Protein forming complex with c-fos protein, nucleic acid encoding the same and method of using the same

Abstract

Proteins that interact with c-Fos, nucleic acids encoding them and inhibitors utilizing them as well as methods for detecting an interaction and screening methods utilizing a protein that interacts with c-Fos are provided. Comprehensive analysis of transcription control factor complexes in a mouse brain cDNA library with c-Fos as a bait by using the cotranslation selection and screening of in vitro virus (IVV) and the C-terminal labeling method are conducted, thereby to analyze proteins unknown so far, proteins known so far, but unknown to form a complex with the c-Fos protein, and so forth.

Description

TECHNICAL FIELD

[0001] The present invention relates to proteins that interact with c-Fos, nucleic acids encoding them and inhibitors utilizing them as well as methods for detecting an interaction and screening methods utilizing a protein that interacts with c-Fos.

[0002] At present, genomic nucleotide sequences of various organisms are going to be elucidated. In researches of genomic sequences, there are expected, as post-sequencing researches of the second act, researches of analyzing meanings of the elucidated genomic information, i.e., structural and functional analyses of genes and proteins (Non-patent documents 1 and 2), analyses of protein/protein and protein/nucleic acid interactions (Non-patent documents 3 and 4) and so forth.

[0003] On the basis of analyses of networks of interactions between protein and protein, protein and nucleic acid and so forth in post-sequencing genomic functional analyses utilizing such techniques as described above, it is expected to create drugs and so forth standing on discoveries of novel functions of known proteins or important biological enzymes such as novel proteins that have been unknown so far.

[0004] As methods for detecting interactions between proteins, the immunoprecipitation (Non-patent document 5), pull-down assay based on a GST fusion protein (Non-patent document 6), TAP method (Non-patent document 7), yeast two-hybrid method (Non-patent document 8) and so forth are known so far. Further, as methods for comprehensive analysis of interactions between proteins in the post-sequencing genomic functional analyses utilizing the "assignment of gene (genotype) and protein (phenotype)" born as a tool of the evolutionary molecular engineering, there are the in vitro virus method (Non-patent documents 9 and 10, Patent documents 1 and 2), STABLE method (Non-patent document 11), phage display method (Non-patent document 12), ribosome display method (Non-patent document 13, Patent document 3), mRNA-peptide fusion method (mRNA display method, Non-patent document 14), and so forth.

[0005] Furthermore, the surface plasmon resonance method, fluorescence resonance energy transfer method, fluorescence depolarization method, evanescent-field imaging method, fluorescence correlation spectroscopy, fluorescent imaging method, enzyme linked immunosorbent assay and so forth are also known. Moreover, methods of modifying C-terminals of proteins in a translation system utilizing a nucleic acid derivatives such as puromycin have been previously proposed (Patent documents 4 and 5). These methods have advantages that functions of proteins are more unlikely to be damaged compared with the conventional chemical modification methods and the fluorescent protein fusion methods.

[0006] In the field of life science, sequence analysis of the human genome was completed, and genome researches rush into functional analyses of genes of the post-genome age. Thus, innovative drug creation based on comprehensive genomic functional analyses and so forth are expected. There is desired a technique that enables comprehensive analysis of genes and proteins, which have been independently studied so far, for example, a technique of analyzing various cofactors of transcription control factors as target proteins of drug creation at once and so forth. As a transcription control factor, the c-Fos protein is known well.

[0007] The v-fos gene was isolated as an oncogene of an FBJ murine osteosarcoma virus (Non-patent document 15). c-fos is a transcription control factor detected in many cell species as a typical immediate early gene in connection with a proliferation stimulus. fra-1 and fra-2 were cloned from the Fos-related antigens (Fra), and fosB was also found as a gene having homology to c-fos in the nucleotide sequence. These constitute the fos family genes together with c-fos. It is known that a chimeric mouse and transgenic mouse expressing c-fos at a high level form chondroma and bone sarcoma, respectively (Non-patent document 16).

[0008] Various genes such as c-jun, junB and junD, which are jun family genes, are known so far as genes of proteins that interact with c-fos (Non-patent document 17), and it was recently found by the two-hybrid method that the transcription control factor Fos/Jun (AP-1) forms a complex with BAF60a of SWI/SNF to induce remodeling of chromatin. Further, it was found that AP-1 binds to NFAT, which is a protein of the cerebral nerve system, to control expression of the IL2 gene. The former is involved in oncogenesis and canceration, and the latter consists of two proteins that are involved in autoimmune diseases and Alzheimer's disease and induce completely different diseases. Thus, comprehensive analyses of various complexes of transcription control factors are very interesting as a new treasury of target proteins for drug creation. However, such a thorough 1:1 molecule analysis technique as the two-hybrid method takes enormous time and labor.

<Non-Patent Document 1>

[0009] Saegusa A., Nature, 401, 6751 (1999) <Non-Patent Document 2> [0010] Dalton R, Abbott A., Nature, 402, 6763 (1999) <Non-Patent Document 3> [0011] Etsuko Miyamoto, Hiroshi Yanagawa, Series Genome Science of Post-sequencing 3, Proteomics, pp. 136-145 (2000) <Non-Patent Document 4> [0012] Etsuko Miyamoto, Hiroshi Yanagawa, PROTEIN, NUCLEIC ACID AND ENZYME, 46(2), pp. 138-147 (2001) <Non-Patent Document 5> [0013] Xiong et al., Nature, 366, 701-704 (1993) <Non-Patent Document 6> [0014] Kaelin, et al., Cell, 64, 521-532 (1991) <Non-Patent Document 7> [0015] Guillaume Rigaut, et al., Nature Biotechnology, 17, 1030 (1999) <Non-Patent Document 8> [0016] Fields S., Song O., Nature, 340, 245 (1989) <Non-Patent Document 9> [0017] Miyamoto-Sato E., et al., Viva Origino, 25, 35 (1997) <Non-Patent Document 10> [0018] Nemoto N., et al., FEBS Lett., 414, 405 (1997) <Patent Document 1> [0019] International Publication WO98/16636 <Patent Document 2> [0020] International Publication WO02/46395 <Non-Patent Document 11> [0021] Doi N., Yanagawa H., FEBS Lett., 457, 227 (1999) <Non-Patent Document 12> [0022] Smith G. P., Science, 228, 1315 (1985) <Non-Patent Document 13> [0023] Mattheakis, L. C. et al., Proc. Natl. Acad. Sci. USA, 91, 9022-9026 (1994) <Patent Document 3> [0024] International Publication WO95/11922 <Non-Patent Document 14> [0025] Roberts R. W. and Szostak J. W., Proc. Natl. Acad. Sci. USA, 94, 12297 (1997) <Patent Document 4> [0026] U.S. Pat. No. 6,228,994 <Patent Document 5> [0027] International Publication WO02/48347 <Non-Patent Document 15> [0028] Curran, T. et al., J. Viol., 44:674-682 (1982) <Non-Patent Document 16> [0029] Agamemunon, E. G. et al., Trends Genet., 11:436-441 (1995) <Non-Patent Document 17> [0030] Yurii Chinenovl and Tom K Kerppola, Oncogene, 20, 2438-2452 (2001)

DISCLOSURE OF THE INVENTION

[0031] An object of the present invention is to provide a complex that interacts with the c-Fos protein, which is well known as a transcription control factor, as a target protein.

[0032] The inventors of the present invention conducted comprehensive analysis of transcription control factor complexes in a mouse brain cDNA library with c-Fos as a bait by using two of techniques, the cotranslation selection and screening of in vitro virus (IVV) and the C-terminal labeling method (U.S. Pat. No. 6,228,994, WO02/48347) named puromycin technologies, which have been researched on the basis of the aforementioned in vitro virus method as a method for comprehensive analysis as a one-to-multiple molecule-analysis method replacing the method, and thereby attempted to analyze proteins unknown so far, proteins known so far, but unknown to form a complex with the c-Fos protein, and so forth. The expression of "a protein that form a complex" used herein refers to a protein that directly or indirectly interacts with the c-Fos protein.

[0033] Further objects of the present invention is to provide a protein that interacts with c-Fos and an inhibitor utilizing it, as well as a method for detecting an interaction and method for screening utilizing a protein that interacts with c-Fos.

[0034] The inventors of the present invention found novel proteins that interact with c-Fos by the cotranslation screening, also found that certain known proteins interacted with c-Fos, and accomplished the present invention. The present invention thus provides the followings.

[0035] 1. A protein of the following (a) or (b):

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 1 to 14,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 1 to 14 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein.

[0036] 2. A protein according to 1, which comprises any one of the amino acid sequences of SEQ ID NOS: 1 to 14.

[0037] 3. A nucleic acid encoding the protein according to 1 or 2.

[0038] 4. A nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 23 to 38,

(b) a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 23 to 38 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0039] 5. A nucleic acid according to 4, which comprises any one of the nucleotide sequences of SEQ ID NOS: 23 to 38.

[0040] 6. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises the protein according to 1 or 2 or a protein translated from the nucleic acid according to any one of 3 to 5 as an active ingredient.

[0041] 7. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is the protein according to 1 or 2 or a protein translated from the nucleic acid according to any one of 3 to 5.

[0042] 8. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to 7 and the step of selecting a prey for which an interaction was detected.

[0043] 9. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 15 to 19,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 15 to 19 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein.

[0044] 10. The inhibitor according to 9, wherein the protein as the active ingredient comprises any one of the amino acid sequences of SEQ ID NOS: 15 to 19.

[0045] 11. The inhibitor according to 9, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 39 to 43,

(b) a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 39 to 43 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0046] 12. The inhibitor according to 11, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 39 to 43.

[0047] 13. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 15 to 19,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 15 to 19 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 39 to 43,

(b') a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 39 to 43 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0048] 14. The method according to 13, wherein the protein comprises any one of the amino acid sequences of SEQ ID NOS: 15 to 19.

[0049] 15. The method according to 13, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 39 to 43.

[0050] 16. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 13 to 15 and the step of selecting a prey for which an interaction is detected.

[0051] 17. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 20 to 22,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 20 to 22 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0052] 18. The inhibitor according to 17, wherein the protein as the active ingredient comprises any one of the amino acid sequences of SEQ ID NOS: 20 to 22.

[0053] 19. The inhibitor according to 17, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 44 to 46,

(b) a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 44 to 46 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0054] 20. The inhibitor according to 19, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 44 to 46.

[0055] 21. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 20 to 22,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 20 to 22 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 44 to 46,

(b') a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 44 to 46 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0056] 22. The method according to 21, wherein the protein comprises any one of the amino acid sequences of SEQ ID NOS: 20 to 22.

[0057] 23. The method according to 21, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 44 to 46.

[0058] 24. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 21 to 23 and the step of selecting a prey for which an interaction is detected.

[0059] 25. A protein of the following (a) or (b):

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 47 to 56,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 47 to 56 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein.

[0060] 26. A protein according to 25, which comprises any one of the amino acid sequences of SEQ ID NOS: 47 to 56.

[0061] 27. A nucleic acid encoding the protein according to 25 or 26.

[0062] 28. A nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 104 to 118,

(b) a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 104 to 118 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0063] 29. A nucleic acid according to 28, which comprises any one of the nucleotide sequences of SEQ ID NOS: 104 to 118.

[0064] 30. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises the protein according to 25 or 26 or a protein translated from the nucleic acid according to any one of 27 to 29 as an active ingredient.

[0065] 31. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is the protein according to 25 or 26 or a protein translated from the nucleic acid according to any one of 27 to 29 as an active ingredient.

[0066] 32. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to 31 and the step of selecting a prey for which an interaction was detected.

[0067] 33. A protein of the following (a) or (b):

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 57 to 76,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 57 to 76 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein.

[0068] 34. A protein according to 33, which comprises any one of the amino acid sequences of SEQ ID NOS: 57 to 76.

[0069] 35. A nucleic acid encoding the protein according to 33 or 34.

[0070] 36. A nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 119 to 140,

(b) a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 119 to 140 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0071] 37. A nucleic acid according to 4, which comprises any one of the nucleotide sequences of SEQ ID NOS: 119 to 140.

[0072] 38. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises the protein according to 33 or 34 or a protein translated from the nucleic acid according to any one of 35 to 37 as an active ingredient.

[0073] 39. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is the protein according to 33 or 34 or a protein translated from the nucleic acid according to any one of 35 to 37 as an active ingredient.

[0074] 40. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to 39 and the step of selecting a prey for which an interaction was detected.

[0075] 41. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 77 to 81,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 77 to 81 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0076] 42. The inhibitor according to 41, wherein the protein as the active ingredient comprises any one of the amino acid sequences of SEQ ID NOS: 77 to 81.

[0077] 43. The inhibitor according to 41, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 141 to 145,

(b) a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 141 to 145 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0078] 44. The inhibitor according to 43, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 141 to 145.

[0079] 45. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 77 to 81,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 77 to 81 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 141 to 145,

(b') a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 141 to 145 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0080] 46. The method according to 45, wherein the protein comprises any one of the amino acid sequences of SEQ ID NOS: 77 to 81.

[0081] 47. The method according to 45, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 141 to 145.

[0082] 48. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 45 to 47 and the step of selecting a prey for which an interaction is detected.

[0083] 49. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 82 to 84,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 82 to 84 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0084] 50. The inhibitor according to 49, wherein the protein as the active ingredient comprises any one of the amino acid sequences of SEQ ID NOS: 82 to 84.

[0085] 51. The inhibitor according to 49, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 146 to 148,

(b) a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 146 to 148 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0086] 52. The inhibitor according to 51, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 146 to 148.

[0087] 53. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 82 to 84,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 82 to 84 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 146 to 148,

(b') a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 146 to 148 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0088] 54. The method according to 53, wherein the protein comprises any one of the amino acid sequences of SEQ ID NOS: 82 to 84.

[0089] 55. The method according to 53, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 146 to 148.

[0090] 56. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 53 to 55 and the step of selecting a prey for which an interaction is detected.

[0091] 57. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 85 or 86,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 85 or 86 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0092] 58. The inhibitor according to 57, wherein the protein as the active ingredient comprises the amino acid sequence of SEQ ID NO: 85 or 86.

[0093] 59. The inhibitor according to 57, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 149 or 150,

(b) a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 149 or 150 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0094] 60. The inhibitor according to 59, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 149 or 150.

[0095] 61. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising the amino acid sequence of SEQ ID NO: 85 or 86,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 85 or 86 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 149 or 150,

(b') a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 149 or 150 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0096] 62. The method according to 61, wherein the protein comprises the amino acid sequence of SEQ ID NO: 85 or 86.

[0097] 63. The method according to 61, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 149 or 150.

[0098] 64. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 61 to 63 and the step of selecting a prey for which an interaction is detected.

[0099] 65. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 87 to 89,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 87 to 89 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0100] 66. The inhibitor according to 65, wherein the protein as the active ingredient comprises any one of the amino acid sequences of SEQ ID NOS: 87 to 89.

[0101] 67. The inhibitor according to 65, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 151 to 153,

(b) a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 151 to 153 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0102] 68. The inhibitor according to 67, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 151 to 153.

[0103] 69. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 87 to 89,

(b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS: 87 to 89 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 151 to 153,

(b') a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 151 to 153 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0104] 70. The method according to 69, wherein the protein comprises any one of the amino acid sequences of SEQ ID NOS: 87 to 89.

[0105] 71. The method according to 70, wherein the nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOS: 151 to 153.

[0106] 72. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 69 to 71 and the step of selecting a prey for which an interaction is detected.

[0107] 73. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 90 or 91,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 90 or 91 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0108] 74. The inhibitor according to 73, wherein the protein as the active ingredient comprises the amino acid sequence of SEQ ID NO: 90 or 91.

[0109] 75. The inhibitor according to 74, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 154 or 155,

(b) a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 154 or 155 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0110] 76. The inhibitor according to 75, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 154 or 155.

[0111] 77. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising the amino acid sequence of SEQ ID NO: 90 or 91,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 90 or 91 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 154 or 155,

(b') a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 154 or 155 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0112] 78. The method according to 69, wherein the protein comprises the amino acid sequence of SEQ ID NO: 90 or 91.

[0113] 79. The method according to 70, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 154 or 155.

[0114] 80. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 77 to 79 and the step of selecting a prey for which an interaction is detected.

[0115] 81. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 92 or 93,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 92 or 93 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0116] 82. The inhibitor according to 81, wherein the protein as the active ingredient comprises the amino acid sequence of SEQ ID NO: 92 or 93.

[0117] 83. The inhibitor according to 82, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 156 or 157,

(b) a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 156 or 157 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0118] 84. The inhibitor according to 83, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 156 or 157.

[0119] 85. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising the amino acid sequence of SEQ ID NO: 92 or 93,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 92 or 93 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 156 or 157,

(b') a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 156 or 157 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0120] 86. The method according to 85, wherein the protein comprises the amino acid sequence of SEQ ID NO: 92 or 93.

[0121] 87. The method according to 85, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 156 or 157.

[0122] 88. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 85 to 87 and the step of selecting a prey for which an interaction is detected.

[0123] 89. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 94 or 95,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 94 or 95 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0124] 90. The inhibitor according to 89, wherein the protein as the active ingredient comprises the amino acid sequence of SEQ ID NO: 94 or 95.

[0125] 91. The inhibitor according to 90, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 158 or 159,

(b) a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 158 or 159 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0126] 92. The inhibitor according to 83, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 158 or 159.

[0127] 93. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising the amino acid sequence of SEQ ID NO: 94 or 95,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 94 or 95 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 158 or 159,

(b') a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 158 or 159 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0128] 94. The method according to 93, wherein the protein comprises the amino acid sequence of SEQ ID NO: 94 or 95.

[0129] 95. The method according to 93, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 158 or 159.

[0130] 96. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 93 to 95 and the step of selecting a prey for which an interaction is detected.

[0131] 97. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 96 or 97,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 96 or 97 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0132] 98. The inhibitor according to 97, wherein the protein as the active ingredient comprises the amino acid sequence of SEQ ID NO: 96 or 97.

[0133] 99. The inhibitor according to 98, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 160 or 161,

(b) a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 160 or 161 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0134] 100. The inhibitor according to 99, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 160 or 161.

[0135] 101. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising the amino acid sequence of SEQ ID NO: 96 or 97,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 96 or 97 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 160 or 161,

(b') a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 160 or 161 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0136] 102. The method according to 101, wherein the protein comprises the amino acid sequence of SEQ ID NO: 96 or 97.

[0137] 103. The method according to 101, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 160 or 161.

[0138] 104. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 101 to 103 and the step of selecting a prey for which an interaction is detected.

[0139] 105. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 98 or 99,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 98 or 99 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0140] 106. The inhibitor according to 105, wherein the protein as the active ingredient comprises the amino acid sequence of SEQ ID NO: 98 or 99.

[0141] 107. The inhibitor according to 98, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 162 or 163,

(b) a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 162 or 163 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0142] 108. The inhibitor according to 107, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 162 or 163.

[0143] 109. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising the amino acid sequence of SEQ ID NO: 98 or 99,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 98 or 99 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 162 or 163,

(b') a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 162 or 163 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0144] 110. The method according to 109, wherein the protein comprises the amino acid sequence of SEQ ID NO: 98 or 99.

[0145] 111. The method according to 109, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 162 or 163.

[0146] 112. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 109 to 111 and the step of selecting a prey for which an interaction is detected.

[0147] 113. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises a protein of the following (a) or (b) as an active ingredient:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 100 or 101,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 100 or 101 including deletion, substitution or addition of one or several amino acid residues and interacts with the c-Fos protein.

[0148] 114. The inhibitor according to 113, wherein the protein as the active ingredient comprises the amino acid sequence of SEQ ID NO: 100 or 101.

[0149] 115. The inhibitor according to 114, wherein the protein is a protein translated from a nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 164 or 165,

(b) a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 164 or 165 under a stringent condition and encodes a protein that interacts with the c-Fos protein.

[0150] 116. The inhibitor according to 115, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 164 or 165.

[0151] 117. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is a protein of the following (a) or (b) or a protein translated from a nucleic acid of the following (a') or (b'):

(a) a protein comprising the amino acid sequence of SEQ ID NO: 100 or 101,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 100 or 101 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein,

(a') a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 164 or 165,

(b') a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 164 or 165 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0152] 118. The method according to 117, wherein the protein comprises the amino acid sequence of SEQ ID NO: 100 or 101.

[0153] 119. The method according to 117, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 164 or 165.

[0154] 120. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to any one of 117 to 119 and the step of selecting a prey for which an interaction is detected.

[0155] 121. A protein of the following (a) or (b):

(a) a protein comprising the amino acid sequence of SEQ ID NO: 102,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 102 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein.

[0156] 122. A nucleic acid encoding the protein according to 102.

[0157] 123. A nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 166,

(b) a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 166 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0158] 124. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises the protein according to 121 or a protein translated from the nucleic acid according to 122 or 123 as an active ingredient.

[0159] 125. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is the protein according to 121 or a protein translated from the nucleic acid according to 122 or 123 as an active ingredient.

[0160] 126. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to 125 and the step of selecting a prey for which an interaction was detected.

[0161] 127. A protein of the following (a) or (b):

(a) a protein comprising the amino acid sequence of SEQ ID NO: 103,

(b) a protein that comprises the amino acid sequence of SEQ ID NO: 103 including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein.

[0162] 128. A nucleic acid encoding the protein according to 127.

[0163] 129. A nucleic acid of the following (a) or (b):

(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 167,

(b) a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 167 under a stringent condition and encodes a protein that interacts with a c-Fos protein.

[0164] 130. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises the protein according to 127 or a protein translated from the nucleic acid according to 128 or 129 as an active ingredient.

[0165] 131. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is the protein according to 127 or a protein translated from the nucleic acid according to 128 or 129 as an active ingredient.

