Mechanism behind Terminator Gene Technology15.5.2012

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WELCOME

Mechanism behind Terminator Gene Technology:

Mechanism behind Terminator Gene Technology Prasad Vitthal Sawant

What is Terminator? :

What is Terminator? Terminator technology refers to plants that have been genetically modified to render sterile seeds at harvest (through an inducible molecular mechanism, which means that the gene for seed sterility or germination can be turned on or off from the outside – by treating the plants with a chemical or other factor). It is technically known as a Genetic Use Restriction Technology or GURTs. Terminator technology was developed by the multinational seed/agrochemical industry and the United States government to prevent farmers from saving and re-planting harvested seed developed by biotechnology and seed corporations.

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'Terminator,' officially named the Technology Protection System (TPS) , incorporates a trait that kills developing plant embryos, so seeds cannot be saved and replanted in subsequent years.

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Genetic use restriction technology ( GURT ), colloquially known as terminator technology or suicide seeds , is the name given to proposed methods for restricting the use of genetically modified plants by causing second generation seeds to be sterile. The technology was developed under a cooperative research and development agreement between the Agricultural Research Service of the United States Department of Agriculture and Delta and Pine Land company in the 1990 s, but it is not yet commercially available.

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Late in 2006, Monsanto acquired Delta and Pine Land company, along with its greenhouse tests of Terminator seeds and rights to its Canadian patent on Terminator granted on October 11 2005 . D&PL has long vowed to commercialize Terminator, targeting rice, wheat and soy in particular. The technology was discussed during the 8th Conference of the Parties to the United Nations Convention on Biological Diversity in Curitiba , Brazil, March 20–31, 2006.

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Several plant patent laws were passed during the last century in an attempt to increase the potential profit to agricultural and horticultural businesses that develop new varieties, but enforcement of the protection can be inconvenient and many companies have not found it profitable when protection cannot be assured. Two biotech protection methods, dubbed 'Terminator' and 'Traitor' by opponents, may allow companies to increase profits on their cultivar development efforts.

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Important landmarks in U.S. patent law Plant Patent Act of 1930 Plant Variety Protection Act of 1970 1980 amendment 1994 amendment Utility Patents

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Who Developed the Technology Protection System? Scientists from the Agricultural Research Service, a subunit of the US Department of Agriculture (USDA-ARS), and Delta and Pine Land Company, a company that develops cotton cultivars, jointly developed the system and on March 3, 1998 , were awarded a joint patent (number 5,723,765) entitled Control of Plant Gene Expression.

Variants (Types) There are conceptually two types of GURT::

Variants (Types) There are conceptually two types of GURT: 1. V-GURT : This type of GURT produces sterile seeds meaning that a farmer that had purchased seeds containing V-GURT technology could not save the seed from this crop for future planting. This would not have an immediate impact on the large number of primarily western farmers who use hybrid seeds , as they do not produce their own planting seeds, and instead buy specialized hybrid seeds from seed production companies.

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However, currently around 80 percent of farmers in both Brazil and Pakistan grow crops based on saved seeds from previous harvests. Consequentially, resistance to the introduction of GURT technology into developing countries is strong. The technology is restricted at the plant variety level , hence the term V-GURT. Manufacturers of genetically enhanced crops would use this technology to protect their products from unauthorised use.

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2. T-GURT : A second type of GURT modifies a crop in such a way that the genetic enhancement engineered into the crop does not function until the crop plant is treated with a chemical that is sold by the biotechnology company. Farmers can save seeds for use each year. However, they do not get to use the enhanced trait in the crop unless they purchase the activator compound. The technology is restricted at the trait level , hence the term T-GURT.

