An academic analysis of the Terminator gene
Matthew Townsend
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How the Terminator terminates:
an explanation for the non-scientist of a remarkable patent
for killing second generation seeds of crop plants
by
Martha L. Crouch, Associate Professor of Biology
Indiana University
Bloomington, Indiana, USA
cro...@indiana.edu
This paper is one in a series of essays meant to stimulate and
inform discussion of the subject. The author invites readers to
correspond with her directly if they have comments or questions
about her interpretation of the Terminator patent.
revised edition©1998
an occasional paper of
The Edmonds Institute
20319-92nd Avenue West
Edmonds, Washington 98020
USA
published with the help of grants from:
The HKH Foundation
The Funding Exchange
C.S. Fund
Introduction
Genetically modified organisms (GMOs) have become a commercial reality in
agriculture. For example, it is estimated that in 1998 over 18 million
acres in the United States will be planted in Roundup Ready® soybeans,
which were first introduced in 1996 (Horstmeier 1998). These soybeans are
engineered by Monsanto Corporation to contain a bacterial gene that confers
tolerance to the herbicide glyphosate, or Roundup® , also made by Monsanto.
Only two years after the introduction of Roundup Ready® soybeans, over 30%
of the corn and soybeans planted in the United States, and close to 50% of
the canola planted in Canada, have been genetically engineered to be either
herbicide or pesticide resistant.
Monsanto and the other companies that have invested heavily in
biotechnology in the last two decades are starting to make some money after
years of promises without products, and they are aggressively protecting
their patented seeds. In a recent issue of the Farm Journal (Monsanto
1997), Monsanto ran a full page advertisement asking farmers to respect the
company's property rights:
It takes millions of dollars and years of research to
develop the biotech crops that deliver superior value to
growers. And future investment in biotech research
depends on companies' ability to share in the added value
created by these crops. Consider what happens if growers
save and replant patented seed. First, there is less
incentive for all companies to invest in future
technology, such as the development of seeds with traits
that produce higher-yielding, higher-value and
drought-tolerant crops....In short, these few growers who
save and replant patented seed jeopardize the future
availability of innovative biotechnology for all growers.
And that's not fair to anyone.
In the future, companies and government breeders who genetically engineer
crops may not have to ask for such compliance. If the procedure outlined in
a recent patent comes to fruition and is widely used, plant variety
protection will be biologically built into the plants themselves.
In March of 1998, a seed company later to be purchased by Monsanto, Delta
and Pine Land Company, in collaboration with the United States Department
of Agriculture, was awarded U.S. Patent Number 5,723,765: Control of Plant
Gene Expression. Although the patent is broad and covers many applications,
one application favored by the patent's authors is a scheme to engineer
crops to kill their own seeds in the second generation, thus making it
impossible for farmers to save and replant seeds.
This "invention" has been dubbed the "Terminator Technology" by Rural
Advancement Foundation International (RAFI), and that group of researchers
have analyzed some of the technology's serious social, economic and
environmental implications (RAFI 1998). However, many of the consequences
of Terminator cannot be fully appreciated without an understanding of the
science behind the invention. In this paper, I outline the steps involved
in engineering Terminator Technology into a specific crop. After explaining
the process, I then discuss which details might have the devil in them.
Overview
To help describe the Terminator procedure, I have confined the explanation
to only one of the many possibilities covered by the patent. The example I
have chosen is cotton seed, which previously has been
genetically-engineered with a unique trait, herbicide tolerance. In my
discussion, I have assumed that to ensure that the descendants of the
herbicide tolerant seeds are not used without compensation to the seed
company, the company has additionally genetically engineered the cotton
with Terminator. Although this is a hypothetical case -- Terminator cotton
is not yet on the market, after all -- all the components of the procedure
have been shown to function, at least in the text of the patent for
Terminator.