[0166] 132. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to 131 and the step of selecting a prey for which an interaction was detected.

BRIEF EXPLANATION OF THE DRAWINGS

[0167] FIGS. 1A and 1B collectively show information of amino acid sequence, gene sequence and so forth of the protein of the present invention. Each of numerals within the parentheses after the DNA sequence numbers indicates the number of amino acid sequence encoded by it. A number having a subnumber means that the DNA encodes the same amino acid sequence, but has a different nucleotide sequence. SEQ ID NOS: 1 to 22 in FIG. 1A: Example 1, SEQ ID NOS: 47 to 76 in FIG. 1A and SEQ ID NOS: 77 to 103 in FIG. 1B: Example 2.

[0168] FIG. 2 shows the outline of the cotranslation screening method using an IVV random library, which is a method for detecting the proteins and genes of the present invention and the nucleotide sequences thereof. An IVV random library of mouse brain and c-Fos as a bait were used to carry out cell-free cotranslation screening, and the library after the screening was amplified by RT-PCR and then subjected to the cell-free cotranslation screening again with the bait. This procedure was repeated 3 times to detect the proteins and genes of the present invention and the nucleotide sequences thereof.

[0169] FIG. 3 shows the random priming library of IVV used for detection of the proteins and genes of the present invention and the nucleotide sequences thereof, and the outline of the method for producing it. Using an RNA library as a template and a random primer including a random sequence consisting of nine nucleotides and a specific sequence (tag2 sequence) in the random priming method, a library of single strand cDNAs (ssDNA) complementary to mRNAs is synthesized by reverse transcription (I). Only RNAs are decomposed in the double strands of cDNA and RNA with RNaseH, at the same time, DNAs complementary to cDNAs are synthesized with DNA polymerase I, and further a nick existing between the DNAs synthesized with DNA polymerase I is modified by using a DNA ligase to synthesized a double strand (dsDNA) library (II). The synthesized double-stranded cDNAs have a phosphate group at the 5' end only on the side synthesized with the DNA polymerase I, and therefore this is utilized to ligate an adaptor having a specific sequence (5' UTR=promoter+enhancer) using a DNA ligase to synthesize a ligated dsDNA library (III). PCR is carried out by utilizing the adaptor and the specific sequence of the random primer to prepare a cDNA library of assigning molecules having the sequences of a promoter and an enhancer on the 5' end side and an A tail on the 3' end side (IVV cDNA library) (IV). Then, the IVV cDNA library is transcribed to form an IVV RNA library (V), a spacer for preparing IVV is ligated (VI), and further it is translated in a cell-free translation system or the like to form a library of assigning molecules (VII).

[0170] FIG. 4 (electrophoretic photographs) shows the result 1 of the verification of the interactions of the proteins and genes of the present invention and the nucleotide sequences thereof.

[0171] A: Proteins having the amino acid sequences of SEQ ID NOS: 2 (Fip-cx), 16 (Eef1dTEF-1), and 22 (Schip1) were confirmed by the C-terminal labeling method in a wheat cell-free translation system. Lane 1 to 4: c-Jun protein, proteins of SEQ ID NO: 2 (Fip-cx), SEQ ID NO: 16 (Eef1dTEF-1), and SEQ ID NO: 22 (Schip1).

[0172] B: IVVs having the amino acid sequences of SEQ ID NOS: 2 (Fip-cx), 16 (Eef1dTEF-1), and 22 (Schip1) were confirmed in a wheat cell-free translation system. Lanes 1 and 2: mRNA and IVV, I to IV: c-Jun, SEQ ID NO: 2 (Fip-cx), SEQ ID NO: 16 (Eef1dTEF-1), and SEQ ID NO: 22 (Schip1).

[0173] C: Interactions were confirmed by the pull-down method utilizing IVVs having the amino acid sequences of SEQ ID NOS: 2 (Fip-cx), 16 (Eef1dTEF-1), and 22 (Schip1). Lanes 1 to 3: IVV, supernatant, and beads. a and b: with and without the bait c-Fos. I to IV: c-Jun, SEQ ID NO: 2 (Fip-cx), SEQ ID NO: 16 (Eef1dTEF-1), and SEQ ID NO: 22 (Schip1).

[0174] FIG. 5 (electrophoretic photographs) shows the result 2 of the verification of the interactions of the proteins and genes of the present invention and the nucleotide sequences thereof.

[0175] A: It was confirmed by the C-terminal labeling method in a wheat cell-free translation system that the proteins of SEQ ID NOS: 48 (Fip-cx.1), 75 (Fip-cx.2), 78 (Optn), 84 (Snapc5), 86 (C130020M04Rik), 88 (FLJ32000), 91 (Rit2), 93 (cytochrome b), 95 (Apoe), 97 (betaAPP), 99 (Hsp40), 101 (Fip-c10), 102 (Fip-c4) and 103 (Fip-c18) (FIGS. 1A and 1B) were expressed form the nucleic acid sequences of SEQ ID NOS: 105, 139, 142, 148, 150, 152, 155, 157, 159, 161, 163, 165, 166 and 167. Lanes 1 to 14: proteins of SEQ ID NOS: 48 (Fip-cx.1), 75 (Fip-cx.2), 78 (Optn), 84 (Snapc5), 86 (C130020M04Rik), 88 (FLJ32000), 91 (Rit2), 93 (cytochrome b), 95 (Apoe), 97 (betaAPP), 99 (Hsp40), 101 (Fip-c10), 102 (Fip-c4) and 103 (Fip-c18).

[0176] B: As a verification experiment of the interactions of the obtained proteins and c-Fos, direct interactions with c-Fos were confirmed by pull-down using C-terminal labeled proteins having the amino acid sequences of the proteins of SEQ ID NOS: 48 (Fip-cx.1) and 75 (Fip-cx.2) based on the nucleic acid sequences of SEQ ID NOS: 105 and 139. Lane 1: SEQ ID NO: 48 (Fip-cx.1), Lane 2: SEQ ID NO: 75 (Fip-cx.2), a and b: with and without bait c-Fos (Lanes 1 and 2: translation product and eluted fraction).

[0177] FIG. 6 shows the result of verification of the concentration rate and indirect interaction of the genes of the present invention. In order to confirm concentrations of 4 kinds of proteins of SEQ ID NOS: 78 (Optn), 84 (Snapc5), 86 (C130020M04Rik) and 88 (FLJ32000), real-time PCR was performed by using the nucleic acid sequences of SEQ ID NO: 142, 148, 150 and 152.

[0178] FIG. 7 shows outlines of the primary screening and secondary screening in the analysis of interaction of IVV with substances or proteins using the proteins and nucleic acid sequences of the present invention. It is possible to detect interactions with the substances and proteins in the primary screening using the proteins and nucleic acid sequences of the present invention, and further analyze the details of the interaction in the secondary screening using FCCS, microarray or the like. Further, the proteins and nucleic acid sequences of the present invention can also be independently used for analysis of interaction with the substances or proteins using FCCS, microarray, or the like as IVV or C-terminal labeled proteins. Furthermore, it is also possible to apply them to the evolutionary molecular engineering using IVV of the proteins or nucleic acid sequences of the present invention, and utilize them to create functional proteins in the primary screening. In such a case, it is also possible to analyze details of interactions of the created functional proteins with a combination of the primary screening and secondary screening.

[0179] FIG. 8 shows configurations of a translation template (A) as well as a coding molecule (B) and spacer molecule (C), which are constituents of the template. The translation template consists of a coding portion derived from the coding molecule and a spacer portion derived from the spacer molecule. F1 and F2 represent a fluorescent dye.

[0180] FIG. 9 shows configurations of a protein modified at the C-terminal (C-terminal labeled protein) (A), translation template of the present invention (B) and modification agent (C).

[0181] FIG. 10 shows outline of formation of a complex by cell-free cotranslation.

[0182] A: Both of bait and prey are translated in a cell-free translation system to interact with each other to form a complex in the cell-free translation system. The prey may exist in a single number (I) or multiple number (II), and it may be a polypeptide itself obtainable by the translation in the cell-free translation system, or an assigning molecule (bound substance).

[0183] B: In the presence of the bait, the prey is translated in a cell-free translation system to interact with the bait and thereby form a complex in the cell-free translation system. The prey may exist in a single number (I) or multiple number (II), and it may be a polypeptide itself obtainable by the translation in the cell-free translation system, or an assigning molecule (bound substance).

[0184] FIG. 11 shows outline of formation of a complex by cell-free cotranslation using a complexed bait.

[0185] Both of bait as a part constituting the complexed bait and prey are translated in a cell-free translation system and interact to form a complex in the cell-free translation system. The prey may exist in a single number (I) or multiple number (II), and it may be a polypeptide itself obtainable by the translation in the cell-free translation system, or an assigning molecule (bound substance). Further, the complexed bait is not limited to the combination of a polypeptide translated in a cell-free translation system and DNA bait shown in the drawing, and it may be, for example, a combination of multiple or single polypeptide translated in a cell-free translation system and multiple or single bait coexisting in the cell-free translation system (e.g., DNA bait etc.), or the like.

[0186] FIG. 12 shows outline of the method of screening for a complex based on cell-free cotranslation.

[0187] By the step of forming a complex on the basis of cell-free cotranslation as shown in FIGS. 10 and 11 (1), the step of screening the prey of the complex (2), and the step of analyzing the prey (3), the cell-free cotranslation and screening can be realized totally in vitro. If the prey is an assigning molecule, and it exists in a multiple number, the screening can be repeated again from the step (1) by reconstructing mRNA or DNA encoding the prey by RT-PCR or PCR. Further, after analyzing the obtained prey, screening can be newly repeated from the step of (1) using the prey as a bait.

BEST MODE FOR CARRYING OUT THE INVENTION

<1>Proteins of the Present Invention

[0188] In this specification, proteins found to interact with c-Fos, including novel proteins, are called "the proteins of the present invention" for convenience of explanation.

[0189] The first group of the proteins of the present invention (SEQ ID NOS: 1 to 14 in FIG. 1A) consists of proteins both of which function of forming a complex with c-Fos and amino acid sequences are novel (Fos interacting protein chromosome X, Fip-cx). These proteins are proteins characterized by showing homology to the nucleotide sequence (275-829 bp) formed by frame shift (+1) of the MAGE-necdin/trophinin complexes gene (AB032477) of the MAGE/necdin homologous region contained in the existing genome sequence WGS supercontig MmX (NW.sub.--042637) at nucleotide sequence level and the amino acid sequence thereof (184aa), and they are different from any of the proteins known so far to form a complex with c-Fos (Yurii Chinenovl and Tom K Kerppola, Oncogene, 20, 2438-2452 (2001)). Further, the MAGE-necdin/trophinin complex is known to be a tumor-related gene existing in the X chromosome discovered as MAGE (melanoma-associated antigen) (Sakura S, et al., J. Biol. Chem., 276, 49378-49389 (2001)). However, as for the present protein Fip-cx, the frame shifts by +1 with respect to the gene sequence of MAGE-necdin/trophinin complex, and any frame shift in the MAGE-necdin/trophinin complex gene is not known. Therefore, the present protein Fip-cx is a protein novel for both of the amino acid sequence and function of interacting with c-Fos. It was confirmed that the amino acid sequence of the present protein Fip-cx contains a leucine zipper and directly interacts with c-Fos (FIG. 2).

[0190] The second group of the proteins of the present invention (SEQ ID NOS: 15 to 19 in FIG. 1A) consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the gene sequence of the existing eukaryotic translation elongation factor-1 delta (Eef1d, TEF-1; NM.sub.--023240) gene at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. The function of forming a complex with c-Fos of the present protein Eef1d was detected by the present invention for the first time. Although Eef1d is known as a protein that controls translation extension, it has recently been shown that it is also a cancer- or tumor-related gene, and it is reported that with increase of the expression amount of Fos, the expression amount of Eef1d also increases as relationship of the translation factor Eef1d and tumor transformation (Joseph P, et al., J. Biol. Chem., 277, 6131-6136 (2002)). The amino acid sequences of these proteins contain a leucine zipper.

[0191] The third group of the proteins of the present invention (SEQ ID NOS: 20 to 22 in FIG. 1A) consists of proteins of which function of interacting with c-Fos is novel. These protein are proteins characterized by showing homology to the existing schwannomin interacting protein 1 (Schip1, NM.sub.--013928) gene sequence at gene sequence level and amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. Although it is known that Schip1 is a cancer- or tumor-related gene (Gouthebroze L, et al., Mol. Cell. Biol., 20, 1699-1712 (2000)), the function of forming a complex with c-Fos was detected for the first time by the present invention. These proteins bind with shwannomin, which is a regulator gene upstream from AP-1 and inhibits AP-1 activity. Further, the amino acid sequences of these proteins contain a leucine zipper.

[0192] The fourth group of the protein of the present invention (SEQ ID NOS: 47 to 56 in FIG. 1A) are proteins both of which function of forming a complex with c-Fos and amino acid sequences are novel (Fos interacting protein chromosome X.1, Fip-cx.1). These proteins are derived from (+1) frame-shifted gene of the Mage-d3 gene (NM.sub.--019548) of the Mage family. The amino acid sequences of these proteins contain a leucine zipper.

[0193] The fifth group of the proteins of the present invention (SEQ ID NOS: 57 to 76 in FIG. 1A) consists of proteins both of which function of forming a complex with c-Fos and amino acid sequences are novel (Fos interacting protein chromosome X.2, Fip-cx.2). These proteins are derived from (+1) frame-shifted gene of the Magphinin gene (AB032477) of the Mage family. The amino acid sequences of these proteins contain a leucine zipper.

[0194] The sixth group of the proteins of the present invention (SEQ ID NOS: 77 to 81 of FIG. 1B) consists of proteins of which function function of interacting with c-Fos is novel. These proteins are proteins characterized by showin homology to the existing Optineurin gene sequence (Optn, NM.sub.--181848) at gene level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. It is said that Optn is a causative gene of visual disturbance called adult-onset primary open-angle glaucoma (Tayebeh Rezaie, et al., Science, 295, 1077-1079 (2002)). The amino acid sequences of these proteins contain a leucine zipper.

[0195] The seventh group (SEQ ID NOS: 82 to 84 of FIG. 1B) of the proteins of the present invention consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the existing Snapc5 (Snpap19, XM.sub.--284503.1) gene sequence at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. Snapc5 is one of the subunits of the SNAP complex that binds to PSE, which is a promoter of snRNA transcribed by pol II and pol III, to regulate transcription (Henry, R. W., Mittal, V., Ma, B., Kobayashi, R., Hernandez, N., Genes Dev. 12:2664-2672 (1998), PubMed ID: 9732265). The amino acid sequences of these proteins contain a leucine zipper.

[0196] The eighth group of the proteins of the present invention (SEQ ID NOS: 85 to 86 in FIG. 1B) consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the existing C130020M04Rik (BC026483) gene sequence at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. C130020M04Rik is a gene of which protein frame is expected, but function is unknown. The annotation thereof is transcription regulatory factor. The amino acid sequences of these proteins contain a leucine zipper.

[0197] The ninth group of the proteins of the present invention (SEQ ID NOS: 87 to 89 in FIG. 1B) consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the existing FLJ3200 (XM.sub.--342896.1) gene sequence at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. FLJ3200 is a gene of which protein frame is expected, but function is unknown. They are proteins having a sequence similar to that of Rattus norvegicus. The amino acid sequences of these proteins contain a leucine zipper.

[0198] The tenth group of the proteins of the present invention (SEQ ID NOS: 90 and 91 in FIG. 1B) consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the existing Rit2 (NM.sub.--009065.2) gene sequence at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. Although Rit2 is an Ras-like protein, and it is a protein of the Ras family, it does not have a typical CAAX box existing at the C-terminal, which is known as a platform for the Ras protein on a membrane. It is known that Ras activates the promoter of the App gene (Ruiz-Leon, Y. and Pascual, A., 2, 278-285 (2001)). Furthermore, it is reported that Ras is involved in the control of the secretion process of the App protein together with Rho (Maillet, M et al., Nat. Cell Biol., 5, 633-639 (2003)). It has been reported that the Rho family consists of small GTP binding proteins and involved in cytoskeleton, transcription, development, transformation and so forth, and the Rho gene may stimulate the activity of AP1 to regulate a transcription factor involved in the activation of T cells (JIN-HONG CHANG, et al., Mil. Cell Biol., 18, 4986-4993 (1998)). The amino acid sequences of these proteins do not contain a leucine zipper.

[0199] The eleventh group of the proteins of the present invention (SEQ ID NOS: 92 to 93 in FIG. 1B) consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the existing cytochrome b (AF540912.1) gene sequence at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. The registered cytochrome b gene is not cloned in the full length. The amino acid sequences of these proteins contain a leucine zipper.

[0200] The twelfth group of the proteins of the present invention (SEQ ID NOS: 94 and 95 in FIG. 1B) consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the existing apolipoprotein E (Apoe; NM.sub.--009696.2) gene sequence at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. Apoe is known as a risk factor gene of Alzheimer's disease, and interacts with APP (David M. Holtzman, et al., PNAS, 97, 2892-97 (2000)). The Apoe gene has an AP1 site and is a gene existing downstream from AP1. The amino acid sequences of these proteins do not contain a leucine zipper.

[0201] The thirteenth group of the proteins of the present invention (SEQ ID NOS: 96 and 97 in FIG. 1B) consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the existing amyloid beta (A4) precursor protein (App; BC005499.1) gene sequence at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. App is known as a risk factor gene of Alzheimer's disease, and interacts with Apoe (David M. Holtzman, et al., PNAS, 97, 2892-97 (2000)). Like the Apoe gene, the App gene has an AP1 site and is a gene existing downstream from AP1. In fact, it has been reported that the first cascade of the series of reactions at the time of formation of memory is expression of Fos/Jun, and App/Apoe is subsequently expressed at an early stage of memory formation (Steven P. R. Rose, Learning & Memory, 7, 1-17 (2000)). Furthermore, it has recently reported that App is folded during translation by cotranslation by means of the chaperon function of Apoe (cotranslational folding, Silke Hab and et al., J. Biol. Chem., 273, 13892-13897 (1998)). It can be said that this is an example of detection of the App/Apoe complex formed during cotranslation of IVV that further forms a complex with a bait Fos. The amino acid sequences of these proteins do not contain a leucine zipper.

[0202] The fourteenth group of the proteins of the present invention (SEQ ID NOS: 98 and 99 in FIG. 1B) consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the existing Dnaja2 (HSP40; BC003420) gene sequence at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. Dnaja2 is a heat shock protein, and it is known that the expression amount thereof increases with increase of those of Fos and Jun when a heat shock is given (Kato N, et. al., Cancer Science, 97, 644-649 (2000)). The amino acid sequences of these proteins do not contain a leucine zipper.

[0203] The fifteenth group of the proteins of the present invention (SEQ ID NOS: 100 and 101 in FIG. 1B) consists of proteins of which function of interacting with c-Fos is novel. These proteins are proteins characterized by showing homology to the existing Fip-c10 (KIAA1209, XM.sub.--136911) gene sequence at gene sequence level and the amino acid sequence thereof, and they are different from any of the proteins that are known so far to form a complex with c-Fos. Fip-c10 is a gene of which protein frame is expected, but function is unknown. The amino acid sequences of these proteins do not contain a leucine zipper.

[0204] The sixteenth group of the proteins of the present invention (SEQ ID NO: 102 in FIG. 1B) consists of a protein both of which function of forming a complex with c-Fos and amino acid sequence are novel (Fos interacting protein chromosome 4.1, Fip-c4). This protein is encoded by a region in a genome sequence for which protein frame has not been expected at all so far. The amino acid sequence of this protein does not contain a leucine zipper.

[0205] The seventeenth group of the protein of the present invention (SEQ ID NO: 103 in FIG. 1B) is a protein both of which function of forming a complex with c-Fos and amino acid sequence are novel (Fos interacting protein chromosome 18, Fip-c18). This protein is encoded by a region in a genome sequence for which protein frame has not been expected at all so far. The amino acid sequence of this protein does not contain a leucine zipper.

[0206] Hereafter, the proteins of the present invention will be further explained.

[0207] Among the proteins of the present invention, the proteins having any one of the amino acid sequences of SEQ ID NOS: 1 to 22 and 47 to 103 are proteins for which it has been found that they interact with the c-Fos protein, i.e., form a complex, as described in the examples mentioned below. For proteins, existence of a mutant having the same function is generally expected. Further, by suitably modifying an amino acid sequence of a protein, a mutant having the same function can be obtained. Therefore, proteins that have any one of the amino acid sequences of SEQ ID NOS: 1 to 22 and 47 to 103 including deletion, substitution or addition of one or several amino acid residues and interact with the c-Fos protein also fall within the scope of the proteins of the present invention. Further, proteins that show a homology of 15% or more to any one of the amino acid sequences of SEQ ID NOS: 1 to 22 and 47 to 103 and interact with the c-Fos protein also fall within the scope of the protein of the present invention. Examples of such proteins of which amino acid residues are modified include, for example, proteins having any one of the amino acid sequences of SEQ ID NOS: 2 to 14 for the protein having the amino acid sequence of SEQ ID NO: 1, proteins having any one of the amino acid sequences of SEQ ID NOS: 16 to 19 for the protein having the amino acid sequence of SEQ ID NO: 15, and proteins having any one of the amino acid sequences of SEQ ID NOS: 21 and 22 for the protein having the amino acid sequence of SEQ ID NO: 20.

[0208] An amino acid sequence of a protein can be modified by modifying a nucleotide sequence of DNA encoding the protein using a well-known means such as site-directed mutagenesis and expressing DNA of which nucleotide sequence is modified. Among such modified proteins, those that interact with the c-Fos protein fall within the scope of the protein of the present invention. The interaction with the c-Fos protein can be measured by a known method for measuring an interaction, and examples include the method of detecting formation of a complex mentioned in the examples described later.