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An Outline of the Terminator Technology The terminator technology consists of three genes with their on/off switches. Before selling to farmers, a seed company treats the seeds with a chemical inducer--probably tetracycline --to initiate the terminator gene interactions. Although the patent covers a number of ways the genes might interact, below is a description of one way the technology could work –

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Terminator Genes in the absence of the Inducer. Terminator Genes in the presence of the Inducer. Gene I: Repressor A repressor gene produces a repressor protein. The same repressor protein is produced. Gene II: Recombinase A recombinase gene is controlled by a promoter. Between the promoter and the recombinase gene, scientists place a DNA fragment which is a binding site for the repressor from Gene I. In the absence of the inducer, the repressor binds to the binding site and the plant cannot produce the recombinase protein, an enzyme that snips out pieces of DNA. The inducer interferes with the repressor attachment to the binding site--thus allowing Gene II to produce recombinase .

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Gene III: Toxin A gene for a toxin lethal to embryos ( Toxin Gene ) is controlled by a late promoter ( LP ), that is active only during the late stage of seed development when the embryo is developing. Between the late promoter and the toxin gene, scientists place a piece of DNA called a Blocker , which interferes with the ability of the promoter to turn on the gene. Without the inducer, there is no recombinase to snip out the blocker. With the blocker in place, no toxin is produced. Recombinase from Gene II snips out the blocker and allows the late promoter to turn on production of the toxin gene late in the season. Thus, by withholding the inducer, seed companies can produce generations of viable seeds.

The terminator technology consists of three genes with their on/off switches ::

The terminator technology consists of three genes with their on/off switches : Gene I: Repressor Gene II: Recombinase Gene III: Toxin

In the absence of the Inducer :

In the absence of the Inducer

In the presence of the Inducer :

In the presence of the Inducer

How Does the Technology Protection System Work?:

How Does the Technology Protection System Work? The Technology Protection System (TPS) inserts half a dozen sequences into the DNA of the parent plant that is slated for protection. These DNA sequences are arranged into a system that kills seeds at a prearranged time in their development. The system can be left inactive while the seed company grows several generations of seeds for sale. The system is switched on by soaking the seeds in a special chemical before the seeds are delivered to the farmer for planting.

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The special chemical triggers a slow cascade of events that lead eventually to the death of progeny seeds developed on the protected plant. For the purpose of preventing replanting, the progeny seeds should be killed only after they have completed production of all commercially valuable products such as oil. Therefore, the system is designed to take effect only after the crop has grown to maturity in the field and the progeny seeds are nearly ripe.

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Many combinations of foreign genes can be used, so the list of all possible genes is quite long. Below is one system that was discussed extensively in the TPS patent.

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name of DNA sequence Source purpose 35S promoter (gene 2) cauliflower mosaic virus switches on the gene connected to it Tn10 tet repressor Escherichia coli , a bacterium prevents recombinase from being made (see " cre recombinase" below in this table) cre recombinase (scissors in diagram) P1 virus cuts out the lox segments that block production of the ribosomal inhibitor (see "lox" below in this table)

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name of DNA sequence Source purpose lox could be native to the host plant or derived from another organism creates a barrier to the production of ribosomal inhibitor; removal of the lox segments allows the production of ribosomal inhibitor late embryogenesis promoter could be native to the host plant or derived from another organism stimulates production of the ribosomal inhibitor when the seeds reach a late stage of development ribosomal inhibitor protein Saponaria officinalis , a plant kills the developing seeds by stalling the essential process of making proteins

ONE WAY TERMINATOR TECHNOLOGY MIGHT WORK:

ONE WAY TERMINATOR TECHNOLOGY MIGHT WORK

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Terminator technology consists of three genes. GENE I Gene I is a repressor gene that produces a repressor protein that interacts with a binding site near Gene II. GENE II Gene II is a recombinase gene that is controlled by a promoter. Between the gene and the promoter is a binding site for the repressor from Gene I. The recombinase gene produces a recombinase protein that is an enzyme and snips out pieces of DNA. GENE III Gene III produces a toxin that is lethal to embryos. The gene is controlled by a late promoter, which is active only during the late stage of seed development when the embryo is developing. Between the late promoter and the toxin gene is a piece of DNA called a blocker, which interferes with the ability of the promoter to turn on the toxin gene.