Cotton is not often sold as a hybrid seed, and is thus a likely candidate
for Terminator protection. By way of contrast, corn is usually planted as a
hybrid, and thus has some measure of variety protection already. This is
because the first generation of a hybrid is genetically fairly uniform, and
has been bred to have desired characteristics that are not present in
either parent alone. When these hybrids make seeds, however, the second
generation is quite variable because of the shuffling of genes that occurs
during sexual reproduction. Industrial agriculture requires uniformity,
because the plants must dovetail with mechanization. Therefore, industrial
farmers who grow corn usually buy new seed every year.
There are several major crops which usually are not grown from hybrid
seeds. These include wheat, rice, soybeans, and cotton. Farmers often save
the seeds from these crops, and may not go back to the seed company for
several years--or longer, in some parts of the world-- to purchase a new
variety.
It would be a big boost to seed company profits if people who now grow
non-hybrid crops would have to buy new seed every year. This may have been
the major incentive for developing the Terminator Technology.
There likely were other reasons for developing Terminator. One reason may
relate to the way in which Terminator's effect differs from hybridization.
When Terminator is used, the second generation is killed. With
hybridization, the second generation is variable, but alive; and any genes
present in the hybrid will be present in the second generation, although in
unpredictable combinations. Therefore, a plant breeder who wanted to use
the genetic material from the hybrid in his or her own breeding program
could retrieve it from these plants. With Terminator, the special genes,
such as the herbicide tolerance of my example, would not be easily
available for use by competitors.
Another reason sometimes cited for using Terminator in combination with a
genetically-engineered variety is to keep the GMOs from "escaping" into the
environment. Many critics of biotechnology cite problems with releasing
GMOs into the wild, noting that their effects on ecosystems and their
members would be difficult to predict (Rissler and Mellon 1996). Having all
of the second generation seeds die would circumvent this problem
altogether.
Rough Sketch and Review
Terminator is a complicated process to understand and it helps to review
beforehand some of the basic information about how genes function during
the life-cycle of a plant. Readers with a good grasp of molecular biology
may want to skip the review section (A simplified version of basic
biological processes) following the general description and proceed
directly to the details of the Terminator Technology.
General Description of Terminator in Cotton
In the cotton example, the goal is to develop a variety of cotton that will
grow normally until the crop is almost mature . Then, and only then, a
toxin will be produced in the (seed) embryos, specifically killing the
entire next generation of seeds.
The system has three key components: 1. A gene for a toxin that will kill
the seed late in development, but that will not kill any other part of the
plant. 2. A method for allowing a plant breeder to grow several generations
of cotton plants, already genetically-engineered to contain the
seed-specific toxin gene, without any seeds dying. This is required to
produce enough seeds to sell for farmers to plant. 3. A method for
activating the engineered seed-specific toxin gene after the farmer plants
the seeds, so that the farmer's second generation will be killed.
These three tasks are accomplished by engineering a series of genes, which
are all transferred permanently to the plant, so that they are passed on
via the normal reproduction of the plant.
A simplified version of basic biological processes
A plant starts life as a single cell, an egg that has been fertilized by
sperm which has been delivered to the egg by the pollen. This first cell
divides many times to form the tissues and organs characteristic of the
species. The process of going from a single cell to an adult is called
development. As development proceeds, cells become different from each
other and change. Cells in the leaf become distinct from cells in the root,
for example. Most of the differences can be attributed to changes in the
kinds and amounts of proteins made in the cells, because many of the
structures in cells are made of proteins, and most of the processes that
occur are influenced by enzymes, which are also proteins. Thus, scientists
who study development spend a lot of effort describing protein patterns.
By studying which proteins are present in different tissues and organs,
biologists have learned that each cell has several thousand different
proteins, but most of the proteins are very rare in the cell. A few hundred
proteins may be moderately abundant, and a few may be quite abundant. Also,
some proteins are found in all kinds of cells and at all times in
development, whereas other proteins are only present in a particular
tissue, or at a specific time. For example, the gluten proteins responsible
for the elasticity of bread dough are found only in the seed,and are
present there in very large amounts. In contrast, the enzyme that splits
glucose as a first step in releasing energy is found in all living cells,
but in fairly small amounts.
