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terminator is interminable
The article deals deals with continued efforts to make something that
most people do not want. Government research grants in this area are
lauded in the article. It makes you wonder why politicians continue load
tax dollars on projects designed to benefit a few companies and USDA ( a
government department bloated on patent revenue and one that mirrors
the multinationals in its approach to the public). The methods of
terminating seed production described below are simple variations on the
theme set forth in the original terminator proposition. The pr people
from industry claim that any technical objections are emotional and
unscientific. However, the introductions are quite threatening many
employing barnase ribonuclease an enzyme highly toxic to mammalian cells
and even at that the human health implications are ignored for the
most part.As well the site specific recombination systems used in many
proposed terminators cry out for safety evaluation.
The research on terminators should not be supported from public taxes,
it should be supported by those profiting from the development.
http://www.isb.vt.edu/news/2002/news02.nov.html#nov0202
EMERGING BIOTECHNOLOGIES: UPGRADING THE TERMINATOR
Zac Hanley and Kieran Elborough
Terminator technology has been a lightning rod for debate and
confrontation on the issue of genetic modification. Most of the
discussion, and all of the hypothesising over potential consequences,
has taken place while Terminator and similar technologies were largely
theoretical concepts. While it is proper to discuss the ethical
ramifications at the early stages, in this case it has often led to the
technical advances being relegated to the introduction or the appendix.
We have previously described the benefits, risks, impacts, and
opportunities of Terminator (more properly known as Genetic Use
Restriction Technologies, or GURTs1). Now, here is the appendix.
Traitors and Terminators
Two types of GURTs have been described.2 The first GURT patent (USP#
5,723,765 - see footnote 3) outlined the concept later described as
Variety-restriction GURT, or V-GURT. This type is the emotively named
`Terminator', as it causes the production of sterile seeds. The
reproductive viability of the plant is under the proprietary control of
the owning company, ensuring that viable seed is not available for the
farmer to harvest. This is achieved via triggering a disrupter gene
prior to the sale of seed, which has a delayed effect, rendering the
next generation non-viable. In `Terminator', this triggering is by
commission, that is, treatment prior to seed sale with an activating
stimulus. In the technology dubbed `Traitor' by critics, this is by
omission; a suppressing stimulus is withdrawn and the disrupter is then
able to act.
The concept of a V-GURT is not tied to any particular disrupter gene;
there are many possibilities. Examples are genes encoding proteins which
break down essential cellular components such as RNA or the cell wall;
or which activate the apoptosis-like programmed cell death pathway in
plants; or which perturb fundamental aspects of metabolism such as
osmolyte balances or proton gradients. The aim is to disrupt the
creation of the next generation or to render that generation incompetent
to grow and survive. This is where real ingenuity is required. It is
vital to arrange the genetic elements so that the disruptor has its
effect in a timely manner.
The essential component is the promoter, which must respond to an
exogenous compound or some triggering stimulus. One interesting example
is the oestrogen receptor transactivation system described by Zuo and
co-workers.4 Their paper discusses the application of this system to
marker excision; marker excision systems are but one application of
GURTs.5 In the oestrogen receptor transactivation system, application of
ß-oestradiol causes the promoter to express strongly; this promoter
could be used to drive a suitable disruptor gene. Without ß-oestradiol,
expression is extremely low and disruption does not occur. However there
are several obstacles to the successful use of exogenous chemical
regulators in GURTs. Firstly, the regulatable promoter must remain
physically linked in the genome to the disrupter gene, else they may on
rare occasions separate during the development of the next generation.
This would lead to viable and fertile trait- and modification-bearing
offspring. Secondly, it is not clear how a chemical treatment can be
100% effective, as some cells or even whole seeds may not be penetrated.
Trait Transfer Termination
The second GURT concept is the Trait-restriction GURT, or T-GURT, where
it is the inheritance of the elite trait differentiating the plant from
other germplasm that is under control, rather than the plant's
reproductive ability. T-GURTs can be described as less crude than
V-GURTs since their effects are localized to one area of the genome, and
such finesse is increasingly desirable given public concerns over the
process of germplasm enhancement. Control over the inheritance of a
trait may occur via regulating the expression of genes conferring the
trait, or ensuring the disruption or loss of these genes in the next
generation (as deployed in marker excision systems such as described in
footnote 4).
To achieve such relative finesse, a different range of disrupter genes
is required. Genes encoding essential components of flowering or
embryogenesis are suitable leverage points, and the inducible promoter
here must drive the expression of an antisense construct. Alternatively,
T-GURTs can employ marker excision-type mechanisms to remove the elite
trait and dispense with disrupter genes altogether. This may be an
important consideration if the plant is part of the human food chain.
The site of action for T-GURTs is the reproductive tissues of the first
generation, so tissue- or developmental stage-specific promoters are
used. The triggering may be by omission or commission, as for V-GURTs
(above).
The next generation of inducible promoters useful for GURTs are likely
to respond to more complex, multi-factorial signals involving
environmental conditions, such as those which initiate flowering in
overwintering plants. Or they may be switched on and off during certain
developmental stages with great precision. Control via such triggers
would circumvent some of the problems inherent in activation via
exogenous chemicals. Early studies on such promoters can be found in the
literature.6
The Third Type?