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According to USDA (1999) and Oliver & Velten (unpub.) , two genes are derived from a bacterium ( Bacillus amyloliquefaciens ) and one from an unspecified plant; however Crouch (1998) states that one is from cotton and another from Saponaria officinalis (Caryophyllaceae).

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INDUCER The inducer is a chemical applied to the seed by the seed company that will initiate the terminator gene interactions. If the seed company does not want to initiate the terminator genes, it will not apply the inducer. This allows the repressor protein to bind to the binding site on Gene II, preventing the production of recombinase. In the absence of recombinase, the blocker on Gene III is not snipped out, and the toxin is not produced. This allows the seed company to raise enough seed to sell to farmers.

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Before the seed company sells the seed to the farmers, it applies the inducer. The inducer blocks the binding site on Gene II preventing the repressor protein to bind. Gene II then produces recombinase which snips out the blocker on Gene III. With the blocker removed, the late promoter is able to turn on production of the toxin gene late in the season.

Technology for hybrid seed production :

Technology for hybrid seed production In case of hybrid seed production, a different strategy, utilizing only two genes (terminator and recombinase), one in each of the two parents of the hybrid are used and no repressor gene is needed. One of the parental lines contains the recombinase gene, which becomes active only after germination, and the other parent contains the lethal (terminator) gene separated from its promoter by a spacer (blocking sequence).

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The hybrid progeny, which is the technology protected hybrid seed bought and planted by the farmer, thus contains both the elements of the system in every cell. The recombinase, expressed right after germination, excises the spacer blocking sequence bringing the promoter and lethal gene together. Since the promoter is embryo specific, the lethal gene does not express till seed development starts. During seed development, the lethal gene expresses during late embryogenesis and kills the embyo. Thus the seeds harvested from the first generation hybrid crop will be normal in all essential respects, except that they will not germinate if sown as a crop.

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Possible advantages 1. Where effective intellectual property protection systems don't exist or are not enforced, GURTs could be an alternative to stimulate plant developing activities by biotech firms. 2. Non-viable seeds produced on V-GURT plants may reduce the propagation of volunteer plants. Volunteer plants can become an economic problem for larger-scale mechanized farming systems that incorporate crop rotation . 3. Under warm, wet harvest conditions non V-GURT grain can sprout, which lowers the quality of grain produced. It is likely that this problem would not occur with the use of V-GURT grain varieties.

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4. Use of V-GURT technology could prevent escape of transgenes into wild relatives and prevent any impact on biodiversity . Crops modified to produce non-food products could be armed with GURT technology to prevent accidental transmission of these traits into crops destined for foods. 5. Seeds could be stored for longer periods of time without affecting their physiology and chemical makeup, which will be an advantage for farmers and food processors.

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Possible disadvantages Initially developed by the United States Department of Agriculture and multinational seed companies, "suicide seeds" have not been commercialized anywhere in the world due to an avalanche of opposition from farmers, indigenous peoples , NGOs , and some governments. 2. In 2000, the United Nations Convention on Biological Diversity recommended a de facto moratorium on field-testing and commercial sale of terminator seeds; the moratorium was re-affirmed in 2006. 3. India and Brazil have already passed national laws to prohibit the technology.

Table : Genetic use restriction technology (GURT): potential economic benefits, costs and risks (Modified from Eaton et al. 2002).:

Table : Genetic use restriction technology (GURT): potential economic benefits, costs and risks (Modified from Eaton et al. 2002). Benefits Costs Risks Farmers Increased productivity from improved genetic inputs due to increased research and development (R&D) investment Increased input costs from seed purchase Misuse of monopoly powers by breeders Reduced seed security and access to genetic improvements (marginalized farmers) Breeders (especially private sector) Increased share of research benefits from new products Increased cost for access to genetic resources of other breeders

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Benefits Costs Risks Governments Reduced investment requirements in breeding Fewer enforcement costs for plant variety protection (PVP) Complementary R&D investment requirements Establishment and enforcement of new regulatory requirements Society Increased agricultural productivity Reduced genetic diversity in fields