[0209] The proteins of the present invention may be fused with another protein and thus provided as a fusion protein.

[0210] The nucleic acids of the present invention are nucleic acids encoding the proteins of the present invention. The nucleic acids are usually RNA or DNA. Examples of the nucleic acids of the present invention include nucleic acids having any one of the nucleotide sequences of SEQ ID NOS: 23 to 40 and 104 to 167. These nucleic acids are nucleic acids of which nucleotide sequences were determined in the examples mentioned below. For a gene, existence of a gene encoding the same product, but having a different nucleotide sequence, or a gene encoding a mutant having the same function is expected. Further, by suitably modifying a nucleotide sequence, a gene encoding the same product or a mutant having the same function can be obtained. Therefore, nucleic acids having a nucleotide sequence similar to any one of the nucleotide sequences of SEQ ID NOS: 23 to 40 and 104 to 167 and encoding a protein that interacts with the c-Fos protein also fall within the scope of the nucleic acids of the present invention. Examples of such nucleic acids having a similar nucleotide sequence include nucleic acids that hybridize with a nucleic acid having a nucleotide sequence complementary to any one of the nucleotide sequences of SEQ ID NOS: 23 to 40 and 104 to 167 under a stringent condition, and nucleic acids having a nucleotide sequence showing a homology of 16% or more to any one of the nucleotide sequences of SEQ ID NOS: 23 to 40 and 104 to 167.

[0211] The stringent condition referred to here corresponds to, for example, that of hybridization using DIG Easy Hyb (Roch Diagnostics) at 42.degree. C. followed by washing in 0.1.times.SSC/0.1% SDS for 15 minutes at 60.degree. C. Homology of nucleotide sequences is obtained as a rate of number of nucleotides matching in alignment of the nucleotide sequences to be compared to the nucleotide number of the chain length of the nucleotide sequences. Further, homology of amino acid sequences is obtained as a rate of number of amino acid residues matching in alignment of the amino acid sequences to be compared to the amino acid number of the chain length of the amino acid sequences.

[0212] Whether a DNA encodes a protein that interacts with the c-Fos protein can be easily confirmed by expressing a protein from that DNA and confirming whether the expressed protein interacts with the c-Fos protein using the aforementioned method.

[0213] The nucleic acids of the present invention can be obtained by a conventional method on the basis of the elucidated nucleotide sequences. For example, it may be synthesized by a chemical synthesis method, or it may be obtained by RT-PCR using suitably designed primers from a mRNA prepared from cells or tissue expressing a protein that interacts with the c-Fos protein.

<2>Use of the Proteins of the Present Inventions and Others.

[0214] The proteins and genes of the present invention can be applied as an inhibitor that blocks transcription function, gene replication function and so forth as for c-Fos in gene therapy etc. by utilizing the novel function obtained by the nucleic acid sequences (function enabling binding with c-Fos in this case). The basis for this originates in the fact that the genes of the proteins of the present invention are detected after undergoing competitive process of screening repeated multiple times. Genes detected by this method exhibit a certain number distribution, and a gene having higher competitive power will be detected in a larger number. This means that a gene of which clones are detected by this method in a larger number should have a higher competitive power, and it more effectively functions as a blocking agent or inhibitor.

[0215] As for use of the proteins of the present invention and genes encoding them, as in vitro applications, they can be applied in, for example, evolutionary molecular engineering using a cell-free protein synthesis system or genomic function analysis by utilizing the novel function provided by the proteins, genes or nucleic acid sequences according to the present invention. In this case, analysis utilizing cotranslation screening and selection of assigning molecules is extremely effective. This is because the cotranslation screening/selection method makes it possible to comprehensively detect proteins that directly or indirectly interact with a bait protein. Furthermore, in analysis of interactions between IVVs, IVV and C-terminal labeled protein etc., they can also be use as a "target molecule (bait protein)". Examples of general methods for analyzing an interaction include, for example, microarray method, fluorescence correlation spectroscopy (FCS/FCCS), fluorescence imaging analysis, fluorescence resonance energy transfer method, evanescent-field molecular imaging method, fluorescence depolarization method, surface plasmon resonance method, enzyme linked immunosorbent assay and so forth. Specific examples of the cell-free protein synthesis system include wheat germ extract, rabbit reticulocyte lysate, Escherichia coli S30 extract and so forth. By adding a protein, gene or nucleic acid sequence as a translation template according to the present invention to any of these cell-free protein synthesis systems, simultaneously adding 1 to 100 .mu.M of modification agent in the case of C-terminal labeling, and maintaining the system at 25 to 37.degree. C. for 1 to several hours, a C-terminal modified protein is synthesized. In the case of assigning, only by adding a protein, gene or nucleic acid sequence as a translation template according to the present invention to the cell-free protein synthesis system and maintaining the system at 25 to 37.degree. C. for 1 to several hours, an assigning molecule is synthesized.

[0216] Further, as for in vivo applications, by utilizing the novel function provided by the proteins, genes or nucleic acid sequences according to the present invention, for example, a protein modified for separation and labeled for detection (double modified protein) synthesized in a cell-free protein synthesis system can be used as it is for a subsequent purification process, detection process, or direct introduction into cells. Specific examples of cell expression system include any kind of cells of from bacteria such as Escherichia coli, Bacillus subtilis and thermophilic bacteria, yeast to cultured cells of insects, mammals and so forth, cells of threadworm, drosophila, zebra fish, mouse and so forth. By directly introducing the aforementioned C-terminal labeled or assigned double modified protein into these cells, an objective protein can be blocked. Alternatively, it is also possible to introduce the aforementioned gene or nucleic acid of the present invention and utilize the gene or nucleic acid as it is as an antisense sequence or RNAi sequence to block expression of an objective nucleic acid, or they can be expressed in a cell and utilized as a protein or assigning molecule to block a protein having an interacting action. When a protein is used in the C-terminal labeling method, by simultaneously introducing 1 to 100 .mu.M modification agent for C-terminal labeling into cells using electroporation, microinjection or the like and maintaining the cells at the optimum growth temperature of the cells for several hours, a modified protein is synthesized. In the case of assigning, by introducing a template of an assigning molecule having the aforementioned gene or nucleic acid sequence of the protein of the present invention into cells and maintaining the cells at the optimum growth temperature of the cells for several hours, an assigning molecule is synthesized. The synthesized double modified protein can be collected by disrupting the cells and used for the subsequent purification process or detection process. Further, it can be used as it is in the cells for the detection process.

[0217] Hereafter, the use of the proteins of the present invention and others will be further explained.

[0218] The detection method of the present invention is a method of utilizing the proteins of the present invention as a bait in detection of interaction between the bait and a prey.

[0219] Preferably, the method is mainly characterized in that the bait and prey are modified for separation and labeled for detection in a specific manner, and the prey is produced by translation in a cell-free translation system in the presence of the bait to contact the bait and prey. In this specification, contacting a bait and a prey by producing the prey by translation in a cell-free translation system in the presence of the bait is also referred to as "cell-free cotranslation".

[0220] In this specification, the terms of "bait" and "prey" have the meanings usually used in the technical field of analysis of interaction between substances. That is, a protein, nucleic acid or the like as a known substance is called "bait", and a protein, nucleic acid or the like as a substance that interacts with the bait is called "prey". In the present invention, the prey is preferably a protein.

[0221] The bait used herein may be the protein of the present invention, or a complex constituted by arbitrary components including protein (including peptide), nucleic acid, or ligand such as antibody and hormone, metal and so forth, so long as it contains the protein of the present invention, and it may be a natural substance or artificial substance. The bait is not particularly limited as for the molecular weight and so forth. Examples include, for example, in the case of protein, a functional domain, a full-length protein containing a functional domain and so forth. If a prey library is used, use of full-length proteins enables comprehensive detection.

[0222] Further, as the prey, a protein is preferably used. The prey is not particularly limited as for the molecular weight and so forth.

[0223] Preferably, the detection method of the present invention is mainly characterized in that, in the detection of an interaction of the bait and prey, the bait and prey are modified for separation and labeled for detection in a specific manner, and cell-free cotranslation is performed as described above. Therefore, a preferred configuration of the detection method of the present invention may be the same as that of a usual method for detecting an interaction between a bait and a prey comprising contacting the bait and prey and detecting a complex formed by the contact, except that the bait and prey are modified for separation and labeled for detection in a specific manner, and cell-free cotranslation is performed.

[0224] Although the modification for separation and labeling for detection of the bait and prey are arbitrarily performed so that they are suitable for the detection of the complex, they should be performed so that both of the bait and prey should not be labeled with a label for detection or modified for separation. Therefore, the prey is used as a fusion protein with a protein that can be used as a label for detection or an assigning molecule, and the bait correspondingly has a modification for separation.

[0225] When the prey is used as a fusion protein, the bait should have a modification for separation. When the bait is a protein, the bait is preferably produced in a cell-free translation system by translation of mRNA encoding a fusion protein containing the bait as a fusion protein with a protein that can be used as a modification for separation in the cell-free translation system.

[0226] Examples of the modification for separation in the case where the bait is a protein include formation of a fusion protein with the GST protein, CBP used for the TAP method etc. (this can be separated by using affinity with calmodulin beads), protein A (this can be separated by using IgG-protein A affinity) as a protein, or any of various antibody tags etc. as an affinity tag. When the bait itself has a property that it can be used as a modification for separation, the bait can be used as it is as a bait having a modification for separation. Examples of the modification for detection of the prey include formation of a fusion protein with a fluorescent protein such as GFP (green fluorescent protein).

[0227] Preparation of mRNA encoding such a fusion protein mentioned above and translation of this mRNA in a cell-free translation system can be performed by usual methods. The mRNA may be mRNA produced by transcription of DNA in a cell-free transcription and translation system.

[0228] When the prey is an assigning molecule, arbitrary modification for separation can be added to the bait. When the bait is a protein, the aforementioned examples of the modification for separation can be used. In addition, when the bait is a nucleic acid, drug or the like, examples of the modification for separation include use of biotin etc. that interact with streptavidin or avidin. When the bait itself has a property that it can be used as modification for separation, the bait can be used it is as a bait having modification for separation.

[0229] An assigning molecule means a molecule assigning a phenotype and a genotype. The assigning molecule is usually a molecule comprising a genotype molecule containing a nucleic acid having a nucleotide sequence reflecting a genotype and a phenotype molecule containing a protein relating to expression of phenotype, which are bound to each other. By using a prey as this protein, the prey can be used as an assigning molecule. Such an assigning molecule can be formed by performing translation of mRNA encoding a prey in a cell-free translation system so that the translated prey should associate with the mRNA, or performing transcription and translation of DNA encoding a prey in a cell-free transcription and translation system so that the translated prey should associate with the DNA. Therefore, by allowing a bait to exist during the production, cell-free cotranslation can be attained. That is, the cell-free cotranslation can be perfomed by the following scheme (1) or (2).

[0230] (1) By performing translation of mRNA encoding the prey in the presence of the bait in a cell-free translation system so that the translated prey should associate with the mRNA, the prey is produced in the cell-free translation system, and thereby the bait and prey are brought into contact with each other.

[0231] (2) By performing transcription and translation of DNA encoding the prey in the presence of the bait in a cell-free transcription and translation system so that the translated prey should associate with the DNA, the prey is produced in the cell-free translation system, and thereby the bait and prey are brought into contact with each other.

[0232] Hereafter, the embodiments of (1) and (2) mentioned above will be explained.

[0233] In the embodiment of (1), the translated prey preferably associates with the mRNA, because the mRNA has a spacer region bound to the 3' end and a peptide acceptor region bound to the spacer region and containing a group that can bind to a peptide by transpeptidation reaction. Examples of the method for detecting an interaction using such an assigning molecule include the in vitro virus method.

[0234] The mRNA is preferably a nucleic acid containing a 5' untranslation region including a transcription promoter and a translation enhancer, an ORF region encoding a prey and binding to the 3' end side of the 5' untranslation region, and a 3' end region including a poly-A sequence and binding to 3' end side of the ORF region. Preferably, an expression amplification sequence containing an SNNS (S is G or C) sequence on the 5' end side of the poly-A sequence (for example, a sequence recognizable by the restriction enzyme XhoI) is further included. The mRNA may or may not have a Cap structure at the 5' end.

[0235] The poly-A sequence is a poly-A continuous chain of at least 2 or more residues comprising dA and/or rA as single kind of residues or mixture of two kinds, and the poly-A chain consists of, preferably 3 or more residues, more preferably 6 or more residues, still more preferably 8 or more residues.

[0236] One of the factors affecting the translation efficiency is a combination of the 5' UTR comprising a transcription promoter and a translation enhancer and the 3' end region including a poly-A sequence. The effect of the poly-A sequence of the 3' end region is usually exerted with a length of ten or less residues. As the transcription promoter of the 5' UTR, T7/T3, SP6, and so forth can be used, and no particular limitation is imposed. SP6 is preferred, and it is particularly preferable to use SP6, especially when a sequence containing an omega sequence or a part of omega sequence is used as the translation enhancer sequence. The translation enhancer is preferably a part of the omega sequence, and as the part of the omega sequence, one containing a part of the omega sequence of TMV (O29, refer to Gallie D. R., Walbot V., Nucleic Acids Res., vol. 20, 4631-4638 (1992), and WO02/48347, FIG. 3) is preferred.

[0237] Further, for the translation efficiency, the combination of the XhoI sequence and a poly-A sequence is preferred in the 3' end region. Furthermore, a combination of the downstream portion of the ORF region, i.e., the upstream region of the XhoI sequence having an affinity tag, and a poly-A sequence is preferred. The affinity tag sequence may be any sequence for utilizing a means that can detect a protein such as an antigen-antibody reaction, and no limitation is imposed. The affinity tag is preferably the Flag-tag sequence or His-tag sequence, which is a tag for affinity separation analysis based on an antigen-antibody reaction. As for the effect of the poly-A sequence, an affinity tag such as the Flag-tag attached with the XhoI sequence and further attached with a poly-A sequence increases the translation efficiency. As for the His-tag, even a His-tag having a configuration not containing the XhoI sequence also exhibits sufficient translation efficiency, and thus is effective.

[0238] Such a configuration effective for improvement of translation efficiency is also effective for assignment efficiency.

[0239] If SP6+O29 and Flag+XhoI+A.sub.n (n=8) or His+A.sub.n (n=8), for example, are used as the 5' UTR and the 3' end region, respectively, the 5' UTR and the 3' end region would have lengths of about 49 bp and about 38 or 26 bp, respectively, and thus they have such a length that they can be incorporated into primers for PCR as an adaptor region. Therefore, a coding region having such a 5' UTR and 3' end region can be easily produced by PCR from any of vectors, plasmids and cDNA libraries. In the coding region, translation may occur beyond the ORF region. That is, there may not be a stop codon at the end of the ORF region.

[0240] The peptide acceptor region is not particularly limited, so long as it can bind to the C-terminal of a peptide. For example, puromycin and 3'-N-aminoacylpuromycin aminonucleosides (PANS-amino acids) including PANS-amino acids corresponding to all amino acids such as PANS-Gly in which the amino acid portion is glycine, PANS-Val in which the amino acid portion is valine, and PANS-Ala in which the amino acid portion is alanine can be utilized. Further, 3'-N-aminoacyladenosine aminonucleosides (AANS-amino acids), in which a 3'-aminoacyladenosine and an amino acid are bonded via an amide bond as a chemical bond formed as a result of dehydration condensation of the amino group of the 3'-aminoacyladenosine and the carboxyl group of the amino acid, corresponding to all amino acids, for example, AANS-Gly in which the amino acid portion is glycine, AANS-Val in which the amino acid portion is valine, AANS-Ala in which the amino acid portion is alanine, and so forth can also be used. Furthermore, nucleosides and nucleosides bound with an amino acid via an ester bond can also be used. In addition, any of substances formed with a bonding scheme that can chemically bond a nucleoside or a substance having a chemical structure similar to that of nucleoside and an amino acid or a substance having a chemical structure similar to amino acid can be used.

[0241] The peptide acceptor region preferably comprises puromycin or a derivative thereof, or puromycin or a derivative thereof and one or two residues of deoxyribonucleotides or ribonucleotides. The term "derivative" used in this case means a derivative that can bind to the C-terminal of peptide in a protein translation system. The puromycin derivative is not limited to those having the total puromycin structure, and includes those having the puromycin structure a part of which is eliminated. Specific examples of the puromycin derivative include PANS-amino acids, AANS-amino acids and so forth.

[0242] Although the peptide acceptor region may have a structure consisting only of puromycin, it preferably has a nucleotide sequence comprising DNA and/or RNA of one or more residues at the 5' end side. As such a sequence, dC-puromycin, rC-puromycin, and so forth, more preferably, a CCA sequence comprising dCdC-puromycin, rCrC-puromycin, rCdC-puromycin, dCrC-puromycin or the like and imitating the 3' end of aminoacyl-tRNA (Philipps, G. R., Nature 223, 374-377 (1969)), is suitable. As for the type of nucleotide, preference is higher in the order of C>(U or T)>G>A.

[0243] The spacer region is preferably a PEG region containing polyethylene glycol as a main component. The spacer region usually contains, in addition to the PEG region, a donor region that can bind to the 3' end of a nucleic acid.

[0244] The donor region that can bind to the 3' end of nucleic acid usually consists of one or more nucleotides. The number of nucleotides is usually 1 to 15, preferably 1 to 2. The nucleotides may be a ribonucleotide or a deoxyribonucleotide. The donor region may have a modification substance.

[0245] The sequence of the 5' end of the donor region affects the ligation efficiency with the coding region encoding the prey. In order to attain ligation of the coding region and the spacer region, it is required to include at least one or more residues, and at least one residue of dC (deoxycytidylic acid) or two residues of dCdC (dideoxycytidylic acid) is preferred for an acceptor having a poly-A sequence. As for the type of nucleotide, preference is higher in the order of C>(U or T)>G>A.

[0246] The PEG region contains polyethylene glycol as a main component. The expression "contains polyethylene glycol as a main component" used herein means that the total number of nucleotides contained in the PEG region is 20 or less, or the average molecular weight of the polyethylene glycol is 400 or more. It preferably means that the total number of nucleotides is 10 or less, or the average molecular weight of the polyethylene glycol is 1000 or more.

[0247] The average molecular weight of the polyethylene glycol in the PEG region is usually 400 to 30,000, preferably 1,000 to 10,000, more preferably 2,000 to 8,000. If the molecular weight of the polyethylene glycol is lower than about 400, a posttreatment for assignment translation may be required for assignment translation of a genotype molecule containing the spacer region (Liu, R., Barrick, E., Szostak, J. W., Roberts, R. W., Methods in Enzymology, vol. 318, 268-293 (2000)). However, if PEG having a molecular weight if 1000 or more, preferably 2000 or more, is used, highly efficient assignment can be attained only by assignment translation, and therefore the posttreatment for the translation becomes unnecessary. Further, when the molecular weight of the polyethylene glycol increases, stability of the genotype molecule tends to increase, and in particular, the stability becomes favorable with a molecular weight of 1000 or more. If the molecular weight is 400 or less, properties thereof are not different so much from those of a DNA spacer, and it may become unstable.

[0248] By having a spacer region containing polyethylene glycol as a main component, it becomes possible to form an assigning molecule not only in a cell-free translation system of rabbit reticulocytes, but also in a cell-free translation system of wheat germ, the stability of the genotype molecule in both translation systems is markedly improved, and it becomes unnecessary to perform any treatment after the translation.

[0249] In the embodiment of (2), it is preferred that DNA encodes a fusion protein of a protein and streptavidin or avidin, DNA is labeled with bibtin, and a translated prey associates with the DNA because transcription and translation is carried out in a state that one DNA molecule is contained in one compartment of emulsion. Examples of the method for detecting an interaction using such an assigning molecule include the STABLE method.

[0250] The emulsion is usually a W/O type emulsion formed by mixing two kinds of surface active agents, mineral oil and a reaction mixture of cell-free transcription and translation system. In order to form a W/O type emulsion, it is usually necessary for the surface active agents to have an HLB (hydrophile-lipophile balance) value of 3.5 to 6. The HLB value of mixed two kinds of surface active agents is calculated from the HLB values of the individual surface active agents by using a simple equation. For example, if Span 85 (HLB=1.8) and Tween 80 (HLB=15.0) are mixed in volumes of 40.2 .mu.l and 9.8 .mu.l, respectively, the mixture has an HLB value of 4.4. The ratio of the surface active agents and mineral oil is usually 1:18 (volume ratio). Further, the ratio of the reaction mixture is 1 to 50% (volume ratio) with respect to the whole emulsion, and it is usually 5%. The emulsion can be formed by adding the reaction mixture as several divided portions to a mixture of the surface active agents and mineral oil at a low temperature with stirring and mixing them. The reactions of transcription and translation can be started by raising the temperature of the emulsion.

[0251] The preparation of DNA encoding a prey, and transcription and translation of such a DNA in a cell-free transcription and translation system can be performed in a usual manner.

[0252] As described above, by labeling the bait and prey for detection and modifying them for separation in particular schemes, a complex formed by cell-free cotranslation can be specifically detected.

[0253] As for the cell-free cotranslation of a bait and a prey, the cell-free translation system (including cell-free transcription and translation system) in which the cell-free cotranslation is performed may be any of systems of E. coli, rabbit reticulocytes, wheat germs and so forth. Although formation of assigning molecules is quite unstable with E. coli in the in vitro virus method, it has been confirmed that it is stable in a system of rabbit reticulocytes (Nemoto N., Miyamoto-Sato E., and Yanagawa H., FEBS Lett. 414, 405 (1997); Roberts R. W., Szostak J. W., Proc. Natl. Acad. Sci. USA, 94, 12297 (1997)), and it has been further confirmed that it is still more stable in a system of wheat germ (Japanese Patent Laid-open No. 2002-176987). For the STABLE method, the system may be any of systems of E. coli, rabbit reticulocyte, wheat germ and so forth.

