Diacylglycerol acyl transferase proteins
AssigneeCalgene, Inc.
InventorsKathryn Dennis Lardizabal, Gregory A. Thompson, Deborah Hawkins
Life of this patent
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Abstract
The invention provides diacylglycerol acyltransferase (DAGAT) proteins, wherein said proteins are active in the formation of triacylglycerol from fatty acyl and diacylglycerol substrates. In one aspect, Mortierella ramanniana DAGAT proteins have been isolated and have molecular weights of between approximately 36 and 37 kDa as measured by SDS-PAGE. The invention also provides novel DAGAT polynucleotide and polypeptide sequences and to methods of producing such polypeptides using recombinannt techniques. In addition, methods are provided for using such sequences to alter triacylglycerol levels in plants and to treat diseases associated with altered DAGAT activity or expression.
Claims48
An isolated DNA sequence encoding an enzyme active in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates.
An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:
- 38.
An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:
- 45.
An isolated polypeptide encoded by the nucleotide sequence as set forth in SEQ ID NO:37.
An isolated polypeptide encoded by the nucleotide sequence as set forth in SEQ ID NO:44.
An isolated polypeptide encoded by the nucleotide sequence as set forth in SEQ ID NO:84.
An isolated polynucleotide selected from the group consisting of:
- a) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:
- 38;
- b) an isolated polynucleotide comprising SEQ ID NO:
- 37;
- c) an isolated polynucleotide comprising a nucleotide sequence which has at least 70% identity to that of SEQ ID NO:
- 37 over the entire length of SEQ ID NO:
- 37;
- d) an isolated polynucleotide comprising a nucleotide sequence which has at least 80% identity to that of SEQ ID NO:
- 37 over the entire length of SEQ ID NO:
- 37;
- e) an isolated polynucleotide comprising a nucleotide sequence which has at least 90% identity to that of SEQ ID NO:
- 37 over the entire length of SEQ ID NO:
- 37;
- f) an isolated polynucleotide comprising a nucleotide sequence which has at least 95% identity to that of SEQ ID NO:
- 37 over the entire length of SEQ ID NO:
- 37;
- g) an isolated polynucleotide that hybridizes, under stringent conditions, to SEQ ID NO:
- 37 or a fragment thereof;
- and
- h) an isolated polynucleotide complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), (f), or (g).
An isolated polynucleotide selected from the group consisting of:
- a) an isolated polynucleotide comprising SEQ ID NO:
- 84;
- b) an isolated polynucleotide comprising a nucleotide sequence which has at least 70% identity to that of SEQ ID NO:
- 84 over the entire length of SEQ ID NO:
- 84;
- c) an isolated polynucleotide comprising a nucleotide sequence which has at least 80% identity to that of SEQ ID NO:
- 84 over the entire length of SEQ ID NO:
- 84;
- d) an isolated polynucleotide comprising a nucleotide sequence which has at least 90% identity to that of SEQ ID NO:
- 84 over the entire length of SEQ ID NO:
- 84;
- e) an isolated polynucleotide comprising a nucleotide sequence which has at least 95% identity to that of SEQ ID NO:
- 84 over the entire length of SEQ ID NO:
- 84;
- f) an isolated polynucleotide that hybridizes, under stringent conditions, to SEQ ID NO:
- 84 or a fragment thereof;
- and
- g) an isolated polynucleotide complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), or (f).
An isolated polynucleotide selected from the group consisting of:
- a) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:
- 45;
- b) an isolated polynucleotide comprising SEQ ID NO:
- 44;
- c) an isolated polynucleotide comprising a nucleotide sequence which has at least 70% identity to that of SEQ ID NO:
- 44 over the entire length of SEQ ID NO:
- 44;
- d) an isolated polynucleotide comprising a nucleotide sequence which has at least 80% identity to that of SEQ ID NO:
- 44 over the entire length of SEQ ID NO:
- 44;
- e) an isolated polynucleotide comprising a nucleotide sequence which has at least 90% identity to that of SEQ ID NO:
- 44 over the entire length of SEQ ID NO:
- 44;
- f) an isolated polynucleotide comprising a nucleotide sequence which has at least 95% identity to that of SEQ ID NO:
- 44 over the entire length of SEQ ID NO:
- 44;
- g) an isolated polynucleotide that hybridizes, under stringent conditions, to SEQ ID NO:
- 44 or a fragment thereof;
- and
- h) an isolated polynucleotide complementary to the polynucleotide sequence of (a), (b), (c), (d), (e), (f) or (g).
An isolated polynucleotide selected from the group consisting of SEQ ID Nos:
- 46 through 67, inclusive.
A Mortierella acyltransferase protein wherein said acyltransferase is active in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates and wherein said acyltransferase is isolated from other proteins indigenous to Mortierella.
An acyltransferase protein, wherein said protein has an apparent molecular mass of about 36 kD on SDS-PAGE, said protein being substantially purified away from membranes and other proteins of the native cell and capable of catalyzing the production of triglycerides from diacylglycerol and fatty acyl substrates.
An acyltransferase protein, wherein said protein has an apparent molecular mass of about 36.5 kD on SDS-PAGE, said protein being substantially purified away from membranes and other proteins of the native cell and capable of catalyzing the production of triglycerides from diacylglycerol and fatty acyl substrates.
A nucleic acid construct comprising as operably linked components in the 5′ to 3′ direction of transcription:
- a transcriptional initiation region;
- and
- a polynucleotide sequence encoding an enzyme active in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates.
A method for producing a recombinant host cell, comprising:
- transforming or transfecting a cell with a nucleic acid construct comprising a transcriptional initiation region and a polynucleotide sequence encoding an enzyme active in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates, such that said host cell, under appropriate culture conditions, produces an acyltransferase protein.
A method of modifying the triacylglycerol composition in a plant cell said method comprising:
- transforming a plant cell with a nucleic acid construct comprising a transcriptional initiation region and a polynucleotide sequence encoding an enzyme active in the formation of triacylglycerol from diacylglycerol and fatty acyl substrates, or fragment or variant thereof
A method of treating or ameliorating a disease or condition associated with altered diacylglycerol acyl transferase (DAGAT) activity, said method comprising the step of:
- administering a therapeutically effective amount of an antagonist of DAGAT activity to a subject in need thereof.
A method of treating or ameliorating a disease or condition associated with altered diacylglycerol acyl transferase (DAGAT) activity, said method comprising the step of:
- administering a therapeutically effective amount of an agonist of DAGAT activity to a subject in need thereof.
A method of diagnosing a disease or susceptibility to a disease associated with DAGAT expression or activity in a patient, said method comprising the step of:
- determining the presence or absence of a mutation in the nucleotide sequence encoding DAGAT in the genome of said patient.
A method of diagnosing a disease or susceptibility to a disease associated with DAGAT expression or activity in a patient, said method comprising the step of:
- detecting the presence or amount of DAGAT or an indicator thereof in a sample obtained from said patient.
A method for identifying a compound which stimulate or inhibit DAGAT expression or activity comprising:
- contacting a composition comprising a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:
- 38 and a polypeptide comprising the ammo acid sequence set forth in SEQ ID NO:
- 45 with a candidate compound;
- and
- detecting an interaction between said polypeptide and said candidate compound.
An agonist of a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:
- 38 and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:
- 45.
An antagonist of a polypeptide selected from the group consisting of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:
- 38 and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:
- 45.
Dependencies are inferred from the claim text.