Some proteins are made in response to environmental changes, such as
increases in temperature, and thus may or may not be present during the
life of a particular plant.
The most common way for a cell to control how much of which kinds of
proteins are present is to control which genes are functioning (Rosenfeld
et al. 1983). Proteins are chains of different amino acids, and the order
of amino acids and the length of the chain are unique for each kind of
protein. Each unique amino acid sequence is specified by a code on a
chromosome in the cell's nucleus. The code is made of DNA. For the purposes
of this discussion, a gene is a piece of DNA that contains the code for a
specific protein. Genes are present in specific places along the length of
the chromosomes.
It turns out that just about every cell has two full sets of genes (one set
of chromosomes from the sperm and one from the egg), which code for the
proteins made in all of the tissues and organs that an individual plant
will need during its life cycle. However, only those genes whose proteins
are needed in a particular cell will be used by that cell. These are the
active genes. The other genes just sit there on the chromosomes, inactive
in that cell, but active somewhere else in the plant.
Whether a gene is active or not depends on complex interactions between the
DNA and other molecules in the cell. Specifically, a typical gene can be
divided into parts. The first part is a stretch of DNA responsible for
interacting with the cell or the environment, and is called the promoter.
The second part actually contains the code for the order of amino acids in
the protein, and is called the coding sequence. When the gene is active,
the promoter is interacting with other molecules in a way that allows the
coding sequence to direct the synthesis of a specific protein (through a
complex set of steps).
Genetic engineering can be defined as the process of manipulating the
pattern of proteins in an organism by altering genes. Either new genes are
added, or existing genes are changed so that they are made at different
times or in different amounts.
Because the genetic code is similar in all species, genes taken from a
mouse can function in a corn plant; and so on. Also, promoters from one
coding sequence can be removed and placed in front of another coding
sequence to change when or where the protein is made. For example, when the
promoter for casein, the major protein in milk, is removed and put in front
of the coding sequence for human growth hormone, it causes human growth
hormone to be made in cow's milk instead of casein. Of course, in order to
make human growth hormone in cow's milk, the engineered gene has to be
incorporated into the genetic material of the cow. There are many ways to
do this. I will not go into the details here.
The general process of moving genes between species is called
transformation, and the result is a transgenic organism. Lately, transgenic
organisms are being called genetically modified organisms, or GMOs.
Details of the Terminator technology
The key to Terminator is the ability to make a lot of a toxin that will
kill cells, and to confine that toxin to seeds.
To accomplish this, in the case of our cotton example, the plan is to take
the promoter from a gene normally activated late in seed development in
cotton, and to fuse that promoter to the coding sequence for a protein that
will kill an embryo going through the last stages of development.
In the Terminator patent, the authors use a promoter from a cotton LEA
(Late Embryogenesis Abundant) gene. This gene is one of the last to be
activated. Its protein is not made until the seed is full-sized, has
accumulated most of its storage oil and protein and is drying down in
preparation for the dormant period in between leaving the parent plant and
germinating in the soil. If the engineered gene has the same pattern of
expression, LEA-promoter-directed proteins should be made in high
quantities, only in seeds, and late in development. It is important for the
cotton seeds to go through most of their growth before the toxin acts,
because the cotton fiber is an outgrowth of the seed coat and is made as
the cotton develops. Further, after the cotton fibers are removed (for
human use), the seed is then crushed for oil and protein, both of which are
eaten by people and livestock. The cotton crop would be of little use to a
farmer if the seeds did not mature normally before dying.
As for a toxin, there are several possibilities discussed in the patent,
but the patent authors recommend a ribosome inhibitor protein (RIP) from
the plant Saponaria officinalis. This protein works is small quantities to
stop the synthesis of all proteins. Since cells need proteins for almost
everything, they die fairly quickly when they can't make proteins.
According to the patent, the RIP is non-toxic to organisms other than
plants.