[0254] The conditions for the translation and transcription in the cell-free cotranslation are suitably selected depending on a cell-free translation system to be used.

[0255] The templates of the bait and prey added to the cell-free translation system may be either RNA or DNA, so long as the cell-free translation system is a cell-free transcription and translation also causing transcription.

[0256] Hereafter, an example of a translation template preferred for use as a bait will be explained.

[0257] As a bait used in the cotranslation screening of this embodiment, used is a translation template characterized by comprising a coding portion having information for translation into a protein and a PEG spacer portion as shown in FIG. 8. The coding portion has information for translation into a protein, and it may be any sequence. However, it is preferably characterized by having an acceptor (A sequence) in a 3' end region of the coding portion, or having an acceptor (A sequence) in a 3' end region of the coding portion and a translation amplification sequence (X sequence) 5'-upstream from the A sequence. It contains a short poly-A sequence as the A sequence of the coding portion. The short poly-A sequence is usually a sequence comprising 2 to 10 nucleotides of A. It is characterized by having a sequence having (C or G)NN(C or G) sequence, for example, a XhoI sequence, as the X sequence. The PEG spacer portion has a PEG region containing polyethylene glycol as a main component, a donor region for ligation with the coding portion, and a CCA region at the 3' end. Although the PEG spacer portion may consist only of the donor region or CCA region, it is preferably has a configuration comprising the PEG region containing polyethylene glycol as a main component. The CCA region is characterized by not having a function of binding by a transpeptidation reaction to a protein translated from the translation template. The PEG region is characterized by having a molecular weight of the polyethylene glycol of 500 or more. Further, it is characterized by containing at least one function-imparting unit (F) in the donor region and/or the CCA region. It is characterized in that the function-imparting unit (F1 and/or F2) immobilizes or labels with fluorescence the translation template and/or a protein translated from the translation template. As the immobilization substance, biotin and so forth are contemplated, and as the fluorescent substance, fluorescein, Cy5, rhodamine green (RhG) and so forth can be contemplated. The present invention relates to these coding portion, translation template, and libraries thereof, as well as a protein translated on a ribosome and library thereof.

[0258] The translation template of a bait (FIG. 8, A) comprises a coding portion derived from a coding molecule (FIG. 8, B) and a PEG spacer portion derived from a PEG spacer molecule (FIG. 8, C). In this embodiment, a PEG spacer portion can be ligated to the coding portion to improve stability thereof, and thus translation efficiency can be improved, basically regardless of the sequence of the coding portion. However, it is further possible to further improve the translation efficiency depending on the configuration of the coding portion or the type of the PEG spacer portion. The details thereof are described below.

[0259] The coding portion of this embodiment (FIG. 8, B) comprises a 5' end region, an ORF region, and a 3' end region, and it may or may not have a Cap structure at the 5' end. Further, the sequence of the coding portion is not particularly limited, and use thereof as one incorporated into any vector or plasmid can be contemplated. The 3' end region of the coding portion includes one having a poly-A x 8 sequence as the A sequence or one having, as the X sequence, the XhoI sequence or a sequence of SNNS(S is G or C) as a sequence of 4 or more nucleotides, and XA as a combination of the A sequence and X sequence. A configuration that a Flag-tag sequence is included as an affinity tag sequence upstream from the A sequence, X sequence, or XA sequence is contemplated. The affinity tag sequence used here may be a sequence for using any means that enables detection or purification of a protein, for example, those utilizing an antigen-antibody reaction such as HA-tag and protein A of IgG (z domain), His-tag, and so forth. As for factors affecting the translation efficiency, the combination of the XA sequence is important. The first four nucleotides of the X sequence are important, and one having a sequence of SNNS is preferred. Further, the 5' end region comprises a transcription promoter and a translation enhancer. As the transcription promoter, T7/T3, SP6, and so forth can be used, and no particular limitation is imposed. However, for a cell-free translation system of wheat, a sequence containing an omega sequence or a part of omega sequence is preferably used as the translation enhancer sequence, and SP6 is preferably used as the promoter. The part of the omega sequence is one containing a part of the omega sequence of TMV (O29, refer to Gallie D. R., Walbot V., Nucleic Acids Res., vol. 20, 4631-4638 (1992) and WO02/48347, FIG. 3). The ORF region of the coding portion may be any sequence comprising DNA and/or RNA. It may be a gene sequence, exon sequence, intron sequence, random sequence, or any natural sequence or artificial sequence, and the sequence is not limited.

[0260] The PEG spacer molecule of this embodiment (FIG. 8, C) comprises a CCA region, a PEG region, and a donor region. The minimum essential component is the donor region. As for the factors affecting the translation efficiency, one having not only the donor region but also the PEG region is preferred, and it preferably has puromycin which does not have an ability to bind with an amino acid. The molecular weight of the polyethylene glycol in the PEG region is 400 to 30,000, preferably 1,000 to 10,000, more preferably 2,000 to 6,000. Further, the CCA region may have a configuration including puromycin or a configuration not including puromycin. As puromycin, puromycin and 3'-N-aminoacylpuromycin aminonucleosides (PANS-amino acids) including PANS-amino acids corresponding to all amino acids such as PANS-Gly in which the amino acid portion is glycine, PANS-Val in which the amino acid portion is valine, and PANS-Ala in which the amino acid portion is alanine can be utilized. Further, 3'-N-aminoacyladenosine aminonucleosides (AANS-amino acids), in which a 3'-aminoacyladenosine and an amino acid is bonded via an amide bond as a chemical bond formed as a result of dehydration condensation of the amino group of the 3'-aminoacyladenosine and the carboxyl group of the amino acid, corresponding to all amino acids, for example, AANS-Gly in which the amino acid portion is glycine, AANS-Val in which the amino acid portion is valine, AANS-Ala in which the amino acid portion is alanine, and so forth can also be used. Furthermore, nucleosides and nucleosides bound with an amino acid via an ester bond can also be used. In addition, any of substances formed with a bonding scheme that can chemically bond a nucleoside or a substance having a chemical structure similar to that of nucleoside and an amino acid or a substance having a chemical structure similar to amino acid can be used. For this translation template, any substances corresponding to the aforementioned puromycin derivatives of which amino group lacks the ability to bind to an amino acid, and a CCA region lacking puromycin are also contemplated. However, by incorporating puromycin that cannot bind with a protein on a ribosome, the translation efficiency can be further enhanced. Although the reason for this is not certain, it may possible that puromycin that cannot bind with a protein stimulates a ribosome to enhance the turnover. A nucleotide sequence comprising DNA and/or RNA of one or more residues is preferably contained on the 5' end side of the CCA region (CCA). As for the type of nucleotide, preference is higher in the order of C>(U or T)>G>A. As such a sequence, dC-puromycin, rC-puromycin, and so forth, more preferably, a CCA sequence comprising dCdC-puromycin, rCrC-puromycin, rCdC-puromycin, dCrC-puromycin or the like and imitating the 3' end of aminoacyl-tRNA (Philipps, G. R., Nature 223, 374-377 (1969)) is suitable. In one embodiment of the present invention, these puromycins are made incapable of binding with an amino acid in a certain manner.

[0261] The PEG spacer portion of this embodiment may have a configuration containing a modification substance (F1 and/or F2). With this characteristic, it can be used as a tag for collection, reuse by purification, or immobilization of translation template. Those comprising at least one residue of nucleotide of DNA and/or RNA incorporated with any of various separation tags such as fluorescent substance, biotin, and His-tag may be possible. Further, if SP6+O29 and Flag+XhoI+An (n=8) are used as the 5' end region and the 3' end region of the coding portion, respectively, for example, the lengths of the 5' end region and the 3' end region are about 60 bp and about 40 bp, respectively, and thus they have such a length that they can be designed in primers for PCR as an adaptor region. This provides a novel advantage. That is, it becomes possible to easily prepare a coding portion having a 5' end region and 3' end region according to this embodiment by PCR from any vector, plasmid, and cDNA library, and by ligating the PEG spacer portion, instead of a 3' UTR, to this coding portion, a translation template showing a high translation efficiency can be obtained.

[0262] The ligation of the PEG spacer molecule and the coding molecule according to this embodiment may be attained any method such as usual methods utilizing a DNA ligase or those based on a photoreaction, and the method is not particularly limited. In the ligation using an RNA ligase, as factors in the coding portion affecting the ligation efficiency, the A sequence of the 3' end region is important. It is a poly-A continuous chain consisting of at least two, preferably 3 or more, more preferably 6 to 8, of single kind or mixed kinds of residues selected from dA and/or rA. The DNA and/or RNA sequence of the 5' end of the donor region of the PEG spacer portion affects the ligation efficiency. In order to ligate the coding portion and PEG spacer portion with an RNA ligase, it is required to contain at least one or more residues, and for an acceptor having a poly-A sequence, at least 1 residue of dC (deoxycytidylic acid) or two residues of dCdC (dideoxycytidylic acid) is preferred. As for the type of nucleotide, preference is higher in the order of C>(U or T)>G>A. Furthermore, it is preferable to add polyethylene glycol of the same molecular weight as the PEG region during the ligation reaction.

[0263] Hereafter, an example of a translation template preferably used as a prey will be explained.

[0264] As a prey in cotranslation screening according to this embodiment, a protein of which C-terminal is modified with a translation template as represented in FIG. 9 (i.e., assigning molecule) is used. The translation template comprises a coding portion having information for translation into a protein and a PEG spacer portion. The coding portion has an A sequence at the 3' end, and the A sequence comprises a short poly-A sequence. The PEG spacer portion is characterized in that polyethylene glycol in the PEG region containing polyethylene glycol as a main component has a molecular weight of 400 or more, and the donor region and/or the CCA region contains at least one modification substance (F1 and/or F2). Further, the CCA region is characterized by having a function of binding by transpeptidation to a protein translated from the translation template, and the CCA region typically has puromycin. Further, it is characterized in that the modification substance (F1 and/or F2) immobilizes or labels with fluorescence the translation template and/or a protein translated from the translation template. As the immobilization substance, biotin and so forth are contemplated, and as the fluorescent substance, fluorescein, Cy5, rhodamine green (RhG) and so forth can be contemplated. The present invention relates to these coding portion, translation template, and libraries thereof, as well as a protein synthesized by translation on a ribosome (i.e., assigning molecule) and a library of such proteins (i.e., assigning molecules).

[0265] The prey is a protein synthesized by translation utilizing the translation template, of which C-terminal is modified with the translation template (FIG. 9, A, assigning molecule), and has characteristics in the translation template (FIG. 9, B) and the configuration of a protein of which C-terminal is modified with PEG (FIG. 9, C). It will be described in detail below.

[0266] The PEG spacer portion of the translation template (FIG. 9, B) is the same as that of the aforementioned translation template preferred for use as an bait except that it is characterized in that puromycin can bind with an amino acid. Further, the coding portion is also the same as that of the aforementioned translation template preferred for use as a bait. However, as for a configuration suitable for assignment, in particular, it is important to use an A sequence as the 3' end region, and this markedly increase the assignment efficiency of the total proteins and markedly decrease the amount of free proteins. Also in this case, if SP6+O29 and Flag+XhoI+An (n=8) are used as the 5' end region and the 3' end region of the coding portion, respectively, for example, the lengths of the 5' end region and the 3' end region are about 60 bp and about 40 bp, respectively, and thus they have such a length that they can be designed in primers for PCR as an adaptor region. This makes it possible to easily prepare a coding portion having a 5' end region and 3' end region according to this embodiment by PCR from any vector, plasmid, and cDNA library, and by ligating the PEG spacer portion, a translation template showing a high assignment efficiency can be obtained.

[0267] When the cording portion of the protein of which C-terminal is modified with PEG according to this embodiment (FIG. 9, C) is not used in detection of an interaction of proteins, i.e., when the protein is use for, for example, FCCS measurement, fluorescence reader, protein chip, and so forth, it may be intentionally cleaved with an RNase A or the like. By the cleavage, difficulty of detection of an interaction between proteins due to inhibition by the coding portion can be eliminated. Further, it is also possible to immobilize such a simple assigning molecule on a plate, bead, or slide glass.

[0268] The cell-free cotranslation will be explained with reference to FIG. 10. As shown in FIG. 10, a prey is translated in vitro in the presence of a bait. As shown in FIG. 10, A and B, there are a case where the bait is a protein, and it is translated simultaneously with the prey in a cell-free translation system, and a case where the bait is a nucleic acid, hormone or the like, and it is added to a cell-free translation system. As shown in FIG. 10, the prey is made into a fusion protein or assigning molecule.

[0269] The complex may be formed by binding of a bait and one prey (I), or by binding of another prey to a prey binding to a bait (II).

[0270] Because the detection method of the present invention enables in vitro formation of the complex, interactions between proteins, nucleic acid and protein, and so forth can be consistently detected in vitro.

[0271] When the bait is a protein, examples of the bait include a protein consisting only of a functional domain for an interaction with an objective protein, a protein including a functional domain, a protein of full length, and so forth. If a protein of full length is used, it is expected that it has multiple functional domains, and therefore it favorably becomes possible to more comprehensively detect preys. The protein of full length may be a single protein having a full length, or an assembly of two or more baits from which a protein of full length can be reconstructed.

[0272] The bait may be a complex as shown in FIG. 11, and this is called "complexed bait". By using such a complex, nonspecific adsorption can be further reduced, and it becomes possible to more comprehensively detect preys as the same effect as that of the full length protein.

[0273] As described above, as a complex contemplated for the cell-free cotranslation, a complex of a single bait and a single prey, a complex of a complexed bait and a prey, a complex of a bait and multiple preys, and a complex of a complexed bait and multiple preys are possible. Therefore, an interaction detectable by the detection method of the present invention includes not only a direct interaction between a bait and a prey, but also an indirect interaction for forming a complex.

[0274] It is considered that the most important factor in the cell-free cotranslation according to the present invention is that a protein is folded in a native state and in an undenatured state immediately after translation, a bait and a prey, a bait and another bait, or a prey and another prey, which should interact with each other, should coexist in a cell-free translation system, and thus they can promptly interact with each other. This is supported by the fact that a more superior result could be obtained by cotranslation compared with separate translation followed by coexistence by mixing. That is, it is considered that this is because a protein translated in vitro in a native folding state can encounter a protein, nucleic acid or the like, and therefore prompt formation of a complex by an interaction becomes possible.

[0275] The conventional methods of detecting an interaction require expression in E. coli and purification of a bait in a large amount. For example, when an interaction of a bait and a prey is expressed in a cell by the TAP method or the like, at least one month of preparation is needed. Further, the mRNA displaying method employing the pull-down method based on a GST fusion protein has problems that it takes at least 2 or 3 weeks because of the large amount expression in E. coli and purification of the bait, a substance that cannot be expressed in E. coli cannot be used as the bait, and so forth, and it further requires addition of bait in an amount 50 to 100 times the amount of prey to cause interaction with the prey. In the cell-free cotranslation, it becomes that only addition of almost equal weight of mRNA or DNA template to a cell-free translation system is sufficient, and it becomes completely unnecessary to express the bait in cells. Thus, the operation time can be markedly shortened. Furthermore, with a complexed bait or full length protein, an interaction of a bait and a prey can be further enhanced and made specific, and thus detection of nonspecific bonds can be avoided. Further, by using a complexed bait, a larger number of preys that interact with the second bait thereof can be comprehensively analyzed.

[0276] Although no system realizing complex formation by an interaction and screening consistently in vitro has existed so far, by performing translation and screening including those for a bait completely in vitro according to the detection method of the present invention described above, a system that can comprehensively detect interactions between proteins or between a protein and a nucleic acid with avoiding nonspecific detection can be constructed. Therefore, the present invention also provides a screening method utilizing the detection method of the present invention.

[0277] The screening method of the present invention is characterized by forming complexes by interactions of a bait and preys during cell-free cotranslation, and analyzing a prey that interacts with the bait by screening of the complexes. Therefore, the screening method of the present invention may be the same as an ordinary screening method for a prey that interacts with a bait comprising the detection step of detecting an interaction between a bait and prey and the selection step of selecting a prey for which an interaction is detected, except that the method comprises the detection step of detecting an interaction between a bait and a prey by the detection method of the present invention.

[0278] The screening method of the present invention further comprises the preparation step of preparing a prey selected in the selection step, and it is preferable to repeat the detection step, selection step and preparation step by using the prepared prey instead of or together with the bait used in the detection step. In this embodiment, the method is constituted by, for example, 1) the step of cell-free cotranslation in a cell-free translation system in which a prey and a bait cause an interaction, 2) the step of screening for detecting a prey interacting with the bait, 3) the step of examining and analyzing the prey, and 4) the step of repeating the steps from 1) by using the prey examined and analyzed in 3), as shown in FIG. 12. The steps of 1) and 2) correspond to the detection step and selection step, and the step of 3) corresponds to the preparation step. That is, the step of contacting a prey to the bait in the detection step corresponds to the step of the cell-free cotranslation, and the steps of detecting and selecting a complex in the detection step correspond to the step of screening.

[0279] In the screening method of the present invention, the prey selected in the selection step may be used again in the detection step.

[0280] In the screening method of the present invention, the cell-free cotranslation may be performed with a bait and a prey library, which is a group of multiple preys, so that two or more preys may be detected in the step of screening.

[0281] As shown in FIG. 11, a complexed bait and a prey may coexist, and a complex of the complexed bait and the prey may be formed by an interaction. By using a prey library in this cell-free cotranslation so that multiple kinds of preys of the prey library should coexist with the bait, and multiple complexes of the preys with the bait should be formed by interactions, multiple kinds of preys that interacts with the bait can be simultaneously and comprehensively detected in the screening. Further, if a full length protein is used as the bait, it becomes possible to comprehensively detect a larger number of preys, because a full length protein generally contains multiple functional domains for interactions.

[0282] Furthermore, as shown in FIG. 11, by forming multiple complexes of preys that interact with a complexed bait, multiple preys that interact with the complexed bait can be detected, and the second bait serves as a reinforcer of the interaction of a bait and a prey to realize a more specific interaction, which makes it possible to avoid nonspecific detection in the comprehensive detection. In evolutionary molecular engineering techniques such as the vitro virus method and the STABLE method, the prey is an assigning molecule (fusion). In the formation of complexes using a prey library or multiple kinds of preys, the preys may or may not directly interact with the bait.

[0283] When the complex obtained by the screening of complexes is an assigning molecule, a prey forming the complex may be detected by RT-PCR or PCR, and the screening may be performed again by using the PCR product as a prey (reconstruction of prey) or by using a prey analyzed from the PCR product as a new succeeding bait, as shown in FIG. 12. The method of performing the screening again by using the PCR product or performing screening by using a prey analyzed from the PCR product as a new succeeding bait can be performed only in the evolutionry molecular engineering techniques such as the in vitro virus method and the STABLE method, and cannot be carried out in a method of directly analyzing a protein such as the pull-down method and the TAP method.

[0284] When an assigning molecule is used, gene sequence of a proteinic prey can be known by RT-PCR or PCR after the screening. As shown in FIGS. 10 and 11, the proteinic prey referred to above is a prey interacting with a bait, a prey interacting that prey or the like, and all multiple kinds of preys interacting a bait can be comprehensively analyzed. When rescreening of a prey is further necessary, a DNA template, which is a product of RT-PCR or PCR, is transcribed, and the same cycles are repeated. Further, when a prey is determined by RT-PCR or PCR and the following sequence, it becomes possible to use that proteinic prey as a bait. If two or more kinds of preys that interact with the first bait are found, it becomes possible to form a complexed bait, and thus it becomes possible to detect a further larger number of preys.

[0285] If the cell-free cotranslation is used, it becomes possible to detect an interaction between proteins consistently in vitro even in the pull-down method or the TAP method. However, the assigning molecule is not formed in the TAP method, and therefore proteins must be directly analyzed in the analysis of preys. Then, if the pull-down method or the TAP method is used as the screening method in the in vitro virus method or the STABLE method, an assigning molecule is formed, and therefore gene sequence of a prey that causes interaction can be easily detected by RT-PCR or PCR in the analysis of the prey. Furthermore, if the cell-free cotranslation is used, it becomes possible to detect interactions between proteins consistently in vitro in the in vitro virus method or the STABLE method. Further, the number of preys is extremely large, the range of candidate preys can be narrowed down by rescreening performed by repeating the cycles. Further, the analyzed prey can be used as a bait in the next analysis, and if the number of preys increases, complexing of the bait advances, which results in detection of further preys. As described above, use of a prey as a bait in the subsequent cycle can be easily realized only in the in vitro virus method, STABLE method and so forth, which use an assigning molecule. However, the mRNA display method and the like requires synthesis in E. coli and purification of a large amount of GST fusion protein as a new bait, and thus preparation of the bait takes time, which makes the method difficult. If the cell-free cotranslation is used, such procedures are unnecessary, and the cycles can be easily repeated.

[0286] In the screening of complexes after the cell-free cotranslation, it is preferred that preys can be screened comprehensively without breaking the complexes produced by the cell-free cotranslation. For this purpose, a device for immobilization may be imparted to the bait with an affinity tag or the like so as to detect a prey that interacts with the bait. Any kind of such a device for immobilization may be used. Examples include, for example, a method of performing two-stage screening using IgG-protein A affinity or calmodulin beads as in the conventional TAP method, and a method of performing one- or two-stage screening using streptavidin or avidin/biotin affinity, GST-tag, Flag-tag, T7-tag, His-tag or the like as in the pull-down method.

