Claims
- 1. An isolated nucleic acid coding for a MMSC2 polypeptide, said polypeptide having the amino acid sequence set forth in SEQ ID NO:3 or a modified form which is functionally equivalent in its interaction with MMAC1.
- 2. The isolated nucleic acid of claim 1, wherein said DNA has the nucleotide sequence set forth in SEQ ID NO:2, its complement or a corresponding RNA.
- 3. The isolated nucleic acid of claim 1, which is a DNA comprising an allelic variant of the nucleotide sequence set forth in SEQ ID NO:2, its complement or a corresponding RNA.
- 4. The isolated nucleic acid of claim 1 coding for a mutated form of the MMSC2 polypeptide set forth in SEQ ID NO:3.
- 5. The isolated nucleic acid of claim 4, which is a DNA comprising a mutated form of the nucleotide sequence set forth in SEQ ID NO:2, its complement or a corresponding RNA.
- 6. The isolated nucleic acid of claim 5, wherein the mutation is selected from the group consisting of a deletion mutation, a nonsense mutation, an insertion mutation, a frameshift mutation and a missense mutation.
- 7. An isolated nucleic acid having at least 15 contiguous nucleotides of a nucleic acid as claimed in claim 1 wherein the nucleic acid sequence suitable for use as a hybridization probe to detect in a sample (i) a DNA having a nucleotide sequence selected from the nucleotide sequence set forth in SEQ ID NO:2, allelic variants thereof and mutated forms thereof or (ii) an RNA corresponding to said DNA.
- 8. A set of nucleic acid probes for use in a microchip assay, wherein each of said nucleic acid probes comprises at least 8 contiguous nucleotides of a nucleic acid as claimed in claim 1 and said set encompasses part or all of said nucleic acid.
- 9. A replicative cloning vector which comprises an isolated nucleic acid as claimed in claim 1 and a replicon operative in a host cell.
- 10. An expression vector which comprises an isolated nucleic acid of claim 1 wherein the coding sequence for the MMSC2 polypeptide or modified form thereof is operably linked to suitable control sequences capable of directing expression of said coding sequence in host cells for said vector.
- 11. Host cells transformed with a vector as claimed in claim 10.
- 12. A method of producing a polypeptide which is the MMSC2 polypeptide having the amino acid sequence set forth in SEQ ID NO:3 or a modified form of said polypeptide as defined in claim 1 which comprises (i) culturing the host cells of claim 11 under conditions suitable for the production of said MMSC2 polypeptide and (ii) recovering said polypeptide.
- 13. A method as claimed in claim 12 which further comprises labeling the recovered polypeptide.
- 14. A preparation of human MMSC2 polypeptide substantially free of other human proteins, said polypeptide having the amino acid sequence set forth in SEQ ID NO:3.
- 15. A preparation of human MMSC2 polypeptide substantially free of other human proteins, the amino acid sequence of said polypeptide having substantial sequence homology with the wild-type MMSC2 polypeptide having the amino acid sequence set forth in SEQ ID NO:3, and said polypeptide having substantially similar function as the wild-type MMSC2 polypeptide.
- 16. A preparation of a polypeptide substantially free of other proteins, said polypeptide being a mutated human MMSC2 polypeptide obtainable by expression of a mutated form of the nucleic acid sequence set forth in SEQ ID NO:2.
- 17. A preparation as claimed in claim 14 wherein said polypeptide is labeled.
- 18. An antibody capable of specifically binding a polypeptide as claimed in claim 14.
- 19. An antigenic fragment of a polypeptide as defined in claim 14 suitable for use as an immunogen to obtain an antibody.
- 20. A polypeptide as defined in claim 14 in the form of a fusion protein.
- 21. Use of a polypeptide as defined in claim 14 as an immunogen for antibody production.
- 22. A use as claimed in claim 21, wherein one or more antibodies products ara subsequently labeled or bound to a solid support.
- 23. A pair of single-stranded oligonucleotide primers for determination of a nucleotide sequence of a MMSC2 gene by a nucleic acid amplification reaction, the sequence of said primers being derived from genomic clones for MMSC2, and the use of said primers in a nucleic acid amplification reaction resulting in the synthesis of DNA or RNA corresponding to all or part of the sequence of the MMSC2 gene.
- 24. A pair of primers as claimed in claim 23 for determination of all or part of the sequence of the MMSC2 gene having the nucleotide sequence set forth in SEQ ID NO:2, allelic variant or a mutated form thereof.