The manipulations of DNA required to engineer a seed-specific
promoter/toxin coding sequence gene are done in test-tubes and bacteria,
and then the altered gene is put into a cotton plant, using one of several
possible well-established methods.
However, this is not all there is to it. It this were all, then as soon as
the transgenic plant went through its life cycle and came around to seed
development, that would be the end of the project. There would soon be no
viable seeds to sell to farmers.
The Terminator patent offers an ingenious method for keeping the toxin gene
from being active until long after the farmers plant their crops. The trick
is accomplished by inserting a piece of DNA in between the seed-specific
promoter and the toxin coding sequence that blocks it from being used to
make protein.
At either end of the blocking DNA are put special DNA pieces that can be
recognized by a particular enzyme, such as the enzyme called recombinase.
Whenever the recombinase encounters these DNA pieces, the DNA is cut
precisely at the outside of each piece, and the cut ends of the DNA fuse
together, with the result that the blocking DNA is removed. When this
happens, the seed-specific promoter is right next to the toxin coding
sequence, and is able to function in making the toxin. But this does not
happen immediately. Toxin will not be produced until the end of the next
round of seed development, because that is when the LEA promoter is active.
Thus, after the recombinase enzyme does its work, the plant grows normally
from germination, through growth of stems, leaves, roots, and all the way
through flower formation, pollination and most of seed development. Then,
on cue, the seeds die.
All this accomplished, there remains one more problem: How to grow several
generations of the genetically-engineered variety so that its seed can be
multiplied to sell to farmers?
The Terminator patent solves the dilemma by preventing recombinase from
acting until just before the farmers plant their seeds. The patent holders
give several possible ways to do this, but concentrate on the following
procedure: They propose putting a recombinase coding sequence next to a
promoter that is always active -- in all cells, at all times -- but that is
repressed. The promoter can be made active again -- derepressed -- by a
chemical treatment. Therefore, the seed sellers can treat the seeds right
before planting, thus allowing the recombinase to be made then, but not
before.
One of the repressible promoter systems they discuss in detail is
controlled by the antibiotic tetracycline. A gene that makes a repressor
protein all of the time would be put into the cotton plant, along with a
recombinase gene that has a promoter engineered to be inactivated by the
repressor protein. Under most conditions, then, the repressor would
interact with the recombinase gene; no recombinase would be made; the toxin
gene would be blocked; and no toxin would be made, even during seed
development when the LEA promoter normally would be active.
To activate the toxin gene, seeds just starting to germinate would be
treated with tetracycline, right before they are sold to farmers. The
tetracycline would interact with the repressor protein, keeping it from
interfering with production of recombinase. Recombinase would be made,
cutting out the blocking DNA from the toxin gene. The toxin gene would now
be capable of making toxin, but would not actually do so until the end of
seed development. The next generation would thus be killed.
To accomplish the Terminator effect in cotton, then, three engineered
components must all be transferred into a cotton plant's DNA:
1. a toxin gene controlled by a seed-specific promoter, but blocked by a
piece of DNA in between the promoter and the coding sequence;
2. a repressor protein coding sequence with a promoter that is active all
of the time; and
3. a recombinase coding sequence, controlled by a promoter that would be
active at all times, except that it is also regulated by repressor protein,
which can be overridden with tetracycline.
The actual transfer of genes into the plant is not a very precise
operation. Any one of a variety of methods can be used: the
genetically-engineered DNA can be injected into the nucleus of a cotton
cell with a tiny needle, or plants cells can be soaked in the DNA and
electrically shocked, or the DNA can be attached to small metal particles
and shot into the cells with a gun, or viruses and bacteria can be
engineered to infect cells with the DNA. In all cases, the
genetically-engineered DNA has to find its way to the nucleus, and become
incorporated into the plant chromosomes. The number of copies of the
inserted genes and their locations on the plant chromosomes are
unpredictable, and how well the new genes will function hangs in the
balance.
