Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-06-24
2002-02-05
McElwain, Elizabeth F. (Department: 1638)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06344548
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of the invention is enzymes, particularly acyltransferases.
2. Background of the Invention
Diacylglycerol O-Acyltransferase (EC 2.3.1.20), also known as diglyceride acyltransferase or DGAT, is a critical enzyme in triacylglycerol synthesis. Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. DGAT catalyzes the rate-limiting and terminal step in triacylglycerol synthesis using diacylglycerol and fatty acyl CoA as substrates. As such, DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for intestinal fat absorption, lipoprotein assembly, fat storage in adipocytes, milk production and possibly egg production and sperm maturation.
Because of its central role in a variety of different processes, including those rocesses listed above, there is intense interest in the characterization of DGAT. To this end, several groups have undertaken the purification of DGAT from a variety of different sources. However, the inventors are not aware of any report in which a polynucleotide encoding DGAT has been identified or cloned.
As such, there is much interest in the identification of polynucleotides encoding proteins having DGAT activity, as well as the proteins encoded thereby.
RELEVANT LITERATURE
References describing at least partial purification of DGAT from naturally occurring sources include: Kamisaka et al., “Purification and Characterization of Diacylglycerol Acyltransferase from the Lipid Body Fraction of Oleaginous Fungus,” J. Biochem (Tokyo) 1997 (6) 1107-1114; Little et al., “Solubilization and Characterization of Diacylglycerol Acyltransferase from Microspore-Derived Cultures of Oilseed Rape,” Biochem J. (Dec. 15, 1994) 304 (Pt 3): 951-958; Andersson et al., “Purification of Diacylglycerol:acyltransferase from Rat Liver to Near Homogeneity,” J. Lipid Res. (March 1994) 35: 535-545; Polokoff & Bell, “Solubilization, Partial Purification and Characterization of Rat Liver Microsomal Diacylglycerol Acyltransferase,” Biochim. Biophys. Acta (1980) 618: 129-142.
References describing the role DGAT plays in various biological processes include: Bell & Coleman, “Enzymes of Glycerolipid Synthesis in Eukaryotes,” Annu. Rev. Biochem. (1980) 49: 459-487; Lehner & Kuksis, “Biosynthesis of Triacylglycerols,” Prog. Lipid Res. (1996) 35: 169-201; Brindley, Biochemistry of Lipids, Lipoproteins and Membranes (eds. Vance & Vance)(Elsevier, Amsterdam)(1991) pp171-203; Haagsman & Van Golde, “Synthesis and Secretion of Very Low Density Lipoproteins by Isolated Rat Hepatocytes in Suspension: Role of Diacylglycerol Acyltransferase,” Arch. Biochem. Biophys. (1981) 208:395-402; Coleman & Bell, “Triacylglycerol Synthesis in Isolated Fat Cells. Studies on the Microsomal Diacylglycerol Acyltransferase Activity Using Ethanol-Dispersed Diacylglycerols,” J. Biol. Chem. (1976) 251:4537-4543.
SUMMARY OF THE INVENTION
Nucleic acid compositions encoding polypeptide products with diglyceride acyltransferase activity, as well as the polypeptide products encoded thereby and methods for producing the same, are provided. Also provided are: methods and compositions for modulating DGAT activity; DGAT transgenic cells, animals and plants, as well as methods for their preparation; and methods for making triglycerides and triglyceride compositions, as well as the compositions produced by these methods. The subject methods and compositions find use in a variety of different applications, including research, medicine, agriculture and industry.
DETAILED DESCRIPTION OF THE INVENTION
Nucleic acid compositions encoding polypeptide products with diglyceride acyltransferase activity, as well as the polypeptide products encoded thereby and methods for producing the same, are provided. Also provided are: methods and compositions for modulating DGAT activity, e.g. in the treatment of disease conditions associated with DGAT activity; DGAT transgenic cells, animals, plants and fungi, and methods for their preparation, e.g. for use in research, food production, industrial feedstock production, etc.; and methods for making triglycerides and triglyceride compositions, e.g. oils. The methods and compositions of the subject invention find use in a variety of different applications and fields, including research, medicine, agriculture and industry.
Before the subject invention is further described, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
N
UCLEIC
A
CID
C
OMPOSITIONS
Nucleic acid compositions encoding polypeptide products, as well as fragments thereof, having diglyceride acetyltransferase (DGAT) activity are provided. By nucleic acid composition is meant a composition comprising a sequence of DNA having an open reading frame that encodes a DGAT polypeptide, i.e. a gene encoding a polypeptide having DGAT activity, and is capable, under appropriate conditions, of being expressed as a DGAT polypeptide. Also encompassed in this term are nucleic acids that are homologous or substantially similar or identical to the nucleic acids encoding DGAT polypeptides or proteins. Thus, the subject invention provides genes encoding mammalian DGAT, such as genes encoding human DGAT and homologs thereof and mouse DGAT and homologs thereof, as well as plant DGAT, such as Arabidopsis thaliana DGAT and homologs thereof. In other words, both animal and plant genes encoding DGAT proteins are provided by the subject invention. The coding sequence of the human DGATgene, i.e. the human cDNA encoding the human DGAT enzyme, comprises the nucleic acid sequence substantially the same as or identical to that identified as SEQ ID NO:01, infra. The coding sequence of the mouse DGAT gene, i.e. the mouse cDNA encoding the mouse DGAT enzyme, has the nucleic acid sequence identified as SEQ ID NO:02, infra. The coding sequence of the A. thaliana DGAT gene, i.e. the A.thaliana cDNA encoding the A. thaliana DGAT enzyme, comprises the nucleic acid sequence identified as SEQ ID NO:03, infra.
The source of homologous genes to those specifically listed above may be any species, including both animal and plant species, e.g., primate species, particularly human; rodents, such as rats and mice, canines, felines, bovines, ovines, equines, yeast, nematodes, etc. Between mammalian species, e.g., human and mouse, homologs have substantial sequence similarity e.g at least 75% sequence identity, usually at least 90%, more usually at least 95% between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990),
J. Mol. Biol
. 215:403-10 (using default settings). The sequences provided herein are essential for recognizing DGAT-related and homologous polynucleotides in database searches.
Nucleic acids encoding the DGAT proteins and DGAT polypeptides of the subject invention may be cDNAs or genomic DNAs, as well as fragments thereof The term “DGAT-gene” shall be intended to mean the
Cases Sylvaine
Erickson Sandra K.
Farese, Jr. Robert V.
Smith Steven
Bozicevic, Field & Francis
Field Bret E.
McElwain Elizabeth F.
The Regents of the University of California
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