Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters fat – fatty oil – ester-type wax – or...
Patent
1994-05-23
1996-12-31
Chereskin, Che S.
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters fat, fatty oil, ester-type wax, or...
4353201, 536 241, A01H 400, C12N 1582
Patent
active
055896146
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a glutathione-S-transferase enzyme and DNA sequences coding for it.
Glutathione-S-transferases (GST) are a family of enzymes which catalyse the conjugation of glutathione, via a sulphydryl group, to a large range of hydrophobic, electrophilic compounds. The conjugation can result in detoxification of these compounds and may result in their removal from tissue.
GST enzymes have been identified in a range of crop plants including maize, wheat, sorghum and peas. GST's comprise from 1 to 2% of the total soluble protein in etiolated maize seedlings.
Three isoforms of GST have been identified: GST-I, GST-II and GST-III. The major isoform in maize tissue, GST-I, is constitutively expressed and is capable of conjugating glutathione with pre-emergent herbicides such as alachlor. Treatment of maize tissues with chemical safeners (for example, N,N-diallyl-2,2-dichloroacetamide) raises the activity of GST-I which participates in the detoxification of the pre-emergent herbicides.
International Patent Application WO 90/08826 describes GST-II (isoform II).
Both GST-I and GST-II proteins have a native molecular weight of approximately 50 kD. As in mammals, maize GST's are dimeric; GST-I has apparently identical polypeptide subunits of 29 kD (GST-I-29), whereas GST-II is a heterodimer of a 29 kD subunit identical to that found in GST-I (GST-I-29) and a novel 27 kD subunit (GST-II-27). GST-II is detected at a very low basal level in the absence of safener, but its expression is enhanced dramatically by safener treatment. Like GST-I, GST-II confers resistance to certain herbicides. GST-II is known to detoxify chloroacetanilide and thiocarbamate herbicides such as alachlor (Mozer et al, 1983, Biochemistry, 22:1068-1072).
A cDNA and a gene corresponding to the 29 kD subunit of GST-I have been cloned previously and sequenced (Wiegand et al, 1986, Plant Mol Biol, 7:235-243). In addition, a cDNA corresponding to a 26 kD subunit of a third, minor component of GST activity in maize seedlings (GST-III-26) has been previously cloned and sequenced (Moore et al, 1986, Nucleic Acid Research, 18:7227-7235). GST-III is a homodimer of these 26 kD subunits. Like GST-I and unlike GST-II, GST-III is constitutively expressed. It is known to detoxify herbicides such as atrazine.
According to the present invention, we provide a genomic DNA sequence encoding the gene promoter for the 27 kD subunit of the glutathione-S-transferase, isoform II, enzyme (GST-II-27), containing the nucleotide sequence shown in FIG. 8 herewith and variants of the said sequence as permitted by the degeneracy of the genetic code.
The genomic DNA was deposited on 14 Jun. 1991 in the National Collections of Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, AB2 1RY, Scotland, UK, as plasmid pGIE7 contained within Escherichia coli, strain XLI-Blue with the accession number NCIMB 40426.
The invention also provides a GST-II-27 enzyme subunit having the amino acid sequence shown in FIG. 3 herewith.
The invention further provides a cDNA sequence encoding this GST-II-27 subunit, having the nucleotide sequence shown in FIGS. 2A-2C herewith and variants of the said sequence as permitted by the degeneracy of the genetic code.
The cDNA was deposited on 19 Apr. 1991 in the National Collections of Industrial and Marine Bacteria (NCIMB), 23 St Machar Drive, Aberdeen, AB2 1RY, Scotland, UK, as plasmid pIJ21 contained within Escherichia coli, strain XLI-Blue with the accession number NCIMB 40413.
International Patent Application WO 90/08826 describes a chemically inducible gene promoter sequence isolated from a 27 kD subunit of the maize GST-II gene (GST-II-27). The said International application is incorporated herein by reference.
In the present application we describe the cloning and sequencing of a complete cDNA corresponding to the 27 kD subunit. This sequence is inducible and specifically recognised by a GST-II-27 specific antiserum. It is partially homologous with other maize GSTs.
By virtue of the present invention, the cDNA for GST-II
REFERENCES:
patent: 5073677 (1991-12-01), Helmer et al.
Biochemistry, vol. 22, No. 5, 1983, pp. 1068-1072, Mozer, T. J., et al, "Purification and characterization of corn glutathione S-transferase" see the whole document.
WO A, 9, 008 826 Aug. 9, 1990, see the whole document.
Plant Molecular Biology, vol. 6, No. 4, 1986, pp. 203-211, Shah et al:, "Structural analysis of glutathione-S-transferase involved in herbicide detoxification" see the whole document.
WO, A, 9 008 830, Aug. 9, 1990, see p. 1-p. 7, see p. 18-p. 25, figures 1, 2.
Biotechnology vol. 3, Jul. 1985, pp. 629-635, Fraley, et al: "The SEV system: a new disarmed Ti plasmid vector system for plant transformation", see p. 632, right col., paragraph 1.
Gene. vol. 76, 1989, pp. 153-160, Wosnick et al: "Total chemical synthesis and expression in Escherichia coli of a maze glutathione-transferase (GST) gene" see the whole documents.
Nucleic Acids Research, vol. 14, No. 18, 1986, pp. 7227-7235 Moore et al: "Cloning and expression of a cDNA encoding a maize gltathione-S-transferase gene in E. coli" see p. 7227 introduction para. 2.
Physiol. Plant. vol. 77, No. 3, 1989 pp. 465-471 Timmerman, "Molecular characterization of corn glutathione S-transferase isozymes involved in herbicide detoxification".
Nucleic Acids Research, vol. 16, No. 2, 1988, pp. 425-438, Grove et al: "Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family" see the whole document.
J. Cell. Biochem. Suppl., Meeting Apr. 10-16, 1992. vol. 16F, 1992, p. 232, Greenland et al: "cloning and analysis of a cDNA clone encoding a glutathione-S-transferase subunit from maize which is induced by treatment with herbicide safeners", see abstract Y412.
Shah, et al (1986) Plant Molecular Biology 6:203-211.
Bridges Ian G.
Bright Simon W. J.
Greenland Andrew J.
Holt David C.
Jepson Ian
Chereskin Che S.
Zeneca Limited
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