Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-05-10
1998-12-08
Fox, David T.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, 4353201, 435419, 5362372-, 800205, C12P 2106, C12N 1542, C12N 1563, C12N 1582, C07H 2104
Patent
active
058467670
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of international application PCT/GB94/02765, filed Dec. 19, 1994.
BACKGROUND OF THE INVENTION
This invention relates generally to the expression of self-processing polyproteins in transgenic plants.
The relative levels of expression of several introduced genes in transgenic plants is notoriously influenced by "position effects" determined by the particular site of transgene integration into the genome. Even when introduced genes are linked on the same T-DNA, driven either by convergent or divergent promoters, they are usually not co-ordinately expressed at similar levels. This poses particular problems when high levels of expression of a number of introduced activities is required, for instance when attempting to express novel biochemical pathways in plants. In an attempt to achieve tissue specific, coordinated expression of two proteins, other researchers have linked genes by co-transference on the same T-DNA. The expression levels of linked nopaline synthase (nos) and octopine synthase (ocs) genes and closely adjacent neomycin phosphotransferase II (NPTII) and chloramphenicol acetyl transferase (CAT) reporter genes were found to vary independently. Another strategy was to link genes via adjacent and divergent promoters. Whereas coordinated expression of the Cab22L and Cab22R genes of petunia was achieved, a similar approach using the CAT and GUS genes produced a high degree of variation of CAT and GUS activities within individual transgenes.
Linking proteins in the form of polyproteins is a strategy adopted in the replication of many viruses. On translation, virus-encoded proteinases mediate extremely rapid intramolecular (cis) cleavages of the polyprotein to yield discrete protein products.
A number of viral proteinases have been partially characterised and are thought to be related, both structurally and in catalytic mechanism, to cellular proteinases. In the picornaviridae a single long open reading frame encodes a polyprotein of some 225kD, but full-length translation products are not normally observed due to extremely rapid "primary" intramolecular (cis) cleavages mediated by virus encoded proteinases. In the case of the entero- and rhinoviruses, a primary cleavage occurs between the P1 capsid protein precursor and the replicative domains of the polyprotein (P2, P3; FIG. 1A). This cleavage is mediated by a virus encoded proteinase (2A.sup.pro), of some 17kDa, cleaving at its own N-terminus.
The aphtho-, or Foot-and-Mouth Disease (FMD), viruses form a distinct group within the picornaviridae. FMDV polyprotein undergoes a primary polyprotein cleavage at the C-terminus of the 2A region between the capsid protein precursor (P1-2A) and replicative domains of the polyprotein 2BC and P3 (FIG. 1B). Precursors spanning the 2A/2B cleavage site are not detected during native polyprotein processing. This situation is somewhat analogous to the 2A cleavage in entero- and rhinoviruses described above. However the 2A region of the FMDV polyprotein was demonstrated to be only 16 amino acids long (FIG. 1,C). The predicted amino acid sequence of this region, is totally conserved amongst all aphthovirus genomic RNAs sequenced to date. The FMDV 2A region also shows high similarity to the C-terminal region of the approximately ten fold larger 2A protein of another genus of the picornaviridae, the cardioviruses.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a method for the expression of multiple proteins in a transgenic plant comprising inserting into the genome of the plant a gene construct comprising a 5'-region which includes a promoter which is capable of initiating transcription of a structural gene under the control thereof, a protein encoding sequence coding for more than one protein and a 3'-terminator region which includes a polyadenylation signal, each of the said protein encoding sequences being separated from an adjacent protein encoding sequence by a DNA sequence which on translation provides a cleavage site whereby the expressed polyprotein is
REFERENCES:
patent: 5162601 (1992-11-01), Slightom
Palmenberg. Proteolytic processing of picornaviral polyprotein. Annual Review of Microbiology. 44:603-623, 1990.
Carrington et al. Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing. The EMBO Journal. 9(5):1347-1353, 1990.
Nucleotide sequence and genome organization of foot-andmouth disease virus. Nucleic Acids Research. 12(16):6587-6600,1984.
The complete nucleotide sequence of the RNA coding for the primary translation product of foot and mouth disease virus. 12(5):2461-2472, 1984.
The 5' untranslated region of PVY RNA, even located in an internal position, enables enitiation of translation. 7(4):367-379, 1993.
Ryan et al: "Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence", J. Gen. Virol., vol. 72, 1991, pp. 2727-2732.
Roosien et al: "Synthesis of foot-and-mouth disease virus capsid proteins in insect cells using baculovirus expression vectors", J. Gen. Virol., vol. 71, 1990, pp. 1703-1711.
Ryan et al: "Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein", The EMBO J., vol. 13, 1994, pp. 928-933.
Halpin Claire
Ryan Martin Denis
Fox David T.
Thomson Marian T.
Wai Thanda
Zeneca Limited
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