[0287] Examples of the prey library include a cDNA library (random priming library, dT priming library), random library, peptide library, hormone library, antibody library, ligand library, pharmaceutical compound library, and so forth, and any kind of library may be used. For example, if a random priming cDNA library is used as the prey library, although a full length prey cannot be expected for this library, a prey containing a functional domain can be expected. If such a library is used especially for screening using combinations with a complexed bait or full length protein, it becomes effective for comprehensive detection of preys.

[0288] Examples of the random priming library include cDNAs obtained by random priming and incorporated into multi-cloning sites (MCS) of vectors having a 5' untranslation region (UTR) containing the promoter of the RNA polymerase of SP6 (SP6) as a transcription promoter and a part of TMV omega sequence (O29) of the tobacco mosaic virus as a translation enhancer on the 5' end side of MCS, and containing a sequence for the Flag-tag, which is a tag for affinity separation analysis based on an antigen-antibody reaction, as an affinity tag sequence on the 3' end side of MCS, so that the Flag-tag should be added to the C-terminal of a protein expressed from an insert sequence incorporated into MCS.

[0289] The aforementioned detection method of the present invention includes the step of contacting a bait and a prey to form a complex. Therefore, a method of forming a complex of a bait and a prey that interacts with the bait is provided according to this step.

[0290] The formation method of the present invention is characterized by using the protein of the present invention as a bait in the formation of a complex of a bait and a prey, which is a protein that interacts with the bait, and preferably further labeling the bait and prey for detection and modifying them for separation in particular schemes, to perform cell-free cotranslation. Therefore, a preferred configuration of the formation method of the present invention may be the same as that of an ordinary method of forming a complex of a bait and a prey comprising contacting a bait and a prey that interacts with the bait, except that the bait and prey are labeled for detection and modified for separation in particular schemes, and the cell-free cotranslation is performed. The labeling for detection and modification for separation of the bait and prey in particular schemes as well as the cell-free cotranslation may be the same as those explained for the detection method of the present invention.

[0291] In the formation method of the present invention, not only a complex of a bait and a prey, for which interaction is known, but also a complex comprising elements for which interaction is unknown can be formed by performing the step of contacting the bait with a prey that interacts with the bait by contacting a bait with a prey library consisting of multiple kinds of preys.

[0292] Other methods for utilizing the protein of the present invention include the followings:

[0293] a method for analyzing an interaction between a protein and a substance, which is performed by fluorescence correlation spectroscopy, fluorescent imaging analysis method, fluorescence resonance energy transfer method, evanescent field molecular imaging method, fluorescence depolarization method, surface plasmon resonance method or enzyme linked immunosorbent assay using the protein of the present invention,

[0294] a method for detecting an interaction between a protein and a substance, which uses the protein of the present invention and detects the interaction by amplification of a nucleotide sequence of a coding portion bound to the C-terminal of the protein of the present invention,

[0295] a method for detecting an interaction between a protein and a substance, which uses the protein of the present invention and uses the cell-free cotranslation method or the cell-free cotranslation screening method,

[0296] a method for detecting an interaction between a protein and a substance, which uses the protein of the present invention and labels the protein with fluorescence and/or immobilizes the protein,

[0297] a method for analyzing an interaction of a protein or substance in vitro by using the protein of the present invention,

[0298] a method for analyzing an interaction of a protein or substance, which uses the protein of the present invention, and utilizes the cotranslation method in vitro,

[0299] a method for analyzing an interaction of a protein or substance in vivo by using the protein of the present invention, and

[0300] the aforementioned methods for analyzing an interaction, which uses a nucleic acid encoding the protein of the present invention.

[0301] Further, the followings are also mentioned:

[0302] a method for analyzing an interaction between a protein and a target molecule, which uses a C-terminal modified protein comprising the protein and a modification agent binding to the C-terminal of the protein. The analysis of the interaction can be carried out by fluorescence correlation spectroscopy, fluorescent imaging analysis method, fluorescence resonance energy transfer method, evanescent field molecular imaging method, fluorescence depolarization method, surface plasmon resonance method or enzyme linked immunosorbent assay. The C-terminal modified protein may be immobilized. The C-terminal modified protein may be added on an array on which the target molecule is immobilized, and then the C-terminal modified protein specifically binding to the target molecule may be detected.

[0303] In the analysis method of this embodiment, the interaction is usually analyzed by contacting the modified protein of the present invention obtained above and the target molecule in a suitable combination selected depending on the type of the modification substance or reaction system and measuring change of a signal generated by the interaction between the modified protein and the target molecule among signals generated by the modified protein or the target molecule. The analysis of the interaction is carried out by, for example, fluorescence correlation spectroscopy, fluorescent imaging analysis method, fluorescence resonance energy transfer method, evanescent field molecular imaging method, fluorescence depolarization method, surface plasmon resonance method or enzyme linked immunosorbent assay. The details of these methods are explained below.

[0304] The "target molecule" means a molecule that interacts with the modified protein of the present invention, and it may be specifically a protein, nucleic acid, sugar chain, low molecular weight compound or the like, preferably a protein or DNA.

[0305] The protein is not particularly limited so long as it has an ability to interact with the modified protein of the present invention, and it may be a protein of full length or a partial peptide containing an active site for binding. Further, it may be a protein of which amino acid sequence or function is known or unknown. It may be a synthesized peptide chain, a protein purified from an organism, a protein obtained by translation from a cDNA library using a suitable translation system and purification, or the like, and they can be used as the target molecule. The synthesized peptide chain may be a glycoprotein consisting of a synthesized peptide chain attached with a sugar chain. Among these, a purified protein of which amino acid sequence is known or a protein obtained by translation from a cDNA library and purification using suitable methods can be preferably used.

[0306] The nucleic acid is not particularly limited so long as it has an ability to interact with the modified protein of the present invention, and either DNA or RNA may be used. Further, it may be a nucleic acid of which nucleotide sequence or function is known or unknown. Preferably, a nucleic acid of which function as a nucleic acid having an ability to bind to a protein or nucleotide sequence is known or a nucleic acid obtained by cleavage with a restriction enzyme or the like and isolation from a genomic library or the like can be used.

[0307] The sugar chain is not particularly limited so long as it has an ability to interact with the modified protein of the present invention, and it may be a sugar chain of which saccharide sequence or function is known or unknown. Preferably, an already isolated and analyzed sugar chain of which saccharide sequence or function is known is used.

[0308] The low molecular weight compound is not particularly limited so long as it has an ability to interact with the modified protein of the present invention. A compound of which function is unknown or a compound of which ability to bind to a protein is already known may also be used.

[0309] The "interaction" caused by these targets molecules with the modified protein of the present invention usually means an action caused by an intermolecular force generated by at least one of covalent bond, hydrophobic bond, hydrogen bond, van der Waals binding and binding caused by electrostatic force between a protein and a target molecule. However, this term should be construed in its broadest sense, and it should not be construed in any limitative way. The covalent bond includes a coordinate bond and dipole bond. The binding caused by electrostatic force includes, besides electrostatic bond, electric repulsion. Further, a bonding reaction, synthetic reaction and decomposition reaction caused as a result of the aforementioned action are also included in the interaction.

[0310] Specific examples of the interaction include association and dissociation of an antigen and an antibody, association and dissociation of a protein receptor and a ligand, association and dissociation of an adhesion molecule and a partner molecule, association and dissociation of an enzyme and a substrate, association and dissociation of a nucleic acid and a protein binding to it, association and dissociation of proteins in an information transmission system, association and dissociation of a glycoprotein and a protein and association and dissociation of a sugar chain and a protein.

[0311] The target molecule to be used may be modified with a modification substance and used depending on embodiments. The modification substance is usually selected from nonradioactive modification substances such as fluorescent substances. The fluorescent substances may be any of various fluorescent dyes of, for example, fluorescein type, rhodamine type, Cy3, Cy5, eosine type, NBD type and so forth, which have a free functional group (e.g., carboxyl group, hydroxyl group, amino group etc.) and can bind to the aforementioned target substance such as proteins and nucleic acids. In addition, other compounds such as dyes may be used, and type and size of the compounds are not critical so long as they enable the modification.

[0312] Among these modification substances, a substance suitable for the method of measurement or analysis of change of signal generated due to an interaction between the target molecule and the modified protein of the present invention is used.

[0313] The aforementioned modification substance can be bound to the target molecule by a suitable method known per se. Specifically, when the target molecule is a protein, the method of modifying the C-terminal described in WO02/48347 or the like may be used. Further, when the target molecule is a nucleic acid, it can by easily modified by a method of performing PCR using an oligo DNA primer bound with a modification substance beforehand via a covalent bond or the like.

[0314] Further, the modified protein of the present invention or the target molecule used for present invention may be bound to a solid phase (i.e., immobilized) depending on the embodiment. As the method for binding to a solid phase, there are a method of binding it via the modification substance and a method of binding it via another portion.

[0315] The modification substance used in binding via the modification substance is usually a molecule specifically binding to a particular polypeptide (henceforth also referred to as a "ligand"), and a particular polypeptide binding to the ligand (henceforth also referred to as an "adaptor protein") is bound to the solid phase. The adaptor protein also includes binding proteins, receptor proteins constituting receptors, antibodies and so forth.

[0316] Examples of combinations of the adaptor protein and the ligand include any of various receptor proteins and a ligand thereof, for example, a biotin or iminobiotin binding protein such as avidin and streptavidin and biotin or iminobiotin, maltose binding protein and maltose, G protein and guanine nucleotide, polyhistidine peptide and metal ion such as nickel or cobalt ion, glutathione-S-transferase and glutathione, DNA binding protein and DNA, antibody and antigen molecule (epitope), calmodulin and calmodulin binding peptide, ATP binding protein and ATP, estradiol receptor protein and estradiol and so forth.

[0317] Among these, preferred combinations of the adaptor protein and the ligand are biotin or iminobiotin binding protein such as avidin and streptavidin and biotin or iminobiotin, maltose binding protein and maltose, polyhistidine peptide and metal ion such as nickel or cobalt ion, glutathione-S-transferase and glutathione, antibody and antigen molecule (epitope) and so forth, and a combination of streptavidin and biotin or iminobiotin is the most preferred. These binding proteins per se are known, and DNAs coding these proteins have already been cloned.

[0318] The adaptor protein can be bound to a solid phase surface by using a method known per se. Specifically, for example, there can be used a method of utilizing tannic acid, formalin, glutaraldehyde, pyruvic aldehyde, bis-diazotized benzizone, toluene-2,4-diisocyanate, amino group, carboxyl group that can be converted into an active ester group, hydroxyl group or amino group that can be converted into phosphoramidite group, or the like.

[0319] When the binding is attained via a portion other than the modification substance, there can be used a known method usually used for binding a protein, nucleic acid, sugar chain or low molecular weight compound to a solid phase. Specifically, there can be used, for example, a method of utilizing tannic acid, formalin, glutaraldehyde, pyruvic aldehyde, bis-diazotized benzizone, toluene-2,4-diisocyanate, amino group, carboxyl group that can be converted into an active ester group, hydroxyl group or amino group that can be converted into phosphoramidite group, or the like.

[0320] The solid phase may be one usually used for immobilizing a protein, nucleic acid or the like, and material and shape thereof are not particularly limited. For example, glass plates, nitrocellulose membranes, nylon membranes, polyvinylidene fluoride membranes, microplates made of plastics and so forth can be used.

[0321] The "measurement" is a means for collecting changes of signals used for analysis, and it should not be construed in any limitative way. As the measurement method used, any of methods that can detect an intermolecular interaction can be used, including fluorescence correlation spectroscopy, fluorescence resonance energy transfer method, evanescent field molecular imaging method, fluorescence depolarization method, fluorescence imaging analysis method, surface plasmon resonance method, enzyme linked immunosorbent assay and so forth.

[0322] The measurement method includes a method comprising adding the modified protein of the present invention onto an array on which a target molecule is immobilized and detecting the modified protein of the present invention specifically binding to the target molecule. The array on which the target molecule is immobilized means a solid phase on which the target molecule is immobilized in an arrangement enabling identification thereof. The method for detecting the modified protein of the present invention specifically binding to the target molecule is not particularly limited, so long as the method enables detection of the modified protein of the present invention specifically binding to the target molecule. However, there is usually used, for example, a method of removing the modified protein of the present invention not binding to the target molecule by washing from the array to which the modified protein of the present invention is added and detecting the remaining modified protein of the present invention.

[0323] Hereafter, examples of the measurement method will be explained.

(1) Fluorescence Correlation Spectroscopy

[0324] The fluorescence correlation spectroscopy (FCS, Eigen, M., et al., Proc. Natl. Acad. Sci., USA, 91, 5740-5747 (1994)) is a method of measuring flow rate, diffusion coefficient, volume shrinkage or the like of particles under a confocal laser microscope or the like. In the present invention, interacting molecules can be measured by measuring change of translational Brownian movement of one original modified molecule of the present invention (C-terminal modified protein) caused by an interaction between the modified protein and a target molecule.

[0325] Specifically, fluorescence emitted from sample particles in a partial volume of a sample solution due to excitation of the sample particles by an excitation light is measured to obtain a photon ratio. This value changes with the number of the particles existing in a space volume observed during a specific period of time. The aforementioned various parameters can be calculated from the change of signals using an autocorrelation function. Apparatuses for carrying out FCS are also marketed from Carl Zeiss and so forth, and analysis can be performed by using these apparatuses also in the present invention.

[0326] When a protein-target molecule interaction is measured or analyzed by using this method, it is required to provide both of the C-terminal modified protein and the target molecule as solutions (liquid phase method). The target molecule does not need to be labeled. Further, a molecule having a molecular weight extremely smaller than that of the C-terminal modified protein of which interaction should be investigated is not suitable for this method, since such a molecule does not affect the Brownian movement of the C-terminal modified protein.

[0327] However, fluorescence cross-correlation spectroscopy (FCCS) using two kinds of fluorescent dyes can detect even an interaction between proteins having molecular weights of similar order, of which detection is difficult by FCS using one kind of fluorescent dye. Although the fluorescence resonance energy transfer (FRET) method is known as another method of using two kinds of fluorescent dyes, two kinds of fluorescent dyes need to approach each other at a distance within 40 to 50 .ANG. in order to cause FRET, and there is a risk in this method that FRET may not be observed depending on sizes of proteins, locations at which the fluorescent dyes are attached or the like, even an interaction occurs. On the other hand, since the detection of cross-correlation does not depend on the distance between the fluorescent dyes in the FCCS method, it does not suffer from such a problem. Further, comparing with the fluorescence depolarization method as another detection system, the FCCS method has advantages of a smaller amount of required sample, shorter detection time, easier automatization for HTS and so forth. Further, since the FCCS method provides extremely fundamental information such as size and number of fluorescence-labeled molecules, it may be used for general purpose like the surface plasmon resonance method. The difference between the both is that, in the surface plasmon resonance method, an interaction is detected in the state that proteins are immobilized, whereas the FCCS method enables observation of interaction in a solution, which is closer to a natural state. In the FCCS method, although proteins do not need to be immobilized, the proteins must be labeled with fluorescent dyes instead. However, it has been made possible by the present invention to overcome this problem.

[0328] Further, the FCCS method enables investigation of a protein-protein interaction or protein-nucleic acid interaction in a state of solution, which is close to the intracellular environment, and enables convenient calculation of dissociation constant (binding constant) by one measurement.

[0329] The method for bringing a target molecule into contact with the C-terminal modified protein in this method may be any method that allows the contact in a sufficient degree such that they can interact with each other. However, it is preferably attained by a method of introducing a solution dissolving the C-terminal modified protein in a buffer usually used for biochemical purpose or the like at an appropriate concentration into a well for measurement in a commercially available FCS apparatus and further introducing a solution dissolving the target molecule in the same buffer at an appropriate concentration into the well.

[0330] In this method, as a method of performing multiple analyses, for example, there is used a method of introducing multiple kinds of different C-terminal modified proteins into wells for measurement in the aforementioned FCS apparatus, respectively, and further introducing a solution of a particular target molecule into the wells, or introducing a particular C-terminal modified protein into wells, and further introducing solutions of multiple kinds of different target molecules into the wells, respectively.

(2) Fluorescence Imaging Analysis Method

[0331] The fluorescence imaging analysis method is a method of bringing a modifying molecule into contact with an immobilized molecule and measuring or analyzing fluorescence emitted by the immobilized modifying molecule remained on the immobilized molecule due to an interaction between the both molecules using a commercially available fluorescence imaging analyzer.

[0332] When a protein-target molecule interaction is measured or analyzed by using this method, one of the C-terminal modified protein or the target molecule must be immobilized by the aforementioned method. When an immobilized target molecule is used, either a modified or unmodified target molecule can be used. Further, when it is used without immobilization, it must be modified with the aforementioned modification substance. Either a C-terminal modified protein immobilized at the modified portion or a C-terminal modified protein immobilized at a portion other than the modified portion may be used.

[0333] As a substrate for immobilizing a C-terminal modified protein or target molecule (solid phase), there can be used glass plates, nitrocellulose membranes, nylon membranes, microplates made of plastics and so forth, which are usually used for immobilizing a protein, nucleic acid or the like. Further, such substrates as mentioned above of which surfaces are bound with various functional groups (amino group, carboxyl group, thiol group, hydroxyl group etc.) or various ligands (biotin, iminobiotin, metal ions such as nickel or cobalt ion, glutathione, saccharides, nucleotides, DNA, RNA, antibody, calmodulin, receptor protein etc.) can also be used.

[0334] The method for bringing a modified target molecule or a C-terminal modified protein into contact with an immobilized molecule in this method may be any method that allows the contact in a sufficient degree such that the both molecules can interact with each other. However, it is preferably attained by a method of preparing a solution dissolving the modified target molecule or the C-terminal modified protein in a buffer usually used for biochemical purpose at an appropriate concentration and bringing the solution into contact with the solid phase surface.

[0335] After bringing the both molecules into contact with each other, a step of washing off excessively existing modified target molecule or C-terminal modified protein with the same buffer or the like is preferably performed, and fluorescence signal emitted from the modification substance of the target molecule or C-terminal modified protein which remained on the solid phase, or a mixed signal of fluorescence emitted from the immobilized modified molecule and fluorescence emitted from the modified molecule remained on the solid phase can be measured or analyzed by using a commercially available imaging analyzer to identify the molecule that interacts with the immobilized molecule.

[0336] In this method, as a method of simultaneously performing multiple analyses, for example, there is used a method of immobilizing multiple kinds of C-terminal modified proteins or modified or unmodified target molecules on the aforementioned solid phase surface with positioning addresses, a method of bringing multiple kinds of non-immobilized C-terminal modified proteins or modified target molecules into contact with one kind of C-terminal modified protein or modified or unmodified target molecule, or the like. When multiple kinds of C-terminal modified proteins or modified target molecules are brought into contact, the molecules remained on the solid phase can be obtained by dissociating them using difference of buffer concentration or the like and analyzed by a known method to identify them.

(3) Fluorescence Resonance Energy Transfer Method

[0337] As another intermolecular interaction detection method using two kinds of fluorescent dyes, the fluorescence resonance energy transfer (FRET) method is well known. FRET means a phenomenon that, if a fluorescence spectrum of one of two kinds of fluorescent dyes (energy donor) and an absorption spectrum of the other (energy receptor) overlap, and the distance between two of the fluorescent dyes is sufficiently small, it becomes more likely that excitation energy of the donor excites the receptor before the donor emits fluorescence. Therefore, two kinds of proteins of which interaction is desired to be detected are labeled with fluorescent dyes serving as the donor and the receptor, respectively, and the donor is excited. When the two kinds of proteins do not interact with each other, FRET is not caused because the distance between the fluorescence dyes is large, and thus fluorescence spectrum of the donor is observed. However, if the two kinds of proteins interact with each other, and hence the distance between the fluorescent dyes becomes smaller, fluorescence spectrum of the receptor is observed due to FRET. Therefore, presence or absence of an interaction between the proteins can be determined on the basis of difference in wavelengths of fluorescence spectra. As for the fluorescent dyes, a combination of fluorescein as the donor and rhodamine as the receptor is frequently used. Further, it is recently attempted to observe FRET in a cell to detect an interaction by using combination of mutant green fluorescence proteins (GFP) emitting fluorescence of different wavelengths. As a drawback of this method, it is mentioned that since two kinds of fluorescent dyes need to approach to each other at a distance within 40 to 50 .ANG. in order to cause FRET, there is a risk that FRET may not be observed depending on sizes of proteins, locations at which the fluorescent dyes are attached or the like, even if an interaction occurs.

(4) Evanescent Field Molecular Imaging Method

[0338] The evanescent field molecular imaging method is a method described in Funatsu, T., et al., Nature, 374, 555-559 (1995) or the like, and it is a method of bringing a second molecule as a solution into contact with a molecule immobilized on a transparent material such as glass, irradiating them with a laser light or the like from a light source at such an angle that an evanescent field should be generated, and measuring or analyzing the generated evanescent light using a detector. These operations can be performed by using an evanescent field fluorescence microscope known per se.

[0339] When a protein-target molecule interaction is measured or analyzed by using this method, one of the C-terminal modified protein or the target molecule must be immobilized by the aforementioned method. When an immobilized target molecule is used, it does not need to be modified. However, when it is used without immobilization, it must be modified with the aforementioned modification substance.

[0340] As the substrate for immobilizing the C-terminal modified protein or target molecule, a substrate made of a material of glass or the like is used, and quartz glass is preferably used. Further, a substrate of which surface is cleaned by ultrasonication is preferred in order to prevent scatter of laser light or the like.

[0341] The method for bringing a non-immobilized C-terminal modified protein or target molecule into contact with an immobilized molecule in this method may be any method that allows the contact in a sufficient degree such that the both molecules can interact with each other. However, a method of preparing a solution dissolving the non-immobilized C-terminal modified protein or modified target molecule in a buffer usually used for biochemical purpose at an appropriate concentration and adding the solution dropwise to the solid phase surface is preferred.

