Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-21
2004-09-14
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S183000, C435S184000, C530S350000, C800S295000, C800S300000
Reexamination Certificate
active
06790619
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The invention relates to nucleic acids which encode plant polypeptides with the biological activity of phosphomevalonate kinases, to the polypeptides encoded by them and their use as targets for herbicides and their use for identifying novel, herbicidally active compounds, and to methods for finding modulators of these polypeptides.
BACKGROUND OF THE INVENTION
Unwanted plant growth can be prevented by using herbicides. The demands made on herbicides with regard to their efficacy, costs and environmental compatibility have been steadily increasing. There exists therefore a demand for new substances which can be developed into potent new herbicides. In general, it is usual to search for such new lead structures in greenhouse tests. However, such tests are laborious and expensive. Accordingly, the number of substances which can be tested in the greenhouse is limited.
Advantageous targets for herbicides are searched for in essential biosynthetic pathways. Thus, the biosynthesis of isoprenoids in plants leads, inter alia, to the synthesis of carotenoids and of the side chains of plastoquinone and of chlorophyll. These products are essential for the photosynthetic growth of plants. The inhibition of one step in this biosynthetic pathway leads to the termination of a plant's growth. Moreover, plant hormones such as gibberellic acid, abscisic acid and brassinosteroids and membrane components (phytosterols), which are also essential for the plant's growth, are formed from isoprenoids.
Isopentyl diphosphate (IPP) is the branching point from which the widest range of isoprenoids are formed. The production of IPP is therefore a critical point in plant metabolism. In plants, IPP is produced via two different metabolic pathways in different compartments. In the endoplasmic reticulum (ER) and in the cytosol, IPP synthesis proceeds via the classic acetate/mevalonate metabolic pathway as it also proceeds in the animal organism. In contrast, IPP is synthesized in chloroplasts via the alternative glyceraldehyde phosphate/pyruvate metabolic pathway. Both metabolic pathways are essential since various isoprenoid metabolites are formed in the different compartments. Moreover, the degree to which the two metabolic pathways are autonomous or to which an exchange of metabolites takes place between the compartments has not been elucidated as yet (Heintze et al., 1990, Kleinig, 1989). (See References section below for full citation to these and other references referred to herein).
Clomazone is a known herbicidal compound which reduces the carotenoid and chlorophyll content in the leaf. For a long time it has been assumed that clomazone acts via the inhibition of the isoprenoid metabolic pathway. Norman et al. (1990) had demonstrated that the site of action would have to be between mevalonate and geranylgeranyl pyrophosphate. This would establish one of the interposed five enzymes, one of which is phosphomevalonate kinase, as the site of action. Somewhat more recent works by Weimer et al. (1992) and Rodney Croteau (1992) suggest, however, that the site of action of clomazone would be found elsewhere.
SUMMARY OF THE INVENTION
Within the context of the present invention, an
Arabidopsis thaliana
cv. Columbia cDNA has been isolated with homology to phosphomevalonate kinase, hereinbelow abbreviated to PMVK, from
Saccharomyces cerevisiae
(FIG.
1
). It was possible to induce this gene in
Arabidopsis thaliana
cv. Columbia by treatment with the herbicide chlorsulfuron (10 g/ha).
The homology between the
Saccharomyces cerevisiae
PMVK (=ERG8) and the cDNA isolated from
A. thaliana
amounts to 44% similarity or 35% identity (see
FIG. 1
, Bestfit with Wisconsin Package Version 10.1). (ERG8 is the name of the gene encoding phosphomevalonate kinase in yeast (
S cerevisiae
)). This corresponds for example to the homology between the
Saccharomyces cerevisiae
mevalonate kinase and the
Arabidopsis thaliana
mevalonate kinase with a similarity of 45% and an identity of 35%. The function was detected for the
Arabidopsis thaliana
mevalonate kinase by complementation of the corresponding mutant from
Saccharomyces cerevisiae.