- 25. A method for identifying a mutant MMSC2 nucleotide sequence in a suspected mutant MMSC2 allele which comprises comparing the nucleotide sequence of the suspected mutant MMSC2 allele with a wild-type MMSC2 nucleotide sequence, wherein a difference between the suspected mutant and the wild-type sequence identifies a mutant MMSC2 nucleotide sequence.
- 26. A method for detecting an alteration in MMSC2 wherein said alteration is associated with cancer in a human, wherein if said alteration is in germline it is associated with predisposition to said cancer and if said alteration is in somatic tissue it indicates that said somatic tissue is cancerous, wherein said method comprises analyzing a MMSC2 gene or a MMSC2 gene expression product from a tissue of said human.
- 27. A method as claimed in claim 26 wherein the sequence of the MMSC2 gene in said sample is compared with the sequence of one or more wild-type MMSC2 gene sequences selected from the sequence set forth in SEQ ID NO:2 and wild type allelic variants thereof.
- 28. The method of claim 26 wherein said expression product is selected from the group consisting of mRNA of the MMSC2 gene and a MMSC2 polypeptide encoded by the MMSC2 gene.
- 29. The method of claim 26 wherein one or more of the following procedures is carried out:
(a) observing shifts in electrophoretic mobility of single-stranded DNA from said sample on non-denaturing polyacrylamide gels; (b) hybridizing a MMSC2 gene probe to genomic DNA isolated from said sample under conditions suitable for hybridization of said probe to said gene; (c) determining hybridization of an allele-specific probe to genomic DNA from said sample; (d) amplifying all or part of the MMSC2 gene from said sample to produce an amplified sequence and sequencing the amplified sequence; (e) determining by nucleic acid amplification the presence of a specific MMSC2 mutant allele in said sample; (f) molecularly cloning all or part of the MMSC2 gene from said sample to produce a cloned sequence and sequencing the cloned sequence; (g) determining whether there is a mismatch between molecules (1) MMSC2 gene genomic DNA or MMSC2 mRNA isolated from said sample, and (2) a nucleic acid probe complementary to the human wild-type MMSC2 gene DNA, when molecules (1) and (2) are hybridized to each other to form a duplex; (h) amplification of MMSC2 gene sequences in said sample and hybridization of the amplified sequences to nucleic acid probes which comprise wild-type MMSC2 gene sequences; (i) amplification of MMSC2 gene sequences in said tissue and hybridization of the amplified sequences to nucleic acid probes which comprise mutant MMSC2 gene sequences; (j) screening for a deletion mutation; (k) screening for a point mutation; (l) screening for an insertion mutation; (m) determining in situ hybridization of the MMSC2 gene in said sample with one or more nucleic acid probes which comprise the MMSC2 gene sequence or a mutant MMSC2 gene sequence; (n) immunoblotting; (o) immunocytochemistry; (p) assaying for binding interactions between MMSC2 protein isolated from said tissue and a binding partner capable of specifically binding the polypeptide expression product of a MMSC2 mutant allele and/or a binding partner for the MMSC2 polypeptide having the amino acid sequence set forth in SEQ ID NO:3; and (q) assaying for the inhibition of biochemical activity of said binding partner.
- 30. The method of claim 29 wherein said alteration of MMSC2 protein is detected by assaying for binding interactions between said MMSC2 protein isolated from said tissue and MMAC1 protein.
- 31. A method for detecting an alteration in MMAC1 wherein said alteration is associated with cancer in a human, wherein if said alteration is in germline it is associated with predisposition to said cancer and if said alteration is in somatic tissue it indicates that said somatic tissue is cancerous, wherein said method comprises analyzing an MMAC1 polypeptide from a tissue of said human by assaying for binding interactions between said MMAC1 polypeptide and MMSC2 or PDZ domain number 7 of said MMSC2.
- 32. A transgenic animal which carries an altered MMSC2 allele in its genome.
- 33. An isolated mutant MMSC2 which cannot form a complex with a wild-type protein with which wild-type MMSC2 does form a complex.
- 34. The isolated mutant MMSC2 of claim 33 wherein said wild-type protein is MMAC1.
- 35. An isolated protein complex comprising MMSC2 and MMAC1.
- 36. The isolated protein complex of claim 35 wherein said MMSC2 contains an alteration.
- 37. The isolated protein complex of claim 35 wherein said MMAC1 contains an alteration.