Tobacco transgene containment has been demonstrated by Kuvshinov and
coworkers,7 devisors of the `Recoverable Block of Function' (RBF) GURT
concept. They attempt to differentiate it from previous GURTs, although
it is conceptually identical to mechanisms described by the Food and
Agriculture Organization working party.2 RBF can be used in T-GURTs or
V-GURTs, depending on the DNA constructs and target genes chosen, and is
applicable to vegetatively propagated plants as well as seed. Kuvshinov
and co-workers give a V-GURT example in their publication.
The RBF is a gene construct that includes a disrupter gene (the
`blocker' of reproductive `function') and a rescue gene, which disrupts
the disrupter and is under the control of an inducible promoter. In
Kuvshinov et al. (2001), the BARNASE protein is expressed during
embryogenesis and destroys most cellular RNA at this critical
developmental stage, leading to the production of sterile seed. The
barstar gene is also present in the same plant and can be triggered by a
particular treatment. The authors used a heat-shock-specific promoter.
On production of the BARSTAR protein, BARNASE is inhibited and viable
seed are formed. However, barnase is reset to be expressed during the
next embryogenesis, so the block is `recovered' and the heat treatment
would be required in the next generation, if it is not to be the last. A
different `blocker' and rescuer combination could control a T-GURT; for
example, an antisense construct could `block' the trait-conferring gene
and be rescued by a gene, which specifically targets the promoter of the
`blocker'.
Trends for Tomorrow
Today's GURTs have been criticized on a number of fronts, and most
commentators have been concerned over their economic, social, and
environmental impacts. These were addressed in our previous article.1
But it is also important to say that ongoing research and development of
new and improved GURTs continues, is exciting, and suggests remedies for
many of the technical objections. However, GURTs do not have quite the
necessary specifications and mostly remain interesting concepts or
elegant creatures residing in the protective environment of the
laboratory or glasshouse. This is the best reason for continuing
research, not inhibiting it. Widespread application in different species
and extensive field testing are required. Government funding should be
placed into programs developing GURTs. Publication in peer-reviewed
journals of new developments, as with the RBF concept, is to be
encouraged and should be recognized by proponents and critics as
laudable. As an aside, the current public mistrust over patent
protection often extends to the misunderstanding that patents are
secrets; the current revolution in public access to patent databases (as
in footnote 3) should help the public image of companies who wish to
protect their investment by publishing their discoveries at the patent
offices. At the toolkit level, more and different promoters are needed
for T- and V-GURT systems, and there are many research groups in
academic institutions that could apply their discoveries here. Lastly, a
demonstration of the RBF system in a T-GURT application would be most
helpful.
Taking insurance and mitigating risk through `belt and braces'
approaches are the best practises where public perceptions of safety,
however distorted for political ends, are a factor. For this reason,
further investment and research into GURTs are needed but emphatically
do not imply that genetic modification is dangerous. GURTs developed as
described herein are one component of several in a portfolio of
technologies that can and should be employed (others include apomixis,
transplastomics, male sterility, and enforced cleistogamy). Such
technologies could be shared between companies. Some customers seek
assurances; GURTs and the biotechnology industry are well on the way to
providing them.
References
1. See for example our article "Re-emerging Biotechnologies:
Rehabilitating the Terminator," in ISB News Report, June 2002.
http://www.isb.vt.edu/news/2002/news02.jun.html#jun0203.
2. Most articles acknowledge the report of the FAO Working Group on
Plant Genetic Resources for Food and Agriculture available online at
http://www.fao.org/waicent/FaoInfo/Agricult/AGP/AGPS/pgr/itwg/pdf/P1W7E.pdf.
3. US Patent No. 5,723,765 entitled `Control of Plant Gene Expression'
granted to Delta and Pine Land Corporation and the USDA. Search at
http://patents.uspto.gov for the full text.
4. The marker excision application of this system can be found in: Zuo
J, Niu Q-W, Moller SG, Chua N-H. 2001. Chemical-regulated, site-specific
DNA excision in transgenic plants. Nature Biotechnology 19(2): 157-61.
5. A recent review: Hare PD and Chua N-H. 2002. Excision of selectable
marker genes from transgenic plants. Nature Biotechnology 20(6): 575-580.
6. Day-length-responsive promoter elements: Kiyosue T and Wada M. 2000.
LKP1 (LOV kelch protein 1): a factor involved in the regulation of
flowering time in Arabidopsis. Plant Journal 23(6): 807-15.
Tissue-specific promoter: Rossak, Smith, and Kunst. 2001. Expression of
the FAE1 gene and FAE1 promoter activity in developing seeds of
Arabidopsis thaliana. Plant Molecular Biology 4(6): 717-25.
7. Kuvshinov V, Koivu K, Kanerva A, and Pehu E. 2001. Molecular control
of transgene escape from genetically modified plants. Plant Science 160:
517-22.
Zac Hanley and Kieran Elborough
Consultants in Plant Biotechnology
New Zealand
biotech@greengenz.com
.