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...
Reexamination Certificate
2000-09-22
2003-07-22
Bui, Phuong T. (Department: 1638)
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...
C435S006120, C435S069100, C435S183000, C435S410000, C435S419000, C435S252300, C435S320100, C530S350000, C530S370000, C536S023200, C536S023600, C536S024100, C536S024300, C536S024330, C800S278000, C800S295000
Reexamination Certificate
active
06596926
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 phosphatidylcholine biosynthetic enzymes in plants and seeds.
BACKGROUND OF THE INVENTION
Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) are the major phospholipids found in plant tissues. The distribution of these lipids among the various organelles of different tissues and among different plants has been comprehensively studied. The pathways by which these lipids are synthesized have also been studied extensively but very few of the plant enzymes involved in these pathways have been purified or their corresponding genes cloned.
The precursor molecule common to the de novo synthesis of all phospholipids in prokaryotes and eukaryotes is phosphatidic acid (PA). Synthesis of PA occurs by the sequential acylation of glycerol-3-phosphate by glycerol-3-phosphate acyltransferase and 1-monoacylglycerol-3-phosphate acyltransferase, both of which utilize acyl-CoA as a source of acyl moieties. PA may be converted to CDP-diacylglycerol by the action of the enzyme CDP-diacylglycerol synthase (E.C. 2.7.7.41; also called CTP:phosphatidate cytidylyltransferase, phosphate cytidylyltransferase, phosphoethanolamine cytidylyltransferase, among others). This enzyme has been characterized in yeast where it has been demonstrated to be highly regulated (Homann et al. (1987)
J. Bacteriol
. 169:3276-3280). While phosphatidate cytidylyltransferase activity has been detected in the chloroplast, mitochondria and microsomes of several plants, no sequence information of plant phosphatidate cytidylyltransferase has been confirmed. The sequence of an
Arabidopsis thaliana
putative phosphoethanolamine cytidylyltransferase was identified when the sequence of the chromosome 2 was determined (Lin et al. (1999)
Nature
402
:
761
-
768
).
In castor bean endosperms PE is sequentially methylated to PC by methyltransferases which utilize S-adenosylmethionine as the methyl donor. PE N-methyltransferase (EC 2.1.1.17) catalyzes the methylation of PE to phosphatidyl methylethanolamine (PME) and phosphatidyl-N-methylethanolamine N-methyltransferase (EC 2.1.1.71; also called phosphatidylethanolamine N-methyltransferase) catalyzes the two methylations necessary to convert PME to PC (McGraw and Henry (1989)
Genetics
122:317-330). The sequence of a plant phosphatidylethanolamine N-methyltransferase has yet to be determined.
Identification of the sequences encoding phosphoethanolamine cytidylyltransferase or phosphatidylethanolamine N-methyltransferase in plants will allow the manipulation of these genes in transgenic plants.
SUMMARY OF THE INVENTION
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 40 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 18, and 20, (b) a second nucleotide sequence encoding a polypeptide of at least 200 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:12, 14, and 18, and (c) a third nucleotide sequence comprising the complement of the first or second nucleotide sequences.
In a second embodiment, it is preferred that the isolated polynucleotide of the claimed invention comprises nucleotide sequence selected from: (a) a first nucleotide sequence of at least 100 nucleotides having at least 80% identity based on the Clustal method of alignment when compared to a nucleotide sequence selected from SEQ ID NOs:1, 3, 5, 7, 9, 17, and 19, and (b) a second nucleotide sequence selected from the group consisting of SEQ ID NOs:11, 13, and 15.
In a third embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 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, and 19 and the complement of such nucleotide sequences.
In a fourth embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.
In a fifth embodiment, the present invention concerns 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.
In a sixth embodiment, the invention also 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.
In a seventh embodiment, the invention concerns a CTP:phosphoethanolamine cytidylyltransferase or a phosphatidylethanolamine N-methyltransferase polypeptide selected from the group consisting of (a) a first polypeptide of at least 40 amino acids comprising at least 80% identity based on the Clustal method of alignment compared to a polypeptide selected from SEQ ID NOs:2, 4, 6, 8, 10, 18, and 20, and (b) a second polynucleotide of at least 200 amino acids comprising at least 80% identity based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:12, 14, and 16.
In an eighth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a CTP:phosphoethanolamine cytidylyltransferase or a phosphatidylethanolamine N-methyltransferase polypeptide or enzyme activity 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 the CTP:phosphoethanolamine cytidylyltransferase or the phosphatidylethanolamine N-methyltransferase polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the CTP:phosphoethanolamine cytidylyltransferase or the phosphatidylethanolamine N-methyltransferase polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of the CTP:phosphoethanolamine cytidylyltransferase or the phosphatidylethanolamine N-methyltransferase polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a ninth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a CTP:phosphoethanolamine cytidylyltransferase or a phosphatidylethanolamine N-methyltransferase, preferably a plant CTP:phosphoethanolamine cytidylyltransferase or phosphatidylethanolamine N-methyltransferase, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 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, and 19, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oli
Famodu Omolayo O.
Kinney Anthony J.
Rafalski J. Antoni
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