocked by a
sequence flanked by sites recognised by a recombinase

  .. anther-specific promoter-s-blocking sequence-s-barnase gene..

The recombinase can be engineered into the same GM line with the barnase
gene for male sterility, or it could be introduced by crossing the GM line
containing barnase with another that contains the recombinase to generate a
hybrid. The recombinase is placed under the control of a promoter that
responds to an external chemical, say, the antibiotic tetracycline.

  ..tet-specific promoter-recombinase gene...

When tetracycline is applied, the recombinase is expressed, and splices out
the blocking sequence in the barnase promoter, so barnase is expressed. By
treating harvested seed with tetracycline before they are sold to the
farmer, the company can ensure that the plants grown from the seeds will be
pollen sterile.

If female-sterility is required, the barnase gene could be placed under the
control of a promoter that works only during ovule development, ie, in the
female part of the flower, and the rest is similar.

Alternatively, the recombinase may be engineered into a GM line with the
gene coding for barstar, which, when crossed with the male sterile GM line
containing barnase, will produce a hybrid. The hybrid, treated with
tetracycline, will produce plants that will still set seed, at least in
theory, because the barstar inactivates the barnase. However, if the
harvested seeds were re-sowed, the farmer will find that only about half
(7/16) [13] of the seeds will have the same characteristics as those
originally purchased from the company, and about one fifth (3/16) of the
seeds may be completely sterile. It could be considerably worse.

Important information concealed

In AgrEvo's application for field trials, only two lines are mentioned.
These are the 'male sterile oilseed rape line' engineered with barnase under
the anther-specific promoter and a gene for phosphinothricin (glufosinate
herbicide) tolerance; and the 'restorer oilseed rape line' engineered with
barstar, also under the anther-specific promoter plus the same gene for
glufosinate tolerance. No detailed genetic map or other molecular genetic
data were supplied with the document, as it was clearly intended for the
public register. Companies are currently allowed to conceal molecular
genetic data under 'commercially sensitive information', and most of them do
so. Does either of these lines contain the site-specific recombinase? Does
the barnase gene exist in a blocked form in another line in which male
fertility can in principle be indefinitely maintained?

If barnase is not blocked, then the 'male-sterile' line cannot possibly be a
true-breeding, uniform line; as it must be fertilised by pollen originating
from non-male sterile oilseed rape. A male-sterile line can only be
heterozygous for barnase and herbicide tolerance.

Terminator crops cannot prevent gene flow and introduce new hazards

The system is ineffective for preventing gene flow for the following
reasons:
a. All gene control systems are known to be 'leaky' in the sense of not
being 100% effective, and the proposed system is no exception, particularly
as so many elements have to be engineered perfectly, which is beyond current
capability. As a result, some fertile pollen/seeds are likely to be
produced.
b. Pollen sterile GM plants can still be fertilised by non-GM pollen, just
as GM pollen from ovule-sterile plants can cross with non-GM plants, thus
enabling gene escape.
c. This system does not at all prevent horizontal gene transfer, a process
whereby the GM DNA is taken up directly into cells of unrelated species and
incorporated into the cell's genome. If anything, horizontal gene transfer
may be enhanced due to the increased structural instability of the
complicated constructs involved. Transfer to bacteria and viruses in all
environments can be envisaged. Plant residues, dust and pollen may all
contribute. Transfer to insect pollinators or feeders could take place; and
these may also become vectors for further horizontal gene transfer.

Significant hazards are introduced by this system, over and above those due
to GM crops in general. First, barnase is a potent RNAse that breaks down
RNA indiscriminately, and is known to be harmful, if not lethal, to all
cells, animals and humans included. When perfused into rat kidneys, barnase
causes kidney damage [14]. It should not be permitted in any GM crop, let
alone GM crop intended for animal feed or human food.

Second, the 'site-specific' recombinases are known not to be 100% specific.
There is already evidence suggesting that unintended rearrangements and
deletions of genomic sequences have resulted from the use of such
recombinases. In other words, the recombinases have the potential to
scramble genomes in unpredictable, harmful ways (see Note 7). This has now
been demonstrated for the first time, basically because some researchers
have finally cared to look for it.

The recombinase Cre is part of the 'site-specific recombination' Cre/lox
system originally isolated from the bacteriophage (bacterial virus) P1. Cre
catalyses recombination between two lox sites, splicing out any stretch of
DNA in between.

The system is not only used in plants, but extensively exploited in
transgenic mice. Studies in the test-tube have shown that Cre recombinase
can catalyze recombination between DNA sequences found naturally in yeast
and mammalian genomes. These 'illegitimate sites' often bear little
similarity to the lox element. However, there have been no reports on such
illegitimate recognition in the animals or plants themselves. And there have
even been pilot studies using the Cre/lox system in human gene therapy.