Moreover, the cDNA isolated within the context of the present invention shows 69% identity with a partial PMVK sequence from
Pinus radiate
in accordance with SEQ ID NO:5, which is of interest for modifying the isoprenoid content, isoprenoid composition and isoprenoid metabolism of plants (WO 00/36 081). Further partial cDNAs from plants (
Medicago trunculata,
Accession Number AA660847, see SEQ ID NO:3 and
Gossypium hirsutum,
Accession Number Al727861, see SEQ ID NO:4) have been isolated as putative PMVKs. Various Arabidopsis spp. sequences (ESTs and genomic sequences) which correspond to the PMVK sequence isolated herein or to parts thereof can be found in databases from various sequencing projects, however, no information is given on the function or importance of these sequences or sequence fragments.
For the first time, the present invention now provides the complete cDNA sequence of a plant phosphomevalonate kinase and describes its use, or the use of the polypeptides encoded thereby, for identifying new herbicidal active compounds.
Subject-matter of the present invention are therefore nucleic acids which encode complete plant phosphomevalonate kinases, with the exception of the partial nucleic acid sequences from
Medicago trunculata
in accordance with SEQ ID NO:3,
Gossypium hirsutum
in accordance with SEQ ID NO:4 and
Pinus radiata
in accordance with SEQ ID NO:5.
Subject-matter of the present invention are, in particular, nucleic acids which encode the
Arabidopsis thaliana
phosphomevalonate kinase.
Subject-matter of the present invention are very particularly nucleic acids which encode the
Arabidopsis thaliana
phosphomevalonate kinase and are described under SEQ ID NO:1 and/or encode a polypeptide in accordance with SEQ ID NO:2 or fragments thereof.
REFERENCES:
patent: 00/15809 (2000-03-01), None
patent: 00/36081 (2000-06-01), None
patent: 00/53782 (2000-09-01), None
patent: 01/14533 (2001-03-01), None
Lange et al. Proc. Natl. Aca. Sci. U.S.A., 1999, vol. 96(24), p. 13714-13719.*
Nucleic Acids Res., vol. 25, No. 17, (month unavailable) 1997, pp. 3389-3402, Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller and David J. Lipman, “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.”
Nucleic Acids Research, vol. 12(22), (month unavailable) 1984, pp. 8711-8721, Michael Bevan, “Binary Agrobacterium vectors for plant transformation.”
Plant Physiol., 98, (month unavailable), 1992, pp. 1515-1517, Rodney Croteau, “Clomazone Does Not Inhibit the Conversion of Isopentenyl Pyrophosphate to Geranyl, Farnesyl, or Geranylgeranyl Pyrophosphate in Vitro.”
Proc. Natl. Acad. Sci., vol. 93, pp. 6025-6030, Jun. 1996, Luda Diatchenko, Yun-Fai Chris Lau, Aaron P. Campbell, Alex Chenchik, Fauzia Mooadam, Betty Huang, Sergey Lukyanov, Konstantin Lukyanov, Nadya Gurskaya, Eugene D. Sverdlov and Paul D. Siebert, “Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries”.
Plant Physiol (month unavailable) 1990, 93, pp. 1121-1127, Adolf Heintze, Jörn Görlach, Carola Leuschner, Petra Hoppe, Petra Hagelstein, Detlef Schulze-Siebert and Gernot Schultz, “Plastidic Isoprenoid Synthesis during Chloroplast Development”.
The Plant Journal, (month unavailable) 1992, 2(3) pp. 417-422, Michèle Minet, Marie-Elisabeth Dufour and Francois Lacroute, Complementation ofSaccharomyces cerevisiaeauxotrophic mutants byArabidopsis thalianacDNAs.
Annu. Rev. Plant Mol. Biol. 40, (month unavailable) 1989, pp. 39-59, H. Kleinig, “The Role of Plastids in Isoprenoid Biosynthesis”.
Gene, 156 (month unavailable) 1995, pp. 119-122, Dominik Mumberg, Rolf Müller and Martin Funk, “Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds”.
Plant Physiol. 94, Jun. 1990, pp. 704-709, M
Lechelt-Kunze Christa
Meissner Ruth
Bayer Aktiengesellschaft
Harmuth Raymond J.
Henderson Richard E. L.
Horlick Kenneth R.
Tung Joyce
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