It takes a lot of effort to locate cells that have incorporated DNA in
significant amounts and in locations that work. Basically, whole plants
have to be regenerated from the cells or tissues that were transformed with
the foreign DNA, and then each plant has to be tested for the presence and
function of the new genes.
After plants with well-functioning new genes are identified, they are then
mated in combinations that result in a line of cotton where both sets of
chromosomes (in all of the offspring) have all the components necessary for
Terminator to function. These plants are mated together to make a large
quantity of seed for sale.
In effect, Terminator Technology gives the seed producer the ability to
determine when to set Terminator in motion. Until the recombinase is made,
the cotton plants grow normally. After recombinase is made, the second
generation of seeds is killed, protecting the patented variety.
Some problems that may crop up with the use of Terminator
The patent on this technology is complex. I have described only one of many
possible applications of the procedure. Clearly, one cannot determine ahead
of time all possible biological ramifications of implementing the patent.
However, potential problems have already been noted (Ho 1998). I deal with
some of them below.
Will the Terminator spread to other plants?
It is likely that Terminator will kill the seeds of neighboring plants of
the same species, under certain conditions. However, the effects will be
confined to the first generation, and will not be able to spread to other
generations. The scenario might go like this: when farmers plant the
Terminator seeds, the seeds already will have been treated with
tetracycline, and thus the recombinase will have acted, and the toxin
coding sequence will be next to the seed-specific promoter, and will be
ready to act when the end of seed development comes around. The seeds will
grow into plants, and make pollen. Every pollen grain will carry a
ready-to-act toxin gene. If the Terminator crop is next to a field planted
in a normal variety, and pollen is taken by insects or the wind to that
field, any eggs fertilized by the Terminator pollen will now have one toxin
gene. It will be activated late in that seed's development, and the seed
will die. However, it is unlikely that the person growing the normal
variety will be able to tell, because the seed will probably look normal.
Only when that seed is planted, and doesn't germinate, will the change
become apparent.
In most cases, the toxin gene will not be passed on any further, because
dead plants don't reproduce. However, under certain conditions I will
discuss later, it is possible for the toxin gene to be inherited.
In any case, dead seeds, where they occur, would be a serious problem for
the farmer whose fields are close to the Terminator crop. How many seeds
die will depend on the degree of cross-pollination, and that is influenced
by the species of plant, the variety of crop, weather conditions, how close
the fields are to each other, and so on. If many seeds die, it will make
saving seed untenable for the adjacent farmer. Even if only a few seeds
die, they will contain the toxin and any other proteins engineered into the
Terminator-protected variety. These new "components" may make the seed
unusable for certain purposes.
Will seeds containing the toxin made by Terminator be safe to eat?
In fact, the effects of the toxin on the uses of the seed are a serious
question. This issue is discussed in the patent at the end of page 8. There
the authors say that "[i]n cotton that would be grown commercially only
selected lethal genes could be used since these proteins could impact the
final quality of seeds....If the seed is not a factor in the commercial
value of a crop (e.g., in forage crops, ornamentals or plants grown for the
floral industry) any lethal gene should be acceptable."
This is dangerously reductionist thinking, because people are not the only
organisms that interact with seeds. In forage crops, for example, not all
of the forage is always harvested before seeds are mature, depending on
conditions. How will a particular toxin affect birds, insects, fungi and
bacteria that eat or infect the seeds? If a forage crop with toxin-laden
seeds is left in the field, and the seeds come in contact with the soil,
how will that affect the ecology of soil organisms? These are important
questions because a variety of specific organisms are necessary for the
healthy growth of plants. Further, a floral or ornamental crop with
Terminator may happen to grow near a related crop where the seeds are used,
and if pollination occurs, the seeds will contain toxin without that farmer
knowing. The toxin could end up in products without anyone's knowledge. For
example, an ornamental sunflower could spread Terminator to an oilseed
variety, and then the toxin could end up in edible oil or in sunflower seed
meal.
Matthew Townsend
Barrister & Accredited Mediator
Lecturer in Environmental Law, Victoria University of Technology
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