There are three key genes critical to the functioning of the Technology
Protection System (TPS) or Genetic Use Restriction Technology:
- Ribosomal inactivating protein gene (the "terminator")
- Cre recombinase gene
- Repressor gene
According to USDA (1999)
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
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
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 &
) when seeds are developing.
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 &
; 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 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
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
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
; Crouch 1998
). About 50 to 100 mg
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.
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
et al. 2007
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
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.