[0342] After bringing the both molecules into contact with each other, fluorescence generated through excitation by the evanescent field illumination can be measured by using a detector such as a CCD camera to identify the molecule that interacts with the immobilized molecule.

[0343] In this method, as a method of simultaneously performing multiple analyses, for example, there is used a method of immobilizing multiple kinds of C-terminal modified proteins or modified target molecules on the aforementioned substrate with positioning addresses, or the like.

(5) Fluorescence Depolarization Method

[0344] The fluorescence polarization method (Perran, J., et al., J. Phys. Rad., 1, 390-401 (1926)) is a method utilizing the fact that a fluorescent molecule excited with a polarized fluorescent light emits fluorescence in the same plane of polarization during the excited state while it maintains a stationary state, whereas the emitted fluorescence has a plane different from that of the excitation light when the excited molecule undergoes rotational Brownian movement or the like during the excited state. The movement of molecule is affected by the size thereof, and when the fluorescent molecule is a macromolecule, the molecule scarcely shows movement during the excited state, and emitted light is maintained to be a polarized light. However, in the case of a low molecular weight fluorescent molecule, since it shows high moving velocity, the emitted light is depolarized. Therefore, if intensity of the fluorescence emitted from a fluorescent molecule excited by a plane polarized light is measured along the original plane and a plane perpendicular thereto, information of motility and existing state of the molecule can be obtained from a ratio of the fluorescence intensities for the both planes. According to this method, behavior of a target molecule that interacts with a fluorescence-modified molecule can be traced without being affected by contaminants, if any. This is because shift of polarization degree is measured only when the fluorescence-modified molecule and the target molecule interact with each other.

[0345] As apparatuses for carrying out this method, BECON (produced by Panyera) and so forth are marketed, and this method can be carried out by using these apparatuses.

[0346] When a protein-target molecule interaction is measured or analyzed by using this method, it is required to provide both of the C-terminal modified protein and the target molecule as solutions. The target molecule does not need to be modified. Further, a molecule having a molecular weight extremely smaller than that of the C-terminal modified protein of which interaction should be investigated is not suitable for this method, since such a molecule does not affect the Brownian movement of the C-terminal modified protein.

[0347] The method for bringing a target molecule into contact with the C-terminal modified protein in this method may be any method that allows the contact in sufficient degree such that they should interact with each other. However, it is preferably attained by a method of introducing a solution dissolving the C-terminal modified protein in a buffer usually used for biochemical purpose at an appropriate concentration into a well for measurement in a commercially available fluorescence depolarization apparatus and further introducing a solution dissolving the target molecule in the same buffer at an appropriate concentration into the well.

[0348] It is expected that specificity of interaction between the C-terminal modified protein and the target molecules to be measured in this method is not necessarily so high as that of an antigen-antibody reaction. Therefore, in order to identify an optimum combination, it is effective that degree of interaction should be numerically defined. As an index representing degree of interaction, for example, a value of the minimum target substance concentration providing the maximum fluorescence polarization degree for a C-terminal modified protein of a fixed concentration or the like can be used.

[0349] In this method, as a method of simultaneously performing multiple analyses, for example, there is used a method of introducing multiple kinds of different C-terminal modified proteins into wells for measurement in the aforementioned fluorescence depolarization apparatus, respectively, and further introducing a solution of a particular target molecule into the wells, or introducing a particular C-terminal modified protein into wells and further introducing solutions of multiple kinds of different target molecules into the wells, respectively.

(6) Surface Plasmon Resonance Method

[0350] The surface plasmon resonance method is a method of measuring surface plasmon excited by a molecule interacting at a metal/liquid interface as change of intensity of reflected light (Cullen, D. C., et al., Biosensors, 3 (4), 211-225 (1987-88)). When a protein-target molecule interaction is measured or analyzed by using this method, the C-terminal modified protein must be immobilized by the aforementioned method, but the target molecule does not need to be modified.

[0351] As a substrate for immobilizing the C-terminal modified protein, a transparent substrate made of glass or the like on which a thin film of metal such as gold, silver or platinum is formed is used. The transparent substrate may be any of those usually used for surface plasmon resonance apparatuses. It generally consists of glass as a substrate consisting of a material transparent to a laser light, and such a substrate having a thickness of about 0.1 to 5 mm is generally used. Further, thickness of the metal thin film is suitably about 100 to 2000 .ANG.. Those marketed as such immobilization substrates for surface plasmon resonance apparatuses can also be used. The C-terminal modified protein can be immobilized on the substrate by the method described above.

[0352] The method for bringing a target molecule into contact with the C-terminal modified protein in this method may be any method that allows the contact in a sufficient degree such that the both molecules can interact with each other. However, a method of bringing the immobilized C-terminal modified protein into contact with a solution dissolving the target molecule in a buffer usually used for biochemical purpose at an appropriate concentration can be preferably used.

[0353] These steps may also be performed by using a commercially available surface plasmon resonance apparatus, for example, BIAcore 2000 (produced by Pharmacia Biosensor). After bringing the both molecules into contact with each other, change with time of relative intensity of each reflected light can be measured by using a surface plasmon resonance apparatus known per se to analyze or measure an interaction of the immobilized C-terminal modified protein and the target molecule.

[0354] In this method, as a method of simultaneously performing multiple analyses, for example, there is used a method of immobilizing multiple kinds of C-terminal modified proteins on a substrate used for the surface plasmon resonance apparatus with positioning addresses, a method of bringing multiple kinds of target molecules into contact with one kind of immobilized C-terminal modified protein, or the like.

(7) Enzyme Linked Immunosorbent Assay

[0355] The enzyme linked immunosorbent assay (ELISA, Crowther, J. R., Methods in Molecular Biology, 42 (1995)) is a method of bringing a solution containing an antibody into contact with an antigen immobilized on a solid phase and measuring or analyzing the antibody remaining on the immobilized antigen due to the interaction between the both molecules (antigen-antibody reaction) on the basis of fluorescence emitted from a modification molecule (IgG etc.) specifically binding to the antibody or a signal emitted by a dye formed from the modification molecule as a substrate using a commercially available detector (ELISA reader).

[0356] When a protein-target molecule interaction is measured or analyzed by using this method, the C-terminal modified protein serving as the antigen must be immobilized by the aforementioned method. Further, the target molecule serving as the antibody must be modified with the aforementioned modification substance.

[0357] As a substrate for immobilizing the C-terminal modified protein serving as the antigen, microplates made of plastics usually used for ELISA and so forth can also be used.

[0358] The method for bringing the modified target molecule serving as the antibody into contact with an immobilized molecule in this method may be any method that allows the contact in a sufficient degree such that the both molecules can interact with each other. However, a method of preparing a solution dissolving the modified target molecule in a buffer usually used for biochemical purpose at an appropriate concentration and introducing the solution into a microplate is preferred.

[0359] After bringing the both molecules into contact with each other, a step of washing off excessively existing modified molecule not binding to the immobilized molecule is preferably performed, and fluorescence emitted from the modified molecule remained on the solid phase can be measured or analyzed by using a commercially available ELISA reader or the like to identify the molecule that interacts with the immobilized antigen molecule.

[0360] In this method, as a method of simultaneously performing multiple analyses, for example, there is used a method of immobilizing multiple kinds of different modified target molecules in each well of the aforementioned microplate.

[0361] Further, the protein of the present invention can also be used for identification of a molecule that causes an interaction.

[0362] When primary structure of a target molecule for which an interaction with a C-terminal modified protein is recognized on the basis of measurement according to any one of the methods described above is unknown, the primary structure can be analyzed by a suitable method known per se. Specifically, when the target molecule for which an interaction is recognized is a protein, its amino acid sequence can be analyzed by using an amino acid analyzer etc. to identify the primary structure. Further, when the target molecule is a nucleic acid, nucleotide sequence can be determined by a nucleotide sequence determination method using an automatic DNA sequencer or the like.

[0363] Furthermore, the protein of the present invention can also be used for analysis of an interaction with a protein library.

[0364] The present invention provides a gene or nucleic acid sequence encoding a novel protein that can form a complex with the c-Fos protein, which was obtained by performing cotranslation selection/screening of IVV using the c-Fos protein as the bait and a mouse brain cDNA library as the prey, and methods for utilizing them. The present invention also provides a method for utilizing a gene or nucleic acid sequence encoding a known protein, which is not known to form a complex with the c-Fos protein.

[0365] The present invention not only enables screening of known gene sequences and known nucleic acid sequences, but also can provide a novel protein having a novel amino acid sequence formed by unexpected frame shift, a novel protein having a nucleic acid sequence for which only the nucleic acid sequence is published on the basis of genome information, or a novel protein having a completely novel nucleic acid sequence, further, a protein that forms a complex by an unexpected indirect interaction in addition to a direct interaction, a gene or nucleic acid sequence encoding the protein, and methods for utilizing them.

EXAMPLES

[0366] Hereafter, the amino acid sequences of the proteins of the present invention and the sequences of the nucleic acids encoding them will be specifically described. However, the following examples should be construed as a mere aid for specifically understanding the present invention, and the scope of the present invention is no way limited by the following examples.

Example 1

[0367] Cotranslation selection/screening of IVV was carried out by using the c-Fos protein as a bait and a mouse brain cDNA library as a prey (FIG. 2), and as a result, genes or nucleic acid sequences encoding novel proteins that can form a complex with the c-Fos protein were obtained.

[0368] The preparation method of the bait, c-Fos protein, was as follows. A DNA template was prepared from a pCMV-FosCBPzz vector (SEQ ID NO: 168) by PCR (primers 5' SP6(O29)T7-FosCBPzz (SEQ ID NO: 169) and 3' FosCBPzz (SEQ ID NO: 170), and PCR program CYCB1 (refer to Table 1)) using TaKaRa Ex Taq (Takara Shuzo). The DNA template was transcribed (37.degree. C., 2 hours) by using RiboMAX.TM. Large Scale RNA Production Systems (Promega) to prepare a mRNA template of the bait c-Fos protein. A bait DNA made to coexist was prepared by PCR (primers 5' DNA (SEQ ID NO: 172) and 3' DNA (SEQ ID NO: 173)) using DNA-Fos/Jun (SEQ ID NO: 171) containing the Fos/Jun binding sequence as a template according to the PCR program V-2 (refer to Table 1).

[0369] The preparation method of the mouse brain cDNA library as the prey was as follows. An IVV random library was prepared as shown in FIG. 3. As an RNA library, a commercially available mouse brain (polyA+) RNA library (obtained by purifying a tissue extracted RNA library in an oligo dT column, Clontech) was purchased. As for design of an adaptor, it was designed so as to add a 5' UTR sequence suitable for the production of assigning molecules (promoter SP6+enhancer O29 or O') to the library as a sequence required for IVV formation. For the mouse brain (polyA+) RNA library, an adaptor having the enhancer O29 was used. The main chain (SEQ ID NO: 174 or 175) and the subchain (gaattcgc or ggaattcg) of the adaptor for the enhancer O29 were each dissolved in the TE buffer (10 mM Tris-Cl, pH8.0, 1 mM EDTA) at a concentration of 100 .mu.M, and 10 .mu.l each of the solutions of the main chain and subchain were mixed so that the main chain and the subchain were mixed in equimolar amounts. The mixture was heated at 90.degree. C. for 2 minutes and at 70.degree. C. for 5 minutes, set on a water bath of 60.degree. C., and then slowly cooled from 60.degree. C. to room temperature by turning off the heater of the bath. The mixture was divided into 5 .mu.l aliquots, and stored at -20.degree. C. Then, the mouse brain (polyA+) RNA library was reverse-transcribed into single stranded DNAs (FIG. 3, I). 0.5 .mu.g of the mouse brain (polyA+) RNA library (1.4 pmole/0.5 .mu.g), 2 pmol of 3' random primer (SEQ ID NO: 176) and DEPC water were added to obtain a volume of 12.0 .mu.l, and the mixture was heated at 70.degree. C. for 10 minutes, and cooled for 1 minute on ice. A reverse transcription reaction was performed at 45.degree. C. for 1 hour by using this mixture and SuperScriptII RT (SuperScript Double-stranded cDNA Synthesis Kit, Invitrogen). Then, the total amount of the single stranded DNAs synthesized by the reverse transcription reaction was used for a reaction with an E. coli DNA ligase, E. coli Polymerase I and E. coli RNase H (SuperScript Double-stranded cDNA Synthesis Kit, Invitrogen) at 16.degree. C. for 2 hours, and the product was blunt-ended with T4 DNA polymerase at 16.degree. C. for 5 minutes to synthesize double-stranded DNAs (FIG. 3, II). Then, the adaptor previously prepared was ligated by taking advantage of the fact that the 5' ends of the double-stranded DNAs were phosphorylated (FIG. 3, III). The synthesized double-stranded DNA library was subjected to ethanol precipitation, and dissolved in 4 .mu.l of DEPC water. 100 .mu.M of the prepared adaptor in a volume of 1.0 .mu.l and 50 .mu.l of Ligation High (TOYOBO) were added thereto, reacted overnight at 16.degree. C., purified (DNA purification kit, QIAGEN), and then adjusted to a volume of 50 .mu.l. Thereafter, PCR (EX Taq Hot Start Version, TaKaRa) was performed (FIG. 3, IV). Out of 50 .mu.l of the ligated double-stranded DNA library, 2 .mu.l was used as a template together with 5' PCR primer (SEQ ID NO: 172) having a specific sequence required for IVV (O29) and 3' PCR primer (SEQ ID NO: 173) to prepare an IVV cDNA library. As for the PCR conditions, the total volume was 100 .mu.l, and 22 cycles of the reactions (each cycle consists of reactions at 94.degree. C. for 30 seconds, at 60.degree. C. for 30 seconds, and at 72.degree. C. for 90 seconds, and the final extension reaction was performed at 72.degree. C. for 180 seconds).

[0370] Cotranslation (26.degree. C., 60 minutes) of the mRNA template of the bait c-Fos protein, the mouse brain cDNA library as the prey, and the bait DNA made to coexist was carried out in a cell-free translation system of wheat (Wheat Germ Extract, Promega) in a volume of 50 .mu.l. To 50 .mu.l of the sample, 50 .mu.l of IgG binding buffer (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.1% NP40) was added to obtain the total volume of 100 .mu.l (cotranslation sample). Then, IgG agarose (Sigma) was washed twice with the IgG binding buffer, and the cotranslation sample (100 .mu.l) was added thereto, and the mixture was stirred by rotation at 4.degree. C. for 2 hours. The IgG agarose was washed 3 times with the binding buffer and once with a TEV cleaving buffer (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.1% NP40, 0.5 mM EDTA, 1 mM DTT), and the bait/prey complex binding to the IgG agarose was cleaved with TEV protease (GIBCO-BRL, 16.degree. C., 2 hours). Further, to 90 .mu.l of the supernatant, 300 .mu.l of a calmodulin binding buffer, 0.3 .mu.l of 1 M CaCl.sub.2 and 50 .mu.l of calmodulin beads washed twice with 500 .mu.l of the calmodulin binding buffer were added, and the mixture was stirred by rotation at 4.degree. C. for 1 hour. After centrifugation, the beads were washed 3 times with 1000 .mu.l the calmodulin binding buffer. 50 .mu.l of a calmodulin elution buffer was added, and the mixture was left on ice for 1 to 2 minutes, and centrifuged to collect 50 .mu.l of a solution. By using the collected solution as a template, RT-PCR (One step RT-PCR kit (QIAGEN), primers: SEQ ID NOS: 177 and 178, program: RT-QH30' (refer to Table 1)). After this screening/selection procedure (FIG. 2) was repeated for 3 rounds, the library was cloned and sequenced to obtain the sequences of SEQ ID NOS: 1 to 14 (amino acid sequences of Fip-cx), SEQ ID NOS: 15 to 19 (amino acid sequences of Eef1dTEF-1), SEQ ID NOS: 20 to 22 (amino acid sequences of Schip1) and nucleic acid sequences corresponding to them (SEQ ID NOS: 1 to 22 in FIG. 1A). The same results were obtained for both of the library prepared by using the sequence of SEQ ID NO: 174 as the main chain of the adaptor for enhancer O29 and the library prepared by using the sequence of SEQ ID NO: 175 as the same.

[0371] All the proteins had a Leu zipper, and they are proteins found by the present invention for the first time to directly interact with c-Fos.

[0372] As a verification experiment of the interactions of the obtained proteins and c-Fos, expression of the proteins of SEQ ID NOS: 2 (Fip-cx), 16 (Eef1dTEF-1) and 22 (Schip1) (FIG. 1A) in a cell-free translation system was experimentally confirmed by using the DNA sequences of SEQ ID NOS: 2-1, 16-1, and 22-1 according to the descriptions of WO02/46395, Example 1, (2) Preparation of coding molecule and (3) Translation of coding molecule, i.e., it was confirmed by the C-terminal labeling method that the proteins were expressed in a wheat cell-free translation system (FIG. 4, A). Further, formation of IVV was also confirmed according to the descriptions of WO02/46395, Example 1, (4) Binding of spacer molecule and coding molecule and (5) Formation of assigning molecule (FIG. 4, B). Furthermore, the interaction with c-Fos was confirmed for those subjected to the first stage pull-down (FIG. 2, IgG+TEV) by 8 M urea/10% SDS-PAGE (FIG. 4, C). As a result, it could be confirmed that the proteins of SEQ ID NOS: 2 (Fip-cx), 16 (Eef1dTEF-1) and 22 (Schip1) interacted with c-Fos.

[0373] Further, the proteins and genes or nucleic acid sequences of the present invention can be used as an inhibitor for blocking transcription, gene duplication and so forth as functions of c-Fos by utilizing the novel function thereof (function of enabling binding with c-Fos in this case). The basis of the above is originates in the fact that the genes detected by the IVV method have been detected through a competitive process constituted by screening repeated multiple times. Therefore, the genes detected by the IVV method show a certain number distribution, and a gene having a stronger competitive power should be detected in a larger number. This suggests that a larger number of clones corresponds to stronger competitive power, and thus such a gene acts more effectively as a blocking agent or inhibitor. In the IVV selection performed in this example, three (/72) of c-Jun well known as a prey were detected for the bait c-Fos. Thus, the numbers of clones (FIG. 1A) detected in the selection indicate that Fip-cx, Eef1 and Schip1 have extremely stronger competitive power compared with known proteins, and they can sufficiently compete, and the proteins can be utilized as an inhibitor for blocking functions of transcription of a complex, gene duplication and so forth by the interaction of c-Jun and a known protein.

Example 2

[0374] A prey IVV library was prepared from the bait c-Fos and a mouse brain cDNA library in the same manner as that used in Example 1, and the screening/selection procedure (FIG. 2) was also performed in the same manner as that used in Example 1. However, in this example, the first stage selection using IgG beads in the two-stage screening was repeated 3 times, and the two-stage selection was performed only for the 4th time to obtain the proteins of SEQ ID NOS: 47 to 56 (amino acid sequences of Fip-cx.1), SEQ ID NOS: 57 to 76 (amino acid sequences of Fip-cx.2), SEQ ID NOS: 77 to 81 (amino acid sequences of Optin), SEQ ID NOS: 82 to 84 (amino acid sequences of Snap19), SEQ ID NOS: 85 and 86 (amino acid sequences of C130020M04Rik), SEQ ID NOS: 87 to 89 (amino acid sequences of FLJ32000), SEQ ID NOS: 90 and 91 (amino acid sequences of Rit2), SEQ ID NOS: 92 and 93 (amino acid sequences of cytocrome b), SEQ ID NOS: 94 and 95 (amino acid sequences of Apoe), SEQ ID NOS: 96 and 97 (amino acid sequences of App), SEQ ID NOS: 98 and 99 (amino acid sequences of Dnaja2), SEQ ID NOS: 100 and 101 (amino acid sequences of Fip-c10), SEQ ID NO: 102 (amino acid sequence of Fip-c4), SEQ ID NO: 103 (amino acid sequence of Fip-c18), and nucleic acid sequences corresponding to these proteins (SEQ ID NOS: 47 to 76 in FIG. 1A, and SEQ ID NOS: 77 to 103 in FIG. 1B). The same results were obtained for both of the library prepared by using the sequence of SEQ ID NO: 174 as the main chain of the adaptor for enhancer O29 and the library prepared by using the sequence of SEQ ID NO: 175 as the same.

[0375] Fip-cx.1, Fip-cx.2, Optin, C130020M04Rik, FLJ32000, and cytocrome b proteins have a Leu zipper, and Rit2, Apoe, App, Dnaja2, Fip-c10, Fip-c4, and Fip-c18 proteins do not have a Leu zipper. All the proteins are proteins found by the present invention for the first time to form a complex with c-Fos.

[0376] As a verification experiment of the interactions of the obtained proteins and c-Fos, expression of the proteins of SEQ ID NOS: 48 (Fip-cx.1), 75 (Fip-cx.2), 78 (Optn), 84 (Snapc5), 86 (C13002004Rik), 88 (FLJ32000), 91 (Rit2), 93 (cytochrome b), 95 (Apoe), 97 (betaAPP), 99 (Hsp40), 101 (Fip-c10), 102 (Fip-c4) and 103 (Fip-c18) (FIG. 1) in a cell-free translation system was experimentally confirmed by using the DNA sequences of SEQ ID NOS: 105, 139, 142, 148, 150, 152, 155, 157, 159, 161, 163, 165, 166 and 167 according to the descriptions of WO02/46395, Example 1, (2) Preparation of coding molecule and (3) Translation of coding molecule, i.e., it was confirmed by the C-terminal labeling method that the proteins were expressed in a wheat cell-free translation system (FIG. 5, A). Further, among those C-terminal labeled proteins for which expression was confirmed, the proteins of SEQ ID NOS: 48 (Fip-cx.1) and 75 (Fip-cx.2), which are completely novel proteins not registered at any database, were used as a prey protein to confirm interactions thereof with the bait c-Fos by pull-down. As for the preparation method of the prey protein, specifically, PCR cloning kit (QIAGEN) was used to extract a sequence cloned in the pDrive vector (SEQ ID NO: 179, QIAGEN) from cells, and a DNA template was prepared by PCR (primers 5' F3 (SEQ ID NO: 180) and 3' R3 (SEQ ID NO: 181), PCR program: ISHI1562 (refer to Table 1), 100 .mu.l scale) using TaKaRa Ex Taq (Takara Shuzo). The DNA template was transcribed (37.degree. C., 2 hours, 50 .mu.l scale) by using RiboMAX.TM. Large Scale RNA Production Systems (Promega) to prepare a mRNA template of the prey protein.

