Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-06-18
2004-02-17
Eyler, Yvonne (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S007210, C435S069100, C435S252300, C435S320100, C436S501000, C514S002600, C530S300000, C530S350000
Reexamination Certificate
active
06693184
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to DNA molecules encoding splice variants of the melanocortin-1 receptor (MC-R1) protein belonging to the rhodopsin sub-family of G-protein coupled receptors, recombinant vectors comprising DNA molecules encoding MC-R1 splice variant proteins, recombinant host cells which contain a recombinant vector encoding MC-R1 splice variants, the human MC-R1 proteins encoded by the DNA molecule, and methods of identifying selective agonists and antagonists of MC-R1 splice variant proteins disclosed throughout this specification.
BACKGROUND OF THE INVENTION
Melanocortin receptors belong to the rhodopsin sub-family of G-protein coupled receptors (GPCR's). Five different subtypes are known. These melanocortin receptors bind and are activated by peptides such as &agr;-, &bgr;-, or &ggr;-melanocyte stimulating hormones (&agr;-, &bgr;-, &ggr;-MSH) derived from the pro-opiomelanocortin (POMC) gene. A wide range of physiological functions are believed to be mediated by melanocortin peptides and their receptors.
U.S. Pat. No. 5,532,347, issued on Jul. 2, 1996, to Cone and Mountjoy discloses and claims human and mouse DNA molecules which encode MC-R1 (also known in the art as &agr;-MSH-R). The expressed human protein contains 317 amino acids.
U.S. Pat. No. 5,849,871, issued on Dec. 15, 1998, to Cone and Mountjoy discloses and claims human and mouse MC-R1. As noted in the previous paragraph, the expressed human protein contains 317 amino acid residues.
Mountjoy, et al. (1992
, Science
257: 1248-1251) describe DNA molecules and the concomitant protein for human MC-R1 and human MC-R2.
Chhajlani, et al. (1992
, FEBS Letters
309: 417-420) also disclose a human DNA molecule comprising an open reading frame which encodes human MC-R1.
Cone et al. (1996
, Recent Progress in Hormone Research
51: 287-318) reviews the state of known mammalian melanocortin receptors, from MC-R1 through MC-R5.
Jackson (1997
, Human Molecular Genetics
6: 1613-24) and Koppula, et al. (1997
, Human Mutation
9:30-36) review the occurrence and potential significance of polymorphisms within the coding sequence of the human MC-R1 form A.
It is desirable to correlate in vivo data with in vitro biochemical activity of compounds.
It is also desirable to select compounds which activate one or more human melanocortin receptor proteins in vitro.
It is further desirable to discover new drugs which effect pathophysiological processes by modulating melanocortin receptor activity, followed by human clinical trials.
The present invention addresses and meets these needs by disclosing isolated nucleic acid molecules which express splice variants of human MC-R1, recombinant vectors which house these nucleic acid molecules, recombinant host cells which expresses these alternative forms of human MC-R1 and/or biologically active equivalents, and pharmacological properties of these human MC-R1 proteins.
SUMMARY OF THE INVENTION
The present invention relates to a series of isolated nucleic acid molecules (polynucleotides) which encode novel variants of the human melanocortin-1 receptor protein, referred to herein as MC-R1B proteins. The nucleic acid molecules of the present invention are substantially free from other nucleic acids. These isolated nucleic acid molecules encode a MC-R1 protein which contains an intracellular domain with an additional 65 amino acid residues in comparison to the previously disclosed human MC-R1, referred to herein also as MC-R1A. Therefore, the present invention relates to isolated nucleic acid molecules (polynucleotides) which encode a mRNA which expresses a novel human MC-R1 protein, these DNA molecules including but by no means being limited to DNA molecules comprising the nucleotide sequence disclosed herein as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25.
The present invention also relates to isolated nucleic acid molecules which represent human genomic clones which comprise at least a single intron within the open reading frame which encodes novel variants of human MC-R1B protein. Therefore, the present invention relates to isolated nucleic acid molecules (polynucleotides) which encode a RNA molecule which is spliced to generate a mRNA molecule which encodes a novel human MC-R1 protein variant, these DNA molecules including but by no means being limited to DNA molecules comprising the nucleotide sequence disclosed herein as SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24. To this end, the present invention also relates to the respective mRNA molecule generated from each of the DNA molecules depicted as SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24.
The isolated nucleic acid molecules of the present invention comprise a 3′ extension in the open reading frame which encodes a 65 amino acid COOH-terminal extension to known MC-R1. Therefore, the present invention relates to isolated nucleic acid molecules, both DNA and RNA molecules, that encode for a splice variant of known MC-R1 which encodes for this 65 amino acid COOH-terminal extension. The totality of nucleic acid molecules of the present invention, including genomic DNA, cDNA, RNA and mRNA, will be referred to herein as “MC-R1 splice variants”, which will identify a disclosed nucleic acid molecule which encodes an protein with melanocortin 1 receptor activity in combination with this additional 3′ exon which encodes a 65 amino acid COOH-terminal extension. These isolated nucleic acid molecules include but are by no means limited to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18; SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:25.
The present invention also relates to biologically active fragments or mutants of MC-R1 splice variants which encodes mRNA expressing a novel human MC-R1. Any such biologically active fragment and/or mutant of the MC-R1 splice variants disclosed herein will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of a wild-type MC-R1 protein and comprises at least a portion of the COOH terminal amino acid extension disclosed as SEQ ID NO:27. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for MC-R1B function.
A preferred aspect of this portion of the present invention is set forth as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18; SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:25, human nucleic acid molecules which comprise the complete open reading frame for the MC-R1B proteins of the present invention.
The isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification, including but not limited to the isolated nucleic acid molecules as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18; SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:25.
The present invention also relates
Howard Andrew D.
MacNeil Douglas J.
Van Der Ploeg Leonardus H. T.
Brannock Michael
Eyler Yvonne
Finnegan Alysia A.
Giesser Joanne
Merck & Co. , Inc.
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