Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
1999-11-12
2002-11-12
Bui, Phuong T. (Department: 1656)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C800S278000, C435S006120, C435S069100, C435S070100, C435S183000, C435S410000, C435S419000, C435S320100, C530S370000, C536S023100, C536S023200, C536S023600, C536S024100, C536S024300, C536S024330
Reexamination Certificate
active
06479733
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in sterol metabolism in plants and seeds.
BACKGROUND OF THE INVENTION
Sterols play major roles in plant growth and development. C-4 methyl sterol oxidase (methyl sterol oxidase) catalyzes the-first of three enzymatic steps in the removal of the two C-4 methyl groups of 4,4-dimethylzymosterol leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. The yeast methyl sterol oxidase ERG25 and its human homologue contain a putative set of metal binding motifs with similarity to that seen in a family of membrane desaturases-hydroxylases. The C-4 methyl sterol oxidase is regulated not by iron but by an end product of the ergosterol pathway, and changes to its activity result in marked changes in lipid metabolism, including the accumulation of fatty acids, triglycerides, methyl sterols, and other sterol precursors (Li and Kaplan (1996)
J. Biol. Chem
. 271:16927-16933).
In plants, the dominant sterols are 24-alkyl sterols, which play multiple roles in plant growth and development, i.e. as membrane constituents and as precursors to steroid growth regulators such as brassinosteroids. The initial step in the conversion of the phytosterol intermediate cycloartenol to the 24-alkyl sterols is catalyzed by S-adenosyl-L-methionine:delta 24-sterol-C-methyl-transferase, a rate-limiting enzyme for phytosterol biosynthesis. The gene encoding the soybean 24-sterol-C-methyl transferase has been identified and is similar to the yeast ERG6 gene. Higher levels of 24-sterol-C-methyl transferase transcript are found in higher abundance in growing vegetative tissues than in mature vegetative tissues. This transcript is highly expressed in flowers and present in very small amounts in young pods and immature seeds (Shi et al. (1996)
J. Biol. Chem
. 271:9384-9389). At least two methyl transferases have been identified in
Arabidopsis thaliana
, two in
Nicotiana tabacum
, and one in
Ricinus communis
(Bouvier-Nave et al. (1997)
Eur. J Biochem
. 246:518-529).
Brassinosteroids are ubiquitously distributed in the plant kingdom, and when applied exogenously at nanomolar to micromolar levels, they exhibit a wide spectrum of physiological effects including promotion of cell elongation and division, enhancement of tracheary element differentiation, retardation of abscission, enhancement of gravitropic-induced bending, promotion of ethylene biosynthesis, and enhancement of stress resistance. The Arabidopsis DEETIOLATED2 (DET2) catalyzes the formation of campestanol from campesterol in brassinosteroid biosynthesis (Fujioka et al (1997)
Plant Cell
9:1951-1962). DET2 is a steroid 5-alpha reductase with biochemical properties similar to the mammalian enzyme which is also called 3-oxo-5-alpha steroid 4-dehydrogenase (EC 1.3.99.5).
Sequences of ESTs which may encode portions of C-4 methyl sterol oxidase are found in the NCBI database having General Identifier Nos. 5069775, 4966861, 4966892, and 6031650. EST sequences which may encode sterol-c-methyl transferases are found in the NCBI database having General Identifier Nos. 5509139, 5753639, 4313622, 5820099, 6070142, and 6070563.
Elucidation of all the genes involved in sterol metabolism will allow the manipulation of the oil and protein content of the grains.
SUMMARY OF THE INVENTION
The present invention relates to an isolated polynucleotide comprising the nucleotide sequence comprising at least 30 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, and 29 or compositions thereof.
The present invention relates to an expression cassette comprising an isolated polynucleotide of the present invention operably linked to a promoter.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 200 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn C-4 methyl sterol oxidase polypeptide of SEQ ID NO:20. The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 90 amino acids that has at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a rice C-4 methyl sterol oxidase polypeptide of SEQ ID NO:22, a soybean C-4 methyl sterol oxidase polypeptide of SEQ ID NO:24, a wheat C-4 methyl sterol oxidase polypeptide of SEQ ID NO:8. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 70 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn steroid 5alpha-reductase polypeptide of SEQ ID NO:10. The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 120 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide a wheat steroid 5alpha-reductase polypeptide of SEQ ID NO:26. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 90 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn sterol-c-methyl transferase polypeptide of SEQ ID NO:28, and a rice sterol-c-methyl transferase polypeptide of SEQ ID NO:16. The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 220 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a soybean sterol-c-methyl transferase polypeptide of SEQ ID NO:30. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotide derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the pr
Famodu Omolayo O.
Rafalski J. Antoni
Bui Phuong T.
E. I. du Pont de Nemours and Company
Spiegler Alexander H.
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