- 38. A protein complex comprising a fragment of MMSC2 and a fragment of MMAC1.
- 39. The protein complex of claim 38 wherein said fragment of MMSC2 comprises PDZ domain number 7.
- 40. The protein complex of claim 38 wherein said MMSC2 comprises an alteration.
- 41. The protein complex of claim 38 wherein said MMAC1 comprises an alteration.
- 42. An isolated antibody immunoreactive with the protein complex of claim 35.
- 43. The antibody of claim 42 wherein said antibody is not immunoreactive with either pure MMSC2 or pure MMAC1.
- 44. The antibody of claim 42 wherein said antibody is a monoclonal antibody.
- 45. A method for supplying a wild-type MMSC2 gene function or a MMSC2 function substantially similar to said wild-type function to a cell which has lost said gene function or has altered gene function by virtue of a mutation in said MMSC2 gene, wherein said method comprises introducing into the cell a nucleic acid which suppresses a transformed state of said cell, said nucleic acid selected from the group consisting of a wild-type MMSC2 gene nucleic acid or a nucleic acid substantially homologous to said wild-type MMSC2 gene nucleic acid, such that said nucleic acid is expressed in said cell.
- 46. The method of claim 45 wherein said nucleic acid is a portion of wild-type MMSC2 gene, said portion encoding a part of said wild-type gene polypeptide which is required for non-neoplastic growth of said cell.
- 47. A method for supplying a wild-type MMSC2 gene function or a MMSC2 function substantially similar to wild-type to a cell which has lost said gene function or has altered gene function by virtue of a mutation in said MMSC2 gene, wherein said method comprises introducing into said cell a molecule which suppresses a transformed state of said cell, said molecule selected from the group consisting of all or a part of a wild-type MMSC2 polypeptide which is required for non-neoplastic growth of said cell, a polypeptide substanially homologous to said wild-type MMSC2 polypeptide and a molecule which mimics the function of said wild-type MMSC2 polypeptide.
- 48. A method for diagnosing a predisposition for cancer in a human wherein said method comprises assaying for the ability of MMSC2 or a fragment of MMSC2 from said human to form a complex with a protein to which wild-type MMSC2 binds wherein an inability to form said complex is indicative of a predisposition to cancer.
- 49. The method of claim 48 wherein said protein is MMAC1.
- 50. The method of claim 48 wherein said assay comprises a yeast two-hybrid assay.
- 51. The method of claim 48 wherein said assay comprises measuring in vitro a complex formed by mixing said protein and MMSC2 purified from said human.
- 52. The method of claim 48 wherein said assay comprises measuring in vitro a complex formed by mixing MMSC2 and said protein purified from said human.
- 53. The method of claim 48 wherein said complex is measured by binding with an antibody specific for a MMSC2-said protein complex.
- 54. The method of claim 48 wherein said assay comprises mixing an antibody specific for a MMSC2-said protein complex with a tissue extract from said person, wherein the lack of formation of a MMSC2-said protein-antibody complex between said antibody and said tissue extract is indicative of a predisposition to cancer.
- 55. A method for determining whether a mutation in a protein to which MMSC2 binds is predispositive for cancer wherein said method comprises binding said protein with said mutation to a wild-type MMSC2 and determining whether a complex forms, wherein the lack of a complex indicates said mutation is predispositive.
- 56. A method for determining whether a mutation in MMSC2 is predispositive for cancer wherein said method comprises binding a MMSC2 with said mutation to a protein to which wild-type MMSC2 binds and determining whether a complex forms, wherein the lack of a complex indicates said mutation is predispositive.
- 57. A method for treating a human with cancer resulting from a mutation in a protein to which MMSC2 binds and wherein said mutation prevents binding of said protein to MMSC2 wherein said method comprises treating said person with a complex of wild-type MMSC2 and wild-type of said protein.
- 58. The method of claim 57 wherein said protein is MMAC1.
- 59. A method of screening for drug candidates useful in treating a cancer resulting from a mutation in MMSC2, wherein said method involves mixing a mutant MMSC2 with a wild-type protein, to which wild-type MMSC2 binds, in both the presence of a drug and the absence of said drug and measuring the amount of binding of said mutant MMSC2 with said wild-type protein, wherein if the amount of said binding is greater in the presence of said drug than in the absence of said drug then said drug is a drug candidate for treating said cancer.
- 60. The method of claim 59 wherein said mutant MMSC2 is a fusion protein and/or said wild-type protein is a fusion protein.