[0377] The preparation method of the bait c-Fos protein is the same as that used for the selection/screening.

[0378] Cell-free translation of the prey template (10 .mu.l scale) was performed for 1 hour by using the C-terminal labeling method to prepare a prey protein in a C-terminal labeled state. At the same time, the translation reaction of the bait c-fos template was performed for 1 hour by the cell-free translation (50 .mu.l scale) to produce the bait protein. After the translation, the both and the binding buffer were mixed (prey: 8 .mu.l, bait: 10 .mu.l, IgG binding buffer: 82 .mu.l), and incubated with 50 .mu.l of IgG agarose beads for 2 hours, and the beads were washed, then added with 20 .mu.l of a buffer containing SDS, boiled at 100.degree. C. for 5 minutes, and eluted. This sample was developed by 17.5% SDS-PAGE, and the FITC fluorochrome was observed by means of a fluorescence imager (FIG. 5, B). In addition, a reaction was also performed without adding the bait c-Fos protein as a control.

[0379] As a result, it could be confirmed that the proteins of SEQ ID NOS: 48 (Fip-cx.1) and 75 (Fip-cx.2) directly interacted with c-Fos.

[0380] Furthermore, as shown in FIG. 6, concentration of genes directly or indirectly interacting with c-Fos was confirmed by real time PCR using the nucleic acid sequences of SEQ ID NOS: 142 (Optn), 148 (Snapc5), 150 (C130020M04Rik) and 152 (FLJ32000). As for the specific method of the real time PCR, primers (SEQ ID NOS: 182 to 189) were designed for four kinds of genes (SEQ ID NOS: 142 (Optn), 148 (Snapc5), 150 (C130020M04Rik) and 152 (FLJ32000)) so that the amplification should be attained in the ranges of sequences obtained by the screening. For preparation of calibration curves, a gene comprising a DNA fragment of positive control incorporated into the pDrive vector was amplified by PCR (5' M13_F primer (SEQ ID NO: 190) and 3' M13_R primer (SEQ ID NO: 191) were used, and the PCR program lightcycler of Table 1 was used), which was controlled so that 1E03, 1E05, 1E07 or 1E09 clones/reaction should be obtained. The measurement was controlled so that each of the library DNA before screening, library DNA in each cycle of screening, and Mock library DNA not added with the bait c-Fos should be in an amount of 5 ng/reaction. The PCR measurement reaction was performed in a scale of 20 .mu.l according to the programs shown in Table 1 by using LightCycler Instrument or LightCycler FastStart DNA Master SYBR Green I (both are produced by Roche Diagnostics).

[0381] Further, the proteins and genes or nucleic acid sequences of the present invention can be used as an inhibitor for blocking transcription, gene duplication and so forth as functions of c-Fos by utilizing the novel function thereof (function of enabling binding with c-Fos in this case). The basis of the above is originates in the fact that the genes detected by the IVV method have been detected through a competitive process constituted by screening repeated multiple times. Therefore, the genes detected by the IVV method show a certain number distribution, and a gene having a stronger competitive power should be detected in a larger number. This suggests that a larger number of clones corresponds to stronger competitive power, and thus such a gene acts more effectively as a blocking agent or inhibitor. In the IVV selection performed in this example, three (/142) of JunD well known as a prey were detected for the bait c-Fos. Thus, the numbers of clones (FIGS. 1A and 1B) detected in the selection indicate that Fip-cx.1, Fip-cx.2, Optn, and so forth have extremely stronger competitive power compared with known proteins, and Snap19, FLJ32000, and so forth can sufficiently compete with known proteins, and thus the proteins can be utilized as an inhibitor for blocking functions of transcription of a complex, gene duplication and so forth by the interaction of c-Jun and a known protein. TABLE-US-00001 TABLE 1 PCR programs Program name: CYCB1 Reaction conditions: ##STR1## Program name: V-2 Reaction conditions: ##STR2## Program name: RT-QH30' Reaction conditions: ##STR3## Program name: ISHI1562 Reaction conditions: ##STR4## Program name: lightcycler Reaction condition: ##STR5##

INDUSTRIAL APPLICABILITY

[0382] Because proteins that interact with c-Fos have been provided, it becomes possible to provide proteins forming a complex with c-Fos by not only a direct interaction, but also an unexpected indirect interaction, and nucleic acids encoding these proteins as well as methods for utilizing them.

Sequence CWU 1

1

191 1 184 PRT Mus musculus 1 Met Pro Leu Arg His Leu Ala Asp Arg Leu Gly His Leu Ala Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Leu Arg 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Leu Arg His Leu 35 40 45 Ala Asp Arg Leu Lys His Leu Thr Ser Arg Leu Gly His Leu Thr Asp 50 55 60 Arg Ser Trp His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu 65 70 75 80 Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Gln Arg Tyr 85 90 95 Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr 100 105 110 Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 115 120 125 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met 130 135 140 His Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Gln Arg His Leu 145 150 155 160 Ala Asp Arg Gln Arg His Leu Ala Asp Arg Leu Arg His Leu Ala Asp 165 170 175 Lys Leu Arg His Gln Leu Gln Leu 180 2 55 PRT Mus musculus 2 Asp Lys Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His Leu 35 40 45 Ala Asp Arg Gln Arg His Asp 50 55 3 55 PRT Mus musculus 3 Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Val Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His Leu 35 40 45 Ala Asp Arg Gln Arg His Asp 50 55 4 38 PRT Mus musculus 4 Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp 1 5 10 15 Arg Leu Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu 20 25 30 Ser His Pro Thr Gln Thr 35 5 45 PRT Mus musculus 5 Asp Arg Leu Gly His Leu Thr Asp Arg Leu Lys His Leu Thr Asp Arg 1 5 10 15 Leu Gly His Leu Thr Asp Arg Leu Val His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Val Arg Gln 35 40 45 6 55 PRT Mus musculus 6 Asp Lys Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His Leu 35 40 45 Ala Asp Arg Arg Arg His Asp 50 55 7 55 PRT Mus musculus 7 Asp Lys Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Pro Arg His Leu 35 40 45 Ala Asp Arg Gln Arg His Asp 50 55 8 55 PRT Mus musculus 8 Asp Lys Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Gly His Leu 35 40 45 Ala Asp Arg Gln Arg His Asp 50 55 9 45 PRT Mus musculus 9 Asp Arg Leu Gly His Leu Thr Asp Arg Leu Lys His Leu Thr Asp Arg 1 5 10 15 Leu Gly His Leu Thr Asp Arg Leu Ile His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Val Arg Gln 35 40 45 10 38 PRT Mus musculus 10 Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His 1 5 10 15 Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Met His Leu Thr 20 25 30 Asp Arg Leu Arg Gln Arg 35 11 31 PRT Mus musculus 11 Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Met His 1 5 10 15 Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Gln Arg His Asp 20 25 30 12 38 PRT Mus musculus 12 Thr Asp Gly Leu Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp 1 5 10 15 Arg Leu Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu 20 25 30 Arg His Leu Ala Asp Gln 35 13 45 PRT Mus musculus 13 Asp Arg Leu Gly His Leu Thr Asp Ile Leu Lys His Leu Thr Asp Arg 1 5 10 15 Leu Gly His Leu Thr Asp Arg Leu Ile His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Val Arg Gln 35 40 45 14 55 PRT Mus musculus 14 Asp Lys Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met 20 25 30 Arg Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His Leu 35 40 45 Ala Asp Arg Gln Arg His Asp 50 55 15 281 PRT Mus musculus 15 Met Ala Thr Asn Phe Leu Ala His Glu Lys Ile Trp Phe Asp Lys Phe 1 5 10 15 Lys Tyr Asp Asp Ala Glu Arg Arg Phe Tyr Glu Gln Met Asn Gly Pro 20 25 30 Val Thr Ser Gly Ser Arg Gln Glu Asn Gly Ala Ser Val Ile Leu Arg 35 40 45 Asp Ile Ala Arg Ala Arg Glu Asn Ile Gln Lys Ser Leu Ala Gly Ser 50 55 60 Ser Gly Pro Gly Ala Ser Ser Gly Pro Gly Gly Asp His Ser Glu Leu 65 70 75 80 Ile Val Arg Ile Thr Ser Leu Glu Val Glu Asn Gln Asn Leu Arg Gly 85 90 95 Val Val Gln Asp Leu Gln Gln Ala Ile Ser Lys Leu Glu Ala Arg Leu 100 105 110 Ser Ser Leu Glu Lys Ser Ser Pro Thr Pro Arg Ala Thr Ala Pro Gln 115 120 125 Thr Gln His Val Ser Pro Met Arg Gln Val Glu Pro Pro Thr Lys Lys 130 135 140 Gly Ala Thr Pro Ala Glu Asp Asp Glu Asp Lys Asp Ile Asp Leu Phe 145 150 155 160 Gly Ser Asp Glu Glu Glu Glu Asp Lys Glu Ala Ala Arg Leu Arg Glu 165 170 175 Glu Arg Leu Arg Gln Tyr Ala Glu Lys Lys Ala Lys Lys Pro Thr Leu 180 185 190 Val Ala Lys Ser Ser Ile Leu Leu Asp Val Lys Pro Trp Asp Asp Glu 195 200 205 Thr Asp Met Ala Gln Leu Glu Thr Cys Val Arg Ser Ile Gln Leu Asp 210 215 220 Gly Leu Val Trp Gly Ala Ser Lys Leu Val Pro Val Gly Tyr Gly Ile 225 230 235 240 Arg Lys Leu Gln Ile Gln Cys Val Val Glu Asp Asp Lys Val Gly Thr 245 250 255 Asp Leu Leu Glu Glu Glu Ile Thr Lys Phe Glu Glu His Val Gln Ser 260 265 270 Val Asp Ile Ala Ala Phe Asp Lys Ile 275 280 16 54 PRT Mus musculus 16 His Ser Glu Leu Ile Val Arg Ile Thr Ser Leu Glu Val Glu Asn Gln 1 5 10 15 Asn Leu Arg Gly Val Val Gln Asp Leu Gln Gln Ala Ile Ser Lys Leu 20 25 30 Glu Ala Arg Leu Ser Ser Leu Glu Lys Ser Ser Pro Thr Pro Arg Ala 35 40 45 Thr Ala Pro Gln Thr Arg 50 17 53 PRT Mus musculus 17 Arg Glu Leu Ile Val Arg Ile Thr Ser Leu Glu Val Glu Asn Gln Asn 1 5 10 15 Leu Arg Gly Val Val Gln Asp Leu Gln Gln Val Ile Ser Lys Leu Glu 20 25 30 Ala Arg Leu Ser Ser Leu Glu Lys Ser Ser Pro Thr Pro Arg Ala Thr 35 40 45 Ala Pro Gln Thr Arg 50 18 54 PRT Mus musculus 18 His Ser Glu Leu Ile Val Arg Ile Asn Ser Leu Glu Val Glu Asn Gln 1 5 10 15 Asn Leu Arg Gly Val Val Gln Asp Leu Gln Gln Ala Ile Ser Lys Leu 20 25 30 Glu Ala Arg Leu Ser Ser Leu Glu Lys Ser Ser Pro Thr Pro Arg Ala 35 40 45 Thr Ala Pro Arg Thr Arg 50 19 54 PRT Mus musculus 19 His Ser Glu Leu Ile Val Arg Ile Thr Ser Leu Glu Val Glu Asn Gln 1 5 10 15 Asn Leu Arg Gly Val Val Gln Asp Leu Gln Gln Ala Ile Ser Arg Leu 20 25 30 Glu Ala Arg Leu Ser Ser Leu Glu Lys Ser Ser Pro Thr Pro Arg Ala 35 40 45 Thr Ala Pro Gln Thr Arg 50 20 268 PRT Mus musculus 20 Met Leu Ser Ala Phe Pro Ala Gln Leu Ala Gln Gln Ser Ser Phe Gly 1 5 10 15 Val Cys Val Leu Gly Cys Thr Glu Met Val His Gln Glu Asn Cys Ser 20 25 30 Tyr Gln Ala Gln Lys Asn Glu Arg Glu Ser Ile Arg Gln Lys Leu Ala 35 40 45 Leu Gly Ser Phe Phe Asp Asp Gly Pro Gly Ile Tyr Thr Ser Cys Ser 50 55 60 Lys Ser Gly Lys Pro Ser Leu Ser Ala Arg Leu Gln Ser Gly Met Asn 65 70 75 80 Leu Gln Ile Cys Phe Val Asn Asp Ser Gly Ser Asp Lys Asp Ser Asp 85 90 95 Ala Asp Asp Ser Lys Thr Glu Thr Ser Leu Asp Thr Pro Leu Ser Pro 100 105 110 Met Ser Lys Gln Ser Ser Ser Tyr Ser Asp Arg Asp Thr Thr Glu Glu 115 120 125 Glu Ser Glu Ser Leu Asp Asp Met Asp Phe Leu Thr Arg Gln Lys Lys 130 135 140 Leu Gln Ala Glu Ala Lys Met Ala Leu Ala Met Ala Lys Pro Met Ala 145 150 155 160 Lys Met Gln Val Glu Val Glu Arg Gln Asn Arg Lys Lys Ser Pro Val 165 170 175 Ala Asp Leu Leu Pro His Met Pro His Ile Ser Glu Cys Leu Met Lys 180 185 190 Arg Ser Leu Lys Pro Thr Asp Leu Arg Asp Met Thr Ile Gly Gln Leu 195 200 205 Gln Val Ile Val Asn Asp Leu His Ser Gln Ile Glu Ser Leu Asn Glu 210 215 220 Glu Leu Val Gln Leu Leu Leu Ile Arg Asp Glu Leu His Thr Glu Gln 225 230 235 240 Asp Ala Met Leu Val Asp Ile Glu Asp Leu Thr Arg His Ala Glu Ser 245 250 255 Gln Gln Lys His Met Ala Glu Lys Met Pro Ala Lys 260 265 21 78 PRT Mus musculus 21 Pro His Thr Pro His Ile Ser Glu Cys Leu Met Lys Arg Ser Leu Lys 1 5 10 15 Pro Thr Asp Leu Arg Asp Met Thr Ile Gly Gln Leu Gln Val Ile Val 20 25 30 Asn Asp Leu His Ser Gln Ile Glu Ser Leu Asn Glu Glu Leu Val Gln 35 40 45 Leu Leu Leu Ile Arg Asp Glu Leu His Thr Glu Gln Asp Ala Met Leu 50 55 60 Val Asp Ile Glu Asp Leu Thr Arg His Ala Glu Arg Glu Gln 65 70 75 22 78 PRT Mus musculus 22 Pro His Met Pro His Ile Ser Glu Cys Leu Met Lys Arg Ser Leu Lys 1 5 10 15 Pro Thr Asp Leu Arg Asp Met Thr Ile Gly Gln Leu Gln Val Ile Val 20 25 30 Asn Asp Leu His Ser Gln Ile Glu Arg Leu Asn Glu Glu Leu Val Gln 35 40 45 Leu Leu Leu Ile Arg Asp Glu Leu His Thr Glu Gln Asp Ala Met Leu 50 55 60 Val Asp Ile Glu Asp Leu Thr Arg His Ala Glu Lys Glu Gln 65 70 75 23 552 DNA Mus musculus 23 atgccattga ggcatctagc agacagattg gggcatctgg cagacagact gaggcatcta 60 acagacagat tgaggcatct agcagacaga ctgaggcatt taacagacag attgaggcat 120 ctagcagaca gattgaggca tctagcagac agactgaaac atcttaccag cagattgggg 180 catctaacag acagatcatg gcatctaaca gacagattgg ggcatctaac agacagattg 240 aggcatctaa cagacagatt ggggcatcta acagacagac agaggtatct agcagacaga 300 ttgaggcatc taacagacag attggggcat ctaacagaca gactgaggca tctaacagac 360 agattggggc atctaacaga cagactgagg catctaacag acagattggg gcatctaaca 420 gacagactga tgcatctaac agacagactg aggcatctag cagacagaca gaggcatcta 480 gcagacagac agaggcatct agcagacaga ctgaggcatc tagcagacaa attgaggcat 540 cagctgcagc tg 552 24 165 DNA Mus musculus 24 gacaaactga ggcatctaac agacagattg gggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgatgcatc taacagacag actgatgcat 120 ctaacagaca gactgaggca tctagcagac agacagaggc acgac 165 25 165 DNA Mus musculus 25 gacaaactga ggcatctaac agacagattg gggcatctaa cagacaggct gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgatgcatc taacagacag actgatgcat 120 ctaacagaca gactgaggca tctagcagac agacagaggc acgac 165 26 165 DNA Mus musculus 26 gacaaactga ggcatctaac agacagattg gggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgatgcatc taacagacag actgatgcat 120 ctaacagaca gactgaggca tctagcagac aggcagaggc acgac 165 27 165 DNA Mus musculus 27 gacagactga ggcatctaac agacagattg gggcatttaa cagacagact gaggcattta 60 acagacagat tggggcatgt aacagacaga ctgatgcatt taacagacag actgatgcat 120 ctaacagaca gactgaggca tttagcagac agacagaggc acgac 165 28 114 DNA Mus musculus 28 acagacagat tggggcatct aacagacaga ctgaggcatc taacagacag attggggcat 60 ctaacagaca gactgatgca tctaacagac agactgagcc atcctacgca gacc 114 29 135 DNA Mus musculus 29 gacagattgg ggcatctaac agacagactg aagcatctaa cagacagatt ggggcatcta 60 acagacagac tggtccatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtta gacag 135 30 165 DNA Mus musculus 30 gacaaactga ggcatctaac agacagattg gggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgatgcatc taacagacag actgatgcat 120 ctaacagaca gactgaggca tctagcagac agacggaggc acgac 165 31 165 DNA Mus musculus 31 gacaaactga ggcatctaac agacagattg gggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgatgcatc taacagacag actgatgcac 120 ctaacagaca gaccgaggca tctagcagac agacagaggc acgac 165 32 165 DNA Mus musculus 32 gacaaactga ggcatctaac agacagattg gggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgatgcatc taacagacag actgatgcat 120 ctaacagaca gactggggca tctagcagac agacagaggc acgac 165 33 135 DNA Mus musculus 33 gacagattgg ggcatctaac agacagactg aagcatctaa cagacagatt ggggcatcta 60 acagacagac tgatccatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 34 114 DNA Mus musculus 34 gggcatctaa cagacagact gaggcatcta acagacagat tggggcatct aacagacaga 60 ctgatgcatc taacagacag actgatgcat ctaacagaca gactgaggca aaga 114 35 93 DNA Mus musculus 35 gggcatctaa cagacagact gatgcatcta acagacagac tgatgcatct aacagacaga 60 ctgaggcatc tagcagacag acagaggcac gac 93 36 114 DNA Mus musculus 36 acagacggat tggggcatct aacagacaga ctgaggcatc taacagacag attggggcat 60 ctaacagaca gactgatgca tctaacagac agactgaggc atctagcaga ccag 114 37 135 DNA Mus musculus 37 gacagattgg ggcatctaac agacatactg aagcatctaa cagacagatt ggggcatcta 60 acagacagac tgatccatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 38 165 DNA Mus musculus 38 gacaaactga ggcatctaac agacagattg gggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgatgcgtc taacagacag actgatgcat 120 ctaacagaca gactgaggca tctagcagac agacagaggc acgac 165 39 843 DNA Mus musculus 39 atggctacaa actttctagc gcatgagaag atctggtttg acaagtttaa atatgatgat 60 gcagaaagga gattctatga gcagatgaac gggcctgtga cctccggctc ccgccaggag 120 aatggtgcca gcgtgatcct ccgagacatt gcaagagcca gagagaacat ccagaaatcc 180 ttggctggaa gctcaggccc tggagcctcc agtggacctg gtggagacca cagtgagctc 240 attgtgagga ttaccagtct ggaagtggag aaccagaacc ttcgaggcgt ggtgcaagat 300 ttgcagcagg ccatttccaa gttggaggcc cggctgagct ctctagagaa gagttcacct 360 actccccgag ccacggcccc acagacccaa catgtctctc ctatgcgtca agtggagccc 420 ccaaccaaga aaggagccac accagcagag gacgatgagg acaaggacat tgacctgttc 480 ggcagtgacg aggaggaaga agataaggag gctgcccgac tacgggagga gaggctacgc 540 cagtacgcag agaagaaggc caagaagccc acactggtgg ccaaatcctc catccttttg 600 gatgttaaac

