Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-10-15
2003-10-07
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C536S023600, C435S419000, C435S468000, C800S278000
Reexamination Certificate
active
06630617
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 squalene metabolism in plants and seeds.
BACKGROUND OF THE INVENTION
The terpenoids constitute the largest family of natural products with over 22,000 individual compounds of this class having been described. The terpenoids play diverse functional roles in plants as hormones, photosynthetic pigments, electron carriers, mediators of polysccharide assembly, and, structural components of membranes. Farnesyl pyrophosphate is converted to squalene in the first dedicated step towards sterol biosynthesis. Squalene is then converted to squalene-2,3-epoxide which, in photosynthetic organisms, is converted cycloarterenol.
Squalene monooxidase (EC 1.14.99.7), also referred to as squalene epoxidase, is an oxidoreductase which acts on paired donors with incorporation of molecular oxygen. This enzyme is located at the endoplasmic reticulum, and catalyzes the conversion of squalene to squalene 2,3-epoxide in the pathway to produce sterol. Squalene monooxygenase may be the rate limiting step in sterol biosynthesis. Oxygen, NADPH, FAD, and a cytosolic protein are required for squalene monooxygenase function. Squalene monooxygenase together with lanosterol synthase was formerly known as squalene oxydocyclase.
Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. This reaction is catalyzed by cycloartenol synthase (EC 5.4.99.8), also called 2,3-epoxysqualene-cycloartenol cyclase.
Sequences encoding peptides with similarities to cycloartenol synthase and squalene monooxygenase are found in the NCBI database having General Identifier Nos. 5566676, 5468642, 5706248, 5608422, 5055959, 2309779, 2310417, 3107671, 702331, and 4874404.
SUMMARY OF THE INVENTION
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 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn cycloartenol synthase polypeptide of SEQ ID NO:2, a rice cycloartenol synthase polypeptide of SEQ ID NO:4, a soybean cycloartenol synthase polypeptide of SEQ ID NO:6, and a wheat cycloartenol synthase polypeptide of SEQ ID NO:8. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 200 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn squalene monooxygenase polypeptide of SEQ ID NO:10, a rice squalene monooxygenase polypeptide of SEQ ID NO:12, a soybean squalene monooxygenase polypeptide of SEQ ID NO:14, and a wheat squalene monooxygenase polypeptide of SEQ ID NO:16. 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, and 15 that codes for a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 40 (preferably at least 30 or at least 15) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 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 or virus. If the host cell is a virus, it is preferably a baculovirus. A virus host cell comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention is most preferred.
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 process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a cycloartenol synthase polypeptide of at least 200 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:1, 4, 6, and 8. The present invention relates to a squalene monooxygenase polypeptide of at least 200 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:10, 12, 14, and 16.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a cycloartenol synthase or a squalene monooxygenase polypeptide in a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a plant cell;
measuring the level a cycloartenol synthase or a squalene monooxygenase polypeptide in the plant cell containing the isolated polynucleotide; and
comparing the level of a cycloartenol synthase or a squalene monooxygenase polypeptide in the plant cell containing the isolated polynucleotide with the level of a cycloartenol synthase or a squalene monooxygenase polypeptide in a plant cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a cycloartenol synthase or a squalene monooxygenase polypeptide gene, preferably a plant cycloartenol synthase or a squalene monooxygenase polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 40 (preferably at least 30 or at least 15) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a cycloartenol synthase or a squalene monooxygenase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a cycloartenol synthase or a squalene monooxygenase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a cycloartenol synthase or a squalene monooxygenase, the method comprising the steps of: (a) transforming a host cell with a nucleic acid fragment encoding; a cycloartenol synthase or a squalene monooxygenase polypeptide preferably operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable fo
Famodu Omolayo O.
Kinney Anthony J.
Rafalski J. Antoni
Baum Stuart
E. I. du Pont de Nemours and Company
McElwain Elizabeth F.
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