- 61. The method of claim 59 wherein said wild-type protein is MMAC1.
- 62. A method for screening for drug candidates useful in treating a cancer resulting from a mutation in a protein, which protein when wild-type binds with wild-type MMSC2, wherein said method involves mixing said protein containing said mutation with wild-type MMSC2 in both the presence of a drug and the absence of said drug and measuring the amount of binding of said protein containing said mutation with said wild-type MMSC2, wherein if the amount of said binding is greater in the presence of said drug than in the absence of said drug then said drug is a drug candidate for treating said cancer.
- 63. The method of claim 62 wherein said wild-type MMSC2 is a fusion protein and/or said mutant protein is a fusion protein.
- 64. The method of claim 62 wherein said protein is MMAC1.
- 65. A method of screening for drug candidates useful in treating a cancer resulting from a mutation in MMSC2, wherein said method involves mixing a mutant MMSC2 with a wild-type protein, to which wild-type MMSC2 binds, in both the presence of a drug and the absence of said drug and measuring the amount of binding of said mutant MMSC2 with said wild-type protein, wherein if the amount of said binding is less in the presence of said drug than in the absence of said drug then said drug is a drug candidate for treating said cancer.
- 66. The method of claim 65 wherein said wild-type MMSC2 is a fusion protein and/or said wild-type protein is a fusion protein.
- 67. The method of claim 65 wherein said wild-type protein is MMAC1.
- 68. A method of screening for drug candidates useful in treating a cancer resulting from a mutation in MMSC2, wherein said method involves mixing a wild-type MMSC2 with a wild-type protein, to which wild-type MMSC2 binds, in both the presence of a drug and the absence of said drug and measuring the amount of binding of said mutant MMSC2 with said wild-type protein, wherein if the amount of said binding is less in the presence of said drug than in the absence of said drug then said drug is a drug candidate for treating said cancer.
- 69. The method of claim 68 wherein said wild-type MMSC2 is a fusion protein and/or said wild-type protein is a fusion protein.
- 70. The method of claim 68 wherein said wild-type protein is MMAC1.
- 71. A method of screening for drug candidates useful in treating a cancer resulting from a mutation in MMAC1, wherein said method involves mixing a wild-type MMSC2 with a wild-type protein, to which wild-type MMSC2 binds, in both the presence of a drug and the absence of said drug and measuring the amount of binding of said mutant MMSC2 with said wild-type protein, wherein if the amount of said binding is less in the presence of said drug than in the absence of said drug then said drug is a drug candidate for treating said cancer.
- 72. The method of claim 71 wherein said wild-type MMSC2 is a fusion protein and/or said wild-type protein is a fusion protein.
- 73. The method of claim 71 wherein said wild-type protein is MMAC1.
- 74. The method of claim 59, wherein said wild-type protein which binds to MMSC2 binds to one or more of the PDZ domains of MMSC2.
- 75. A method of screening for drug candidates useful in treating a cancer resulting from a mutation in MMSC2, wherein said method comprises treating an animal which is homozygous for MMSC2 containing said mutation with a drug wherein if said animal does not develop cancer said drug is a drug candidate for treating said cancer.
- 76. A method of screening for drug candidates useful in treating a cancer resulting from a mutation in MMSC2, wherein said method comprises treating an animal which has a tumor and which is homozygous for MMSC2 containing said mutation with a drug wherein if said tumor regresses said drug is a drug candidate for treating said cancer.
- 77. The method of claim 75 wherein said animal is transgenic for MMSC2 with said mutation.
- 78. A method of screening for drug candidates useful in treating a cancer resulting from a mutation in MMSC2, wherein said method comprises the steps of:
(a) growing a cell culture of cells which are homozygous for MMSC2 containing said mutation in the presence of a drug, (b) growing a cell culture of cells which contain a wild-type MMSC2 gene, and (c) growing a cell culture of cells which are homozygous for MMSC2 containing said mutation in the absence of said drug, wherein if the cells in step (a) behave more like the cells in step (b) than like the cells in step (c) then said drug is a drug candidate for treating said cancer.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is related to U.S. provisional patent application Ser. No. 60/084,740, filed May 8, 1998, incorporated herein by reference.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60084740 |
May 1998 |
US |
Divisions (1)
|
Number |
Date |
Country |
Parent |
09306998 |
May 1999 |
US |
Child |
09922101 |
Aug 2001 |
US |