Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-12-15
2003-04-08
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S069100, C435S183000, C435S410000, C435S419000, C435S252300, C435S320100, C530S350000, C530S370000, C536S023600, C536S024100, C536S024330, C800S295000
Reexamination Certificate
active
06545200
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 sterol biosynthetic enzymes in plants and seeds.
BACKGROUND OF THE INVENTION
The C-8 sterol isomerase enzyme is involved in plant, animal, and fungal sterol biosynthesis. The product of the ERG2 gene is also called delta 8-delta 7-sterol isomerase, C-8,7 sterol isomerase or D8AE7 isomerase and is non-essential in yeast. This membrane-bound enzyme is required for the isomerization of the delta 8 double bond to the delta 7 position in the distal portion of the ergosterol biosynthesis pathway (Ashman et al. (1991)
Lipids
26:628-632).
Brassinosteroids are plant enzymes important in growth promotion. Steroid 22-alplha-hydroxylase (DWF4) is a brassinosteroid biosynthetic enzyme, a member of the cytochrome P450 superfamily. The
Arabidopsis thaliana
DWF4 is a cytochrome P450 monooxygenase with significant similarity to the brassinosteroid enzyme CPD. DWF4 contains the four domains characteristic of cytochrome non-A P450 enzymes and is involved in plant sterol biosynthesis functioning as a 22 alpha-hydroxylase in what is probably the rate limiting step in brassinosteroid biosynthesis (Choe t al. (1998)
Plant Cell
10:231-243).
3-beta hydroxy-delta-5-steroid dehydrogenase (EC 1.1.1.145) is also called progesterone reductase. It is an oxidoreductase which acts on the CH—OH group of donors with NAD+ or NADP+ as acceptors in the C-21 steroid metabolism and the androgen and estrogen metabolisms. The enzyme converts 3 beta hydroxy-5-ene-steroids into 3-keto-4-ene derivatives and interconvers 3 beta-hydroxy and 3-keto-5 alpha-androstane steroids (Labrie et al. (1992)
J. Steroid Biochem. Mol. Biol.
41:421-435).
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 40 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a C-8,7 sterol isomerase polypeptide of SEQ ID NOs:2, 4, 6, 8, 22, 24, 26, and 28. 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 polypeptide of at least 70 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a steroid 22-alpha-hydroxylase polypeptide of SEQ ID NOs:10, 12, 14, 30, 32, 34, 36, 38, and 40. 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 polypeptide of at least 80 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide of SEQ ID NOs:16, 18, 20, 42, and 44. 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, 29, 31 33, 35, 37, 39, 41, and 43 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, 30, 32, 34, 36, 38, 40, 42, and 44. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43 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 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 C-8,7 sterol isomerase polypeptide of at least 40 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 22, 24, 26, and 28.
The present invention relates to a Steroid 22-alpha-hydroxylase polypeptide of at least 70 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:10, 12, 14, 30, 32, 34, 36, 38, and 40.
The present invention relates to a 3-beta-Hydroxy-delta-5-steroid dehydrogenase polypeptide of at least 80 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 42, and 44.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a sterol biosynthetic enzyme polypeptide in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level of a C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of a sterol biosynthetic enzyme polypeptide in the host cell containing the isolated polynucleotide with the level of a sterol biosynthetic enzyme polypeptide in the host 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 C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide gene, preferably a plant C-8,7 sterol isomerase, steroid 22-alpha-hydroxylase or 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43 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 C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase 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 C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogena
Cahoon Rebecca E.
Famodu Omolayo O.
McGonigle Brian
Rafalski J. Antoni
Sakai Hajime
Bui Phuong T.
E. I. du Pont de Nemours and Company
LandOfFree
Sterol biosynthetic enzymes does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Sterol biosynthetic enzymes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Sterol biosynthetic enzymes will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3059924