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
1998-03-20
2001-09-11
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S252300, C435S254110, C435S320100, C536S023500
Reexamination Certificate
active
06287805
ABSTRACT:
BACKGROUND OF THE INVENTION
The chemokine family of peptides is defined on the basis of sequence homology and on the presence of variations on a conserved cysteine motif. Schall (1996)
Cytokine
3:165-183; and Oppenheim et al. (1991)
Annu. Rev. Immunol.
9:617-648. The family can be subdivided on the basis of this motif into two major subfamilies, in which members of each contain four characteristic cysteine residues. This subdivision therefore defines the CC or &bgr;-chemokine family in which the first two cysteines are juxtaposed, and the CXC or &agr;-chemokine family in which there is an intervening amino acid between the first two cysteines. Two other subfamilies have subsequently been described which have variations in the number of amino acids between the first two cysteine residues. Kelner et al. (1994)
Science
266:1395-1399; Domer et al. (1997)
J. Biol. Chem.
272:8817-8823; and Bazan et al. (1996)
Nature
385:640-644.
Chemokines display a range of in vitro and in vivo functions ranging from proinflammatory activities on a range of cell types to proliferative regulatory activities. All of the functions of the chemokine family are believed to be signaled into a responsive cell using members of the G protein-coupled heptahelical receptor family. To date several &agr;-chemokine and &bgr;-chemokine receptors have been described. See, for e.g., Neote et al. (1993)
Cell
72:415-425; Ponath et al. (1996)
J. Exp. Med.
183:2437-2448; and Power et al. (1995)
J. Biol. Chem.
270:19495-19500.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of novel molecules of the G protein-coupled heptahelical receptor superfamily, referred to herein as “D6” nucleic acid and protein molecules. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding D6 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of D6-encoding nucleic acids.
In one embodiment, a D6 nucleic acid molecule is at least about 60-65%, more preferably at least about 70-75%, even more preferably at least about 78-80%, 80-85%, and most preferably at least about 90-95% or more homologous to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or a complement thereof. In yet another embodiment, a D6 nucleic acid molecule is at least about 60-65%, more preferably at least about 70-75%, even more preferably at least about 80-85%, and most preferably at least about 88-95% or more homologous to the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6, or a complement thereof. In a preferred embodiment, the isolated nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:1, SEQ ID NO:3, or a complement thereof. In another preferred embodiment, the isolated nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6, or a complement thereof.
In another preferred embodiment, the isolated nucleic acid molecule comprises nucleotides 1-858 of SEQ ID NO:1 or nucleotides 1-846 of SEQ ID NO:3. In another embodiment, the isolated nucleic acid molecule comprises nucleotides 955-1172 of SEQ ID NO:1 or nucleotides 943-1160 of SEQ ID NO:3. In yet another embodiment, the isolated nucleic acid molecule comprises nucleotides 1-117 or nucleotides 1395-1664 of SEQ ID NO:4.
In another embodiment, a D6 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:5. In another preferred embodiment, a D6 nucleic acid molecule include a nucleotide sequence encoding a protein having an amino acid sequence 65-70%, preferably at least about, 71-75%, 75-80%, more preferably at least about 85-90%, and most preferably at least about 95% or more homologous to the amino acid sequence of SEQ ID NO:2. In another embodiment, a D6 nucleic acid molecule include a nucleotide sequence encoding a protein having an amino acid sequence 65-70%, preferably at least about, 71-75%, 75-80%, more preferably at least about 85-90%, and most preferably at least about 90-95% or more homologous to the amino acid sequence of SEQ ID NO:5. In yet another embodiment, the nucleic acid molecule encodes the amino acid sequence of human or murine D6.
In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein, preferably a D6 protein, which includes at least two, three, preferably four conserved cysteine residues, and is membrane bound. In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein, preferably a D6 protein, which includes at least one G-protein docking motif, at least two conserved cysteine-residues, and is membrane bound. In a further embodiment, an isolated nucleic acid molecule of the present invention encodes a protein, preferably a D6 protein, which has seven transmembrane domains. In yet another embodiment, a D6 nucleic acid molecule encodes a D6 protein and is a naturally occurring nucleotide sequence.
Another embodiment of the invention features nucleic acid molecules, preferably D6 nucleic acid molecules, which specifically detect D6 nucleic acid molecules relative to nucleic acid molecules encoding non-D6 proteins. For example, in one embodiment, such a nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotides 1-858, 1-69, 955-969, or 955-1172 of the nucleotide sequence shown in SEQ ID NO:1, or to nucleotides 1-846, 1-57, 943-957 or 943-1160 of nucleotide sequence shown in SEQ ID NO:3. In another embodiment, the nucleic acid molecule is at least 215 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, respectively, or a complement thereof. In yet another embodiment, a D6 nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotides 1-117 of the nucleotide sequence shown in SEQ ID NO:4, or to nucleotides 1395-1664 of nucleotide sequence shown in SEQ ID NO:4. In a further embodiment, the nucleic acid molecule is at least 117 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:4 or SEQ ID NO:6, respectively, or a complement thereof.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a D6 nucleic acid molecule.
Another aspect of the invention provides a vector comprising a D6 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. The invention also provides a method for producing a protein, preferably a D6 protein, by culturing in a suitable medium, a host cell of the invention containing a recombinant expression vector such that a D6 protein is produced.
Another aspect of this invention features isolated or recombinant D6 proteins and polypeptides. In one embodiment, an isolated protein, preferably a D6 protein, has at least two, three, preferably four conserved cysteine residues, and is membrane bound. In another embodiment, an isolated protein, preferably a D6 protein, has at least one G-protein docking motif, at least two conserved cysteine-residues, and is membrane bound. In a further embodiment, an isolated protein, preferably a D6 protein, includes seven transmembrane domains. In yet another embodiment, an isolated protein, preferably a D6 protein, has an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:5. In a preferred embodiment, a protein, preferably a D6 protein, has an amino acid sequence at least about 65-70%, preferably at least about 71-75%, 75-80%, even more preferably at least about 85-90%, and most preferably at least about 95% or more homologous to the amino acid sequence of SEQ
Gonzalo Jose-Angel
Graham Gerard J.
Gutierrez-Ramos Jose-Carlos
Nibbs Robert J. Benjamin
Kunz Gary L.
Laccotripe Maria C.
Lahive & Cockfield LLP
Landsman Robert S.
Mandragouras Amy E.
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