cttgggatga tgagactgac atggcccagc tagagacttg tgtgcgttcc 660 atccaattgg acgggctggt ttggggggcc tccaagcttg tgcctgttgg ctatggcatc 720 cggaagctgc agatccagtg tgtggtggag gatgacaaag tgggcaccga cttgctcgag 780 gaggagatca ccaaatttga ggagcatgtg cagagtgtcg acatcgcagc tttcgacaag 840 atc 843 40 162 DNA Mus musculus 40 cacagtgagc tcattgtgag gattaccagt ctggaagtgg agaatcagaa ccttcgaggc 60 gtggtgcaag atttgcagca ggccatttcc aagttggagg cccggctgag ctctctagag 120 aagagttcac ctactccccg agccacggcc ccacagaccc ga 162 41 159 DNA Mus musculus 41 cgtgagctca ttgtgaggat taccagtctg gaagtggaga atcagaacct tcgaggcgtg 60 gtgcaagatt tgcagcaggt catttccaag ttggaggccc ggctgagctc tctagagaag 120 agttcaccta ctccccgagc cacggcccca cagacccga 159 42 162 DNA Mus musculus 42 cacagtgagc tcattgtgag gattaacagt ttggaagtgg agaatcagaa ccttcgaggg 60 gtggtgcaag atttgcagca ggccatttcc aagttggagg cccggctgag ctctttagag 120 aagagttcac ctactccccg agccacggcc ccacggaccc ga 162 43 162 DNA Mus musculus 43 cacagtgagc tcattgtgag gattaccagt ctggaagtgg agaatcagaa ccttcggggc 60 gtggtgcaag atttgcagca ggccatttcc aggttggagg cccggctgag ctctctagag 120 aagagttcac ctactccccg agccacggcc ccacagaccc ga 162 44 804 DNA Mus musculus 44 atgctcagcg ctttccctgc gcagctcgcc cagcagtcca gctttggggt ctgcgtccta 60 ggatgtactg agatggtaca tcaggagaac tgctcgtacc aggcacagaa gaatgagaga 120 gagtctatca gacagaagtt ggcactcgga agcttctttg acgatggccc aggaatctat 180 accagctgca gcaaaagtgg gaagccaagc ctttctgcaa gactacagag cgggatgaac 240 ctccagatat gctttgtcaa tgacagcggc agtgacaagg acagcgatgc agatgacagt 300 aagacggaaa ccagcttgga cacgcccttg tcccccatga gcaagcagag ttcttcctat 360 tcggatagag acacaactga ggaggagtct gaatccctgg atgacatgga cttcctcaca 420 aggcaaaaga agctacaagc tgaagccaaa atggctctgg ccatggccaa accaatggcc 480 aaaatgcaag tagaagtgga aagacagaac aggaaaaagt ctcccgtcgc tgatcttctc 540 ccacacatgc ctcacataag cgaatgtttg atgaaaagaa gcttaaagcc caccgacctg 600 agagacatga ctatcgggca gctacaagtg atcgtcaatg acctccactc ccagattgaa 660 agtttgaatg aagagttggt ccagctgctc cttattcgag atgagctgca cacagaacaa 720 gatgccatgc tggtggacat tgaagacttg actagacacg ctgagagtca gcagaagcac 780 atggctgaga aaatgcccgc gaag 804 45 234 DNA Mus musculus 45 ccacacacgc ctcacataag cgaatgtttg atgaaaagaa gcttaaagcc caccgacctg 60 agagacatga ctatcgggca gctacaagtg atcgtcaatg acctccactc ccagattgaa 120 agtttgaatg aagagttggt ccagctgctc cttattcgag atgagctgca cacagaacaa 180 gatgccatgc tggtggacat tgaagacttg actagacacg ctgagaggga gcag 234 46 234 DNA Mus musculus 46 ccacacatgc ctcacataag cgaatgtttg atgaaaagaa gcttaaagcc caccgacctg 60 agagacatga ctatcgggca gctacaagtg atcgtcaatg acctccactc ccagattgag 120 cgtttgaatg aagagttggt ccagctgctc cttattcgag atgagctgca cacagaacaa 180 gatgccatgc tggtggacat tgaagacttg actagacacg ctgagaagga gcag 234 47 191 PRT Mus musculus 47 Met Pro Leu Arg His Leu Ala Asp Arg Leu Gly His Leu Ala Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Leu Arg 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Leu Lys His Leu 35 40 45 Ala Asp Arg Leu Lys His Leu Thr Asp Arg Leu Gly His Leu Thr Asp 50 55 60 Arg Ser Trp His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu 65 70 75 80 Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Gln Arg Tyr 85 90 95 Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr 100 105 110 Asp Lys Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 115 120 125 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met 130 135 140 His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His Leu 145 150 155 160 Ala Asp Arg Gln Arg His Leu Ala Asp Arg Gln Arg His Leu Ala Asp 165 170 175 Arg Leu Arg His Leu Ala Asp Lys Leu Arg His Gln Leu Gln Leu 180 185 190 48 71 PRT Mus musculus 48 Ile Glu Ala Ser Asn Arg Gln Ile Gly Ala Ser Asn Arg Gln Thr Glu 1 5 10 15 Ala Ser Asn Arg Gln Ile Gly His Leu Thr Asp Arg Leu Arg His Leu 20 25 30 Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp 35 40 45 Arg Leu Met His Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Gln 50 55 60 Arg His Leu Ala Asp Arg Leu 65 70 49 55 PRT Mus musculus 49 Asp Lys Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His Leu 35 40 45 Ala Asp Arg Gln Arg His Asp 50 55 50 45 PRT Mus musculus 50 Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg 1 5 10 15 Leu Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Val Arg Gln 35 40 45 51 45 PRT Mus musculus 51 Asp Arg Leu Gly Arg Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg 1 5 10 15 Leu Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Val Arg Gln 35 40 45 52 45 PRT Mus musculus 52 Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg Tyr Leu Thr Asp Arg 1 5 10 15 Leu Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Val Arg Gln 35 40 45 53 45 PRT Mus musculus 53 Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg 1 5 10 15 Leu Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Thr 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Val Arg Gln 35 40 45 54 45 PRT Mus musculus 54 Asp Arg Leu Gly His Leu Thr Asp Arg Leu Lys His Leu Thr Asp Arg 1 5 10 15 Leu Gly His Leu Thr Asp Arg Leu Ile His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Val Arg Gln 35 40 45 55 45 PRT Mus musculus 55 Gly Arg Leu Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg 1 5 10 15 Leu Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Met 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Val Arg Gln 35 40 45 56 38 PRT Mus musculus 56 Asp Arg Leu Arg His Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Ala Asp Arg Leu Lys His Leu Ala Asp Arg Leu Lys 20 25 30 His Leu Thr Asn Arg Lys 35 57 184 PRT Mus musculus 57 Met Pro Leu Arg His Leu Ala Asp Arg Leu Gly His Leu Ala Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Leu Arg 20 25 30 His Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Leu Arg His Leu 35 40 45 Ala Asp Arg Leu Lys His Leu Thr Asp Arg Leu Gly His Leu Thr Asp 50 55 60 Arg Ser Trp His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu 65 70 75 80 Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Gln Arg Tyr 85 90 95 Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr 100 105 110 Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg 115 120 125 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met 130 135 140 His Leu Thr Asp Arg Leu Arg His Leu Ala Asp Arg Gln Arg His Leu 145 150 155 160 Ala Asp Arg Gln Arg His Leu Ala Asp Arg Leu Arg His Leu Ala Asp 165 170 175 Lys Leu Arg His Gln Leu Gln Leu 180 58 79 PRT Mus musculus 58 Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser Thr 1 5 10 15 Ala Asn Thr Leu Arg Glu Gln Val Ala Leu Leu Lys Gln Lys Val Met 20 25 30 Asn His Val Asn Ser Gly Cys Gln Leu Met Leu Thr Gln Gln Leu Gln 35 40 45 Thr Phe Trp Glu Gln Thr Val Arg Ala Glu Gly Gln Trp Lys Lys Lys 50 55 60 Asn Asn Arg Asp Lys Leu Glu Asn Leu Thr Gly Cys Asp Arg Glu 65 70 75 59 76 PRT Mus musculus 59 Arg Ile Lys Ala Glu Arg Lys Arg Met Arg Asn Arg Ile Ala Ala Ser 1 5 10 15 Glu Cys Arg Lys Arg Lys Leu Glu Arg Ile Ala Arg Leu Glu Glu Lys 20 25 30 Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn 35 40 45 Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val Met Asn His 50 55 60 Val Asn Ser Gly Cys Gln Leu Met Leu Thr Gln Gln 65 70 75 60 49 PRT Mus musculus 60 Gly His Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg Leu Arg His 1 5 10 15 Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg Leu Arg His Leu Ala 20 25 30 Asp Arg Leu Arg His Leu Ala Asp Arg Leu Lys His Leu Thr Asp Arg 35 40 45 Tyr 61 56 PRT Mus musculus 61 His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu 1 5 10 15 Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Arg His Leu Thr Asp 20 25 30 Arg Leu Met His Leu Thr Asp Arg Leu Gly His Leu Ala Asp Arg Gln 35 40 45 Arg His Leu Ala Asp Arg Gln Arg 50 55 62 44 PRT Mus musculus 62 Ala Asp Arg Leu Gly His Leu Ala Asp Arg Leu Arg His Leu Thr Asp 1 5 10 15 Arg Leu Arg His Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg Leu 20 25 30 Arg His Leu Ala Asp Arg Leu Arg His Leu Ala Asp 35 40 63 27 PRT Mus musculus 63 Ala Asp Arg Leu Arg His Leu Thr Asp Arg Leu Arg His Leu Ala Asp 1 5 10 15 Arg Leu Arg His Leu Thr Asp Arg Leu Arg His 20 25 64 53 PRT Mus musculus 64 Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp 1 5 10 15 Arg Leu Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu 20 25 30 Gly His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His 35 40 45 Leu Ala Asp Arg Pro 50 65 46 PRT Mus musculus 65 Arg Gln Arg His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu 1 5 10 15 Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His 20 25 30 Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg Pro 35 40 45 66 39 PRT Mus musculus 66 Asp Arg Leu Ser His Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg 20 25 30 His Leu Ala Asp Arg Gln Arg 35 67 39 PRT Mus musculus 67 Asp Arg Leu Arg His Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg 20 25 30 His Leu Ala Asp Arg Gln Arg 35 68 39 PRT Mus musculus 68 Gly Arg Leu Arg His Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg 1 5 10 15 Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg 20 25 30 His Leu Ala Asp Arg Gln Arg 35 69 43 PRT Mus musculus 69 Tyr Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu 1 5 10 15 Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp 20 25 30 Arg Leu Arg His Leu Thr Asp Arg Leu Gly Gln 35 40 70 43 PRT Mus musculus 70 Tyr Leu Ala Asp Arg Leu Arg His Leu Thr Asp Arg Leu Arg His Leu 1 5 10 15 Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp 20 25 30 Arg Leu Arg His Leu Thr Asp Arg Leu Gly Gln 35 40 71 76 PRT Mus musculus 71 Arg Ile Lys Ala Glu Arg Lys Arg Met Arg Asn Arg Ile Ala Ala Ser 1 5 10 15 Lys Cys Arg Lys Arg Lys Leu Glu Arg Ile Ala Arg Leu Glu Glu Lys 20 25 30 Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn 35 40 45 Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val Met Asn His 50 55 60 Val Asn Ser Gly Cys Gln Leu Met Leu Thr Gln Gln 65 70 75 72 44 PRT Mus musculus 72 Leu Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly 1 5 10 15 His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His Leu 20 25 30 Ala Asp Arg Gln Arg His Leu Ala Asp Arg Gln Lys 35 40 73 36 PRT Mus musculus 73 Leu Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly 1 5 10 15 His Leu Thr Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His Leu 20 25 30 Ala Asp Thr Gln 35 74 44 PRT Mus musculus 74 Gly His Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His 1 5 10 15 Leu Thr Asp Arg Leu Arg His Leu Thr Asp Arg Leu Gly His Leu Thr 20 25 30 Asp Arg Leu Met His Leu Thr Asp Arg Leu Arg His 35 40 75 51 PRT Mus musculus 75 Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg His 1 5 10 15 Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Met His Leu Thr 20 25 30 Asp Arg Leu Arg His Leu Ala Asp Arg Gln Arg His Leu Ala Asp Arg 35 40 45 Gln Arg His 50 76 51 PRT Mus musculus 76 Arg His Leu Thr Asp Arg Leu Gly His Leu Thr Asp Arg Leu Arg His 1 5 10 15 Leu Thr Asp Arg Leu Gly Arg Leu Thr Asp Arg Leu Met His Leu Thr 20 25 30 Asp Arg Leu Arg His Leu Ala Asp Arg Gln Arg His Leu Ala Asp Arg 35 40 45 Gln Arg His 50 77 584 PRT Mus musculus 77 Met Ser His Gln Pro Leu Ser Cys Leu Thr Glu Lys Gly Asp Ser Pro 1 5 10 15 Cys Glu Thr Pro Gly Asn Gly Pro Ser Asn Met Val His Pro Ser Leu 20 25 30 Asp Thr Phe Thr Pro Glu Glu Leu Leu Gln Gln Met Lys Glu Leu Leu 35 40 45 Val Glu Asn His Gln Leu Lys Glu Ala Met Lys Leu Asn Asn Gln Ala 50 55 60 Met Lys Gly Arg Phe Glu Glu Leu Ser Ala Trp Thr Glu Lys Gln Lys 65 70 75 80 Glu Glu Arg Leu Leu Phe Glu Met Gln Ser Lys Glu Val Lys Glu Arg 85 90 95 Leu Lys Ala Leu Thr His Glu Asn Glu Arg Leu Lys Glu Glu Leu Gly 100 105 110 Lys Phe Lys Glu Lys Ser Glu Lys Pro Leu Glu Asp Leu Thr Gly Gly 115 120 125 Tyr Arg Tyr Pro Arg Ala Leu Glu Glu Glu Val Glu Lys Leu Lys Thr 130 135 140 Gln Val Glu Gln Glu Val Glu His Leu Lys Ile Gln Val Met Arg Leu 145 150 155 160 Arg Ala Glu Lys Ala Asp Leu Leu Gly Ile Val Ser Glu Leu Gln Leu 165 170 175 Lys Leu Asn Ser Gly Gly Ser Ser Glu Asp Ser Phe Val Glu Ile Arg 180 185 190

Met Thr Glu Gly Glu Thr Glu Gly Ala Met Lys Glu Met Lys Asn Cys 195 200 205 Pro Thr Pro Thr Arg Thr Asp Pro Ile Ser Leu Ser Asn Cys Thr Glu 210 215 220 Asp Ala Arg Ser Cys Ala Glu Phe Glu Glu Leu Thr Val Ser Gln Leu 225 230 235 240 Leu Leu Cys Leu Arg Glu Gly Asn Gln Lys Val Glu Arg Leu Glu Val 245 250 255 Ala Leu Arg Glu Ala Lys Glu Arg Ile Ser Asp Phe Glu Lys Lys Ala 260 265 270 Asn Gly His Ser Ser Thr Glu Lys Gln Thr Ala Arg Arg Ala Asp Arg 275 280 285 Glu Lys Glu Asp Lys Gly Gln Glu Ser Val Gly Ser Glu Val Glu Thr 290 295 300 Leu Ser Ile Gln Val Thr Ser Leu Phe Lys Glu Leu Gln Glu Ala His 305 310 315 320 Thr Lys Leu Ser Glu Ala Glu Leu Met Lys Lys Arg Leu Gln Glu Lys 325 330 335 Cys Gln Ala Leu Glu Arg Lys Asn Ser Ala Thr Pro Ser Glu Leu Asn 340 345 350 Glu Lys Gln Glu Leu Val Tyr Ser Asn Lys Lys Leu Glu Leu Gln Val 355 360 365 Glu Ser Met Arg Ser Glu Ile Lys Met Glu Gln Ala Lys Thr Glu Glu 370 375 380 Glu Lys Ser Arg Leu Ala Thr Leu Gln Ala Thr His Asn Lys Leu Leu 385 390 395 400 Gln Glu His Asn Lys Ala Leu Lys Thr Ile Glu Glu Leu Thr Lys Gln 405 410 415 Gln Ala Glu Lys Val Asp Lys Met Leu Leu Gln Glu Leu Ser Glu Lys 420 425 430 Leu Glu Leu Ala Glu Gln Ala Leu Ala Ser Lys Gln Leu Gln Met Asp 435 440 445 Glu Met Lys Gln Thr Leu Ala Lys Gln Glu Glu Asp Leu Glu Thr Met 450 455 460 Ala Val Leu Arg Ala Gln Met Glu Val Tyr Cys Ser Asp Phe His Ala 465 470 475 480 Glu Arg Ala Ala Arg Glu Lys Ile His Glu Glu Lys Glu Gln Leu Ala 485 490 495 Leu Gln Leu Ala Ile Leu Leu Lys Glu Asn Asn Asp Ile Glu Glu Gly 500 505 510 Gly Ser Arg Gln Ser Leu Met Glu Met Gln Cys Arg His Gly Ala Arg 515 520 525 Thr Ser Asp Ser Asp Gln Gln Thr Tyr Leu Phe Gln Arg Gly Ala Glu 530 535 540 Asp Arg Ser Trp Gln His Gly Gln Gln Pro Arg Ser Ile Pro Ile His 545 550 555 560 Ser Cys Pro Lys Cys Gly Glu Val Leu Pro Asp Ile Asp Thr Leu Gln 565 570 575 Ile His Val Met Asp Cys Ile Ile 580 78 108 PRT Mus musculus 78 Leu Lys Thr Gln Val Glu Gln Glu Val Glu His Leu Lys Ile Gln Val 1 5 10 15 Met Arg Leu Arg Ala Glu Lys Ala Asp Leu Leu Gly Ile Val Ser Glu 20 25 30 Leu Gln Leu Lys Leu Asn Ser Gly Gly Ser Ser Glu Asp Ser Phe Val 35 40 45 Glu Ile Arg Met Thr Glu Gly Glu Thr Glu Gly Ala Met Lys Glu Met 50 55 60 Lys Ser Cys Pro Thr Pro Thr Arg Thr Asp Pro Ile Ser Leu Ser Asn 65 70 75 80 Cys Thr Glu Asp Ala Arg Ser Cys Ala Glu Phe Glu Glu Leu Thr Val 85 90 95 Ser Gln Leu Leu Leu Cys Leu Arg Glu Gly Asn Gln 100 105 79 62 PRT Mus musculus 79 His Leu Lys Ile Gln Val Met Arg Leu Arg Ala Glu Lys Ala Asp Leu 1 5 10 15 Leu Gly Ile Val Ser Glu Leu Gln Leu Lys Leu Asn Ser Gly Gly Ser 20 25 30 Ser Glu Asp Ser Phe Val Glu Ile Arg Met Thr Glu Gly Glu Thr Glu 35 40 45 Gly Ala Met Lys Glu Met Lys Asn Cys Pro Thr Pro Thr Arg 50 55 60 80 62 PRT Mus musculus 80 His Leu Lys Ile Gln Val Met Arg Leu Arg Ala Glu Lys Ala Asp Leu 1 5 10 15 Leu Gly Ile Val Ser Glu Leu Gln Leu Lys Leu Asn Ser Gly Gly Ser 20 25 30 Ser Glu Asp Ser Phe Val Glu Ile Arg Met Thr Glu Gly Glu Thr Glu 35 40 45 Gly Ala Met Lys Glu Met Lys Asn Cys Pro Ala Pro Thr Arg 50 55 60 81 62 PRT Mus musculus 81 His Leu Lys Ile Gln Val Met Arg Leu Arg Ala Glu Lys Ala Asp Leu 1 5 10 15 Leu Gly Ile Val Ser Glu Leu Arg Leu Lys Leu Asn Ser Gly Gly Ser 20 25 30 Ser Glu Asp Ser Phe Val Glu Ile Arg Met Thr Glu Gly Glu Thr Glu 35 40 45 Gly Ala Met Lys Glu Met Lys Asn Cys Pro Thr Pro Thr Arg 50 55 60 82 102 PRT Mus musculus 82 Met Leu Ser Arg Leu Gln Glu Leu Arg Lys Glu Glu Glu Thr Leu Leu 1 5 10 15 Arg Leu Lys Ala Ala Leu His Asp Gln Leu Asn Arg Leu Lys Val Glu 20 25 30 Glu Leu Ala Leu Gln Ser Met Ile Asn Ser Arg Gly Arg Thr Glu Thr 35 40 45 Leu Ser Ser Gln Pro Ala Pro Glu Gln Leu Cys Asp Met Ser Leu His 50 55 60 Val Asp Asn Glu Val Thr Ile Asn Gln Thr Thr Leu Lys Leu Ser Thr 65 70 75 80 Arg Ser Pro Met Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu 85 90 95 Glu Glu Glu Ser Asp Ser 100 83 56 PRT Mus musculus 83 Arg Cys Arg Gln Lys Arg Lys Leu Trp Val Ser Ser Leu Glu Lys Lys 1 5 10 15 Ala Glu Glu Leu Thr Ser Gln Asn Ile Gln Leu Ser Asn Glu Val Thr 20 25 30 Leu Leu Arg Asn Glu Val Ala Gln Leu Lys Gln Leu Leu Leu Ala His 35 40 45 Lys Asp Cys Pro Val Thr Ala Gln 50 55 84 79 PRT Mus musculus 84 Arg Lys Trp Lys Gly Thr Leu Ser Arg Leu Gln Glu Leu Arg Lys Glu 1 5 10 15 Val Glu Thr Pro Leu Arg Leu Lys Ala Ala Leu His Asp Gln Leu Asn 20 25 30 Arg Leu Lys Val Glu Glu Leu Ala Leu Gln Ser Met Ile Asn Ser Arg 35 40 45 Gly Arg Thr Glu Thr Leu Ser Ser Gln Pro Ala Pro Glu Gln Leu Cys 50 55 60 Asp Met Ser Leu His Val Asp Asn Glu Val Thr Ile Asn Gln Thr 65 70 75 85 413 PRT Mus musculus 85 Met Gly Asp Asp Arg Pro Phe Val Cys Ser Ala Pro Gly Cys Gly Gln 1 5 10 15 Arg Phe Thr Asn Glu Asp His Leu Ala Val His Lys His Lys His Glu 20 25 30 Met Thr Leu Lys Phe Gly Pro Ala Arg Thr Asp Ser Val Ile Ile Ala 35 40 45 Asp Gln Thr Pro Thr Pro Thr Arg Phe Leu Lys Asn Cