Plant Science Portal 
Genetic Use Restriction Technology: How it works

There are three key genes critical to the functioning of the Technology Protection System (TPS) or Genetic Use Restriction Technology:
  1. Ribosomal inactivating protein gene (the "terminator")
  2. Cre recombinase gene
  3. Repressor gene
According to USDA (1999) and Oliver & Velten (unpub.), two 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).

In an email interview conducted with Dr. Adam Dimech (6 September 2001), Dr. Melvin Oliver said that the developers designed the TPS as a ‘constructed gene set’; selecting genes and arranging them to meet the requirements of the technology. The source of each gene was not divulged as it was deemed commercially-sensitive.

The genes are introduced into separate transgenic founder lines which were then cross-pollinated to provide a genome with the full suite of TPS genes in the target crop. The method of transformation wasn't specified.

Multiple Gene Constructs

The TPS genes are regulated by the Late Embryogenesis Abundant (LEA) promoter.

A promoter is a sequence of DNA at the start of a gene which determines how or when a particular gene is switched on and transcribed. Genes under the control of the LEA promoter are only transcribed (activated) during late embryogenesis (Crouch 1998; Hundertmark & Hincha 2008) when seeds are developing.

The Ribosomal Inactivating Protein gene encodes the production of Ribosome Inactivating Protein (RIP; otherwise known as saporin).

Ribosomes are organelles in plant cells where proteins are synthesised. The Ribosome Inactivating Protein functions as a toxin; working in small concentrations to prevent plant cells from synthesising proteins (Nielsen & Boston 2001; Jiang et al. 2008). An inability to synthesise proteins is fatal (Crouch 1998).

Saporin is considered non-toxic to organisms other than plants (Oliver et al. 1998), though Crouch (1998) has doubts about this. John Radin (pers.comm., 6 September 2001) told Dr. Adam Dimech that Ribosomal Inactivating Protein is of bacterial origin and is not normally produced in plants.

On either side of the Ribosomal Inactivating Protein gene is a LOX sequence, which acts as a spacer beside the Late Embryogenesis Abundant (LEA) promoter. This permits control over the activation of the Technology Protection System.

To be a commercial success, seed companies need to harvest large quantities of TPS-inactivated seed before activating the TPS and selling it to farmers. The presence of the LOX sequence on either side of the Ribosomal Inactivating Protein gene blocks its function, and thus prevents the transcription of the "terminator" Ribosomal Inactivating Protein gene (Gupta 1998). This allows normal seed set because the ribosomes are functioning normally.

Further down the chromosome from the Ribosomal Inactivating Protein gene is the recombinase gene, which is blocked by a tet repressor protein.

The tet repressor protein is produced by a tet repressor gene (Gupta 1998, Oliver & Velten unpub.). The repressor gene is preceded by a tetracyline-responsive promoter which is sensitive to the “chemical stimulant” tetracycline (Crouch 1998).
Figure 1: A schematic diagram of the Technology Protection System.
Figure 1: A schematic diagram of the Technology Protection System. A Late Embryogenesis Abundant (LEA) promoter regulates the expression of a LOX sequence (itself containing a recognised blocking sequence), followed by a Ribosomal Inactivating Protein ("terminator") gene. Further down the chromosome is a Cre Recombinase gene, under the control of a tet promoter. A tet repressor gene prevents transcription of the Recombinase gene by synthesising a blocking protein. When tetracycline is applied, repressor gene transcription is stopped. This causes transcription of the Cre Recombinase gene, which produces Cre. Cre recognises the Cre blocking sequence in the LOX sequence and splices LOX from the genome, thus placing the Ribosomal Inactivating Protein under the direct control of the Late Embryogenesis Abundant promoter. During late embryogenesis, the Ribosomal Inactivating Protein "Terminator gene" is expressed, leading to the abortion of all embryos.
Activating the Technology Protection System

When a seed company wants to sell the seed, it is treated with tetracycline (the “chemical stimulant”) which is absorbed by the plant tissue (Rakshit 1998; Gupta 1998; Crouch 1998). About 50 to 100 mg mL-1 of tetracycline is used (Oliver & Velten unpub.). Tetracycline is an antibiotic derived originally from Streptomyces spp. (Perdue 1996).

An application of tetracycline activates the tet promoter, causing the cessation of repressor gene transcription.
Figure 2: Tetracycline chemical structure
Figure 2: The chemical structure of tetracycline.
When the repressor gene transcription stops, the repressor is no longer synthesised (Gupta 1998), causing the recombinase gene to be transcribed (activated). This produces an enzyme called Cre recombinase, which has the abbreviation Cre.

At each end of the LOX sequence is a DNA excision sequence which is recognised by Cre (Hills et al. 2007). When Cre comes into contact with these DNA pieces, the LOX sequence is spliced from the genome. The DNA is cut precisely on the outside of the LOX genes and the spliced ends of the remaining DNA fuse together, placing the LEA promoter beside the Ribosomal Inactivating Protein gene (Crouch 1998).

(The Cre/LOX system is used in many applications to activate transgenes in a tissue specific manner (Odell, Hoopes & Vermerris 1994) and is vital for the operation of TPS).

In the absence of the LOX sequence, the Ribosomal Inactivating Protein gene is under the direct influence of the LEA promoter (Gupta 1998).

When the plants reach late embryogenesis (the final developmental phase of seed formation), the Ribosomal Inactivating Protein gene is activated by the Late Embryogenesis Abundant promoter and saporin is produced, killing all of the embryos (Crouch 1998; Gupta 1998).

A physiologically-normal seed will be produced under TPS, the only difference being that the embryo is dead.

TPS seeds contain endosperm and hence can still be harvested for food in the case of wheat, or fibres in the case of cotton.


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