Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2002-04-12
2004-12-28
Wax, Robert A. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C435S325000, C536S023100
Reexamination Certificate
active
06835556
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the identification of a human gene family expressed in metabolically relevant tissues. The genes encode a group polypeptides referred to as “Protein Cluster V” which are predicted to be useful in the diagnosis of metabolic diseases, such as obesity and diabetes, as well as in the identification of agents useful in the treatment of the said diseases.
BACKGROUND ART
Metabolic diseases are defined as any of the diseases or disorders that disrupt normal metabolism. They may arise from nutritional deficiencies; in connection with diseases of the endocrine system, the liver, or the kidneys; or as a result of genetic defects. Metabolic diseases are conditions caused by an abnormality in one or more of the chemical reactions essential to producing energy, to regenerating cellular constituents, or to eliminating unneeded products arising from these processes. Depending on which metabolic pathway is involved, a single defective chemical reaction may produce consequences that are narrow, involving a single body function, or broad, affecting many organs and systems.
One of the major hormones that influence metabolism is insulin, which is synthesized in the beta cells of the islets of Langerhans of the pancreas. Insulin primarily regulates the direction of metabolism, shifting many processes toward the storage of substrates and away from their degradation. Insulin acts to increase the transport of glucose and amino acids as well as key minerals such as potassium, magnesium, and phosphate from the blood into cells. It also regulates a variety of enzymatic reactions within the cells, all of which have a common overall direction, namely the synthesis of large molecules from small units. A deficiency in the action of insulin (diabetes mellitus) causes severe impairment in (i) the storage of glucose in the form of glycogen and the oxidation of glucose for energy; (ii) the synthesis and storage of fat from fatty acids and their precursors and the completion of fatty-acid oxidation; and (iii) the synthesis of proteins from amino acids.
There are two varieties of diabetes. Type I is insulin-dependent diabetes mellitus (IDDM), for which insulin injection is required; it was formerly referred to as juvenile onset diabetes. In this type, insulin is not secreted by the pancreas and hence must be taken by injection. Type II, non-insulin-dependent diabetes mellitus (NIDDM) may be controlled by dietary restriction. It derives from insufficient pancreatic insulin secretion and tissue resistance to secreted insulin, which is complicated by subtle changes in the secretion of insulin by the beta cells. Despite their former classifications as juvenile or adult, either type can occur at any age; NIDDM, however, is the most common type, accounting for 90 percent of all diabetes. While the exact causes of diabetes remain obscure, it is evident that NIDDM is linked to heredity and obesity. There is clearly a genetic predisposition to NIDDM diabetes in those who become overweight or obese.
Obesity is usually defined in terms of the body mass index (BMI), i.e. weight (in kilograms) divided by the square of the height (in meters). Weight is regulated with great precision. Regulation of body weight is believed to occur not only in persons of normal weight but also among many obese persons, in whom obesity is attributed to an elevation in the set point around which weight is regulated. The determinants of obesity can be divided into genetic, environmental, and regulatory.
Recent discoveries have helped explain how genes may determine obesity and how they may influence the regulation of body weight. For example, mutations in the of gene have led to massive obesity in mice. Cloning the of gene led to the identification of leptin, a protein coded by this gene; leptin is produced in adipose tissue cells and acts to control body fat. The existence of leptin supports the idea that body weight is regulated, because leptin serves as a signal between adipose tissue and the areas of the brain that control energy metabolism, which influences body weight.
Metabolic diseases like diabetes and obesity are clinically and genetically heterogeneous disorders. Recent advances in molecular genetics have led to the recognition of genes involved in IDDM and in some subtypes of NIDDM, including maturity-onset diabetes of the young (MODY) (Velho & Froguel (1997) Diabetes Metab. 23 Suppl 2:34-37). However, several IDDM susceptibility genes have not yet been identified, and very little is known about genes contributing to common forms of NIDDM. Studies of candidate genes and of genes mapped in animal models of IDDM or NIDDM, as well as whole genome scanning of diabetic families from different populations, should allow the identification of most diabetes susceptibility genes and of the molecular targets for new potential drugs. The identification of genes involved in metabolic disorders will thus contribute to the development of novel predictive and therapeutic approaches.
The &bgr;3-adrenergic receptor (AR) represents one of a number of potential anti-obesity drugs targets for which selective agonists have been developed. In rodents, &bgr;3-AR mRNA is abundant in white adipose tissue (WAT) and brown adipose tissue (BAT). It has been demonstrated that mice lacking endogenous &bgr;3-adrenoreceptors have a slight increase in body fat, but otherwise appear normal (Susulic V. S., et al. (1995) J. Biol. Chem. 270(49). 29483-29492). These-mice are completely resistant to the specific &bgr;3-agonist CL-316,243, which has been shown to increase lipolysis, energy expenditure and affect insulin and leptin levels. When the &bgr;3-AR was ectopically expressed in white and brown adipose tissue or brown adipose tissue only, it was recently demonstrated that the anorectic and insulin secretagogue effects appeared to be mediated by white adipose tissue (Grujic D, et al. (1997) J Biol Chem. 272(28): 17686-93). How these effects are mediated by &bgr;3-AR agonists remains poorly understood.
Lardizabal, K. D. et al. (J. Biol. Chem. 276: 38862-38869) and Cases, S. et al. (J. Biol. Chem. 276: 38870-38876; both papers published Jul. 31, 2001) disclose a new gene family, including members in fungi, plants and animals, which encode proteins corresponding to the “Cluster V” proteins according to the present invention. The proteins were shown to have acyl CoA:diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) function. The gene family is unrelated to the previously identified DGAT(1) family and was designated DGAT2. DGAT2 was shown to have high expression levels in liver and white adipose tissue, suggesting that it may play a significant role in mammalian triglyceride metabolism.
DISCLOSURE OF THE INVENTION
According to the present invention, a family of genes and encoded homologous proteins (hereinafter referred to as “Protein Cluster V”) has been identified. Consequently, the present invention provides an isolated nucleic acid molecule selected from:
(a) nucleic acid molecules comprising a nucleotide sequence as shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, or 19.
(b) nucleic acid molecules comprising a nucleotide sequence capable of hybridizing, under stringent hybridization conditions, to a nucleotide sequence complementary to the polypeptide coding region of a nucleic acid molecule as defined in (a); and
(c) nucleic acid molecules comprising a nucleic acid sequence which is degenerate as a result of the genetic code to a nucleotide sequence as defined in (a) or (b).
The nucleic acid molecules according to the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. RNA transcribed from DNA is also encompassed by the present invention.
The term “stringent hybridization conditions” is known in the art from standard protocols (e.g. Ausubel et al., supra) and could be understood as e.g. hybridization to filter-bound DNA in 0.5 M NaHPO
4
, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at +65° C., and washing in 0.1×SSC/0.1% SDS at +68° C.
In a
Pharmacia AB
Wax Robert A.
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