Some burning issues & future prospectus of terminator gene technology:

Some burning issues & future prospectus of terminator gene technology 1) Hybrid varieties vs. terminator technology and IPRs In order to get a fair return on their investment, individual seed companies developing new varieties have been interested in biological protection of their varieties to prevent re-use of seeds harvested by farmers. At present, they intend to achieve this through the grant of patents and plant breeders rights, which have been revised in 1991 under the umbrella of UPOV to provide better control of the breeder/seed companies over the protected seed material. In case of hybrid varieties (e.g. maize, sunflower, etc.), there is a built-in protection, which forces the farmers to purchase hybrid seeds every year, because the increased yield is exhibited only in the F1 seed that is sold to the farmers and the performance declines in F2 and subsequent generations. This discourages the farmers to harvest and re-use the harvested seed of hybrid varieties for sowing the next crop.

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In several self-pollinated crops like wheat, rice, barley, beans, etc., on the other hand, the commercially grown cultivars are actually ‘pure lines’ so that the yield does not decline and harvested seeds can be used for sowing the next crop. For these inbreds, the patents/PBRs are available, but their implementation seems to be really difficult (if re-use of seed is not allowed), particularly in the developing world having millions of small resource-poor farmers. In countries like USA also, private companies have to hire investigators and detectives to root out farmers, who are saving seeds from these companies for re-plantation. In view of these inherent difficulties in the implementation of patents/PBRs in these self-pollinated crops, the termination technology is intended to be used.

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2) ‘Verminator’, an alternative to ‘terminator’ More recently, Zeneca of UK indicated that it will seek patents in more than 50 countries for its improved plant germplasm invention. This technology will prevent plant growth , rather than killing the seed. The technology makes use of a gene from the fat tissue of a rat, which will block the normal plant growth , unless the blocking process is deactivated by a chemical. The technique has been dubbed as ‘verminator’ by RAFI and appears to be wider and more flexible than the ‘terminator’, though intended to serve the same purpose.

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R AFI (2001 c ) provided a comprehensive list of companies with ‘terminator’ patents, shown below: Figure 4: A schedule of companies with patents on Genetic Use Restriction Technologies.

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Summary The Technology Protection System (TPS) is a genetic technology that prevents plants from producing viable seed. It is classified as a Genetic Use Restriction Technology (GURT). The TPS system requires three key genes as well as a number of specific promoters in order to function properly. This principally relies on the Cre/LOX system. The seeds are exposed to tetracycline to activate a toxin (saporin) which kills the embryos of the seeds. Without exposure to tetracycline, the GURT genes remain inactive. TPS was developed by Delta & Pine Land Company and the United States Department of Agriculture for the purposes of protecting the intellectual property of seed companies who develop new cultivars, as well as preventing the spread of genes from transgenic to wild plants.

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Delta & Pine Land Company (now Monsanto) and the USDA are not the only companies that have produced Genetic Use Restriction Technologies; other companies have produced similar systems. Some farmers are concerned that Genetic Use Restriction Technology may harm them, particularly in third-world countries where non-GURT crops may be contaminated with GURT genes and have reduced seed viability. Others are concerned about the ethics of the technology. GURTs are indirectly supported in Australia through Plant Breeders Rights laws, which discourage farmers from collecting and sowing the seeds of cultivars. Many lobby groups have expressed concern about Genetic Use Restriction Technologies, however a number of them demonstrate a lack of understanding of the technology and the science behind it.

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Genetic Use Restriction Technologies can benefit the environment and plant breeders, but also has the potential to be used as a monopolising mechanism by companies to ensure continued profit. As national communities, people need to have a debate about how far this contentious technology should be used in world agriculture and whether the government, companies and farmers will be involved in, and benefit from, the introduction of TPS or a similar technology. There is currently a moratorium on the use of Genetic Use Restriction Technologies.

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Thank you Thank you