In a study just published [15], researchers in the United States showed that
high levels of Cre expression in the spermatids of heterozygous transgenic
mice leads to 100% sterility in the males, despite the absence of any lox
sites. Heterozygous mice carry only one copy of the Cre recombinase gene.

The sterility is caused directly by the recombinase enzyme scrambling the
genome, essentially by breaking and rejoining DNA at inappropriate sites on
the same or different chromosomes. The researchers have pinpointed the
genome-scrambling event to the time at which the two 'daughter' spermatids
and their paired chromosomes have just separated from each other, but are
still joined by a 'cytoplasmic bridge'. This is enough to allow the enzyme
to pass from the spermatid containing the recombinase gene to the other
which does not, thereby to scramble up the chromosomes of both the
transgenic and nontransgenic spermatid. The result is 100% sterility.
Embryos fertilized by these sperms arrest predominantly at the 2-cell stage,
and do not go beyond the 4-cell stage.

The researchers warn: "These results indicate that Cre can catalyze
illegitimate recombination having overt pathological consequences in
animals." A similar recombination system is found in animals containing the
RAG recombinases. Illegitimate recombinations in somatic cells are linked to
human leukemias.

The greatest danger of terminator crops stems from the spread of the genes
and constructs, not only to related species by out-crossing but by
horizontal transfer to unrelated species. The increased complication of the
GM constructs involved will only increase structural instability and hence
the tendency to horizontal gene transfer and recombination. Transfer of both
the terminator gene barnase and the recombinase will have drastic,
potentially fatal effects on agriculture and on biodiversity.

It is high time to stop these killing crops once and for all.

References

1. Application for field trials from AgrEvo (now Aventis) March 1999 "Part
B: Information about the release application to be included on the public
register".
2. www.nbiap.vt.edu/cfdocs/fieldtests3.cfm
3. "Environmental Assessment and Finding of No Significant Impact" Prepared
by Biotechnology Permits, Biotechnology, Biologics, and Environmental
Protection Animal and Plant Health Inspection Service, U.S. Department of
Agriculture, Permit Number 92-017-01: rapeseed; male sterility; restorer
gene www.nbiap.vt.edu/biomon/relea/9201701r.eaa.
4. www.delphion.com.
5. US04673640 06/16/1987 Regulated protein production using
site-specific recombination.
6. See "Terminator in different guises" ISIS News #3, December 1999
www.i-sis.org/i-sisnews3.htm#terminator
7. ISIS has warned of this more than once. See "Why patents on life-forms
and living processes should be rejected from TRIPS - Scientific briefing on
TRIPS Article 27.3(b)" by Mae-Wan Ho And Terje Traavik, TWN and ISIS Report,
1999 www.i-sis.org/trips99.shtml; "Terminator in different guises" by
Mae-Wan Ho, ISIS News #3, December 1999
www.i-sis.org/i-sisnews3.htm#terminator; "Terminator gene product alert" by
Joe Cummins, ISIS News#6, September 2000 www.i-sis.org/i-sisnews6.htm#term.
8. "GM 'poison' allegation denied"
http://uk.news.yahoo.com/001205/79/ar32n.html.
9. "Terminator alert: UK GM field trials contain 'terminator' crops" ISIS
Press Release 6.12.2000; "Terminator recombinase does scramble genomes"
ISIS Press Release 8.12.2000 www.i-sis.org/terminatorrecomb-pr.shtml
10. Personal communication by e-mail to ISIS from Dr. Ian Woiwod, 8.12.2000.
11. Firbank, L.G. et al, (1999). Nature 399, 727-8.
12. Critical Issues on Biosafety, Third World Network Workshop during the
first meeting of the Intergovernmental Committee on the Cartagena Protocol
on Biosafety, 11 Dec., Le Corum, Montpellier
13. It deviates from the usual 9/16 Mendelian ratio for the inheritance of
two genes, barnase and barstar. On account of a one-to-one complex between
barnase and barstar; plants in which there are two copies of barnase to one
copy of barstar will be expected to be partially sterile.
14. Ilinskaya, O and Vamvakas, S (1997). Nephrotic effect of bacterial
ribonucleases in the isolated and perfused rat kidney. Toxicology 120, 55-63
 15. Schmidt, E.E., Taylor, D.S., Prigge, J.R., Barnett, S. and Capecchi,
M.R. (2000). Illegitimate Cre-dependent chromosome rearrangements in
transgenic mouse spermatids. PNAS 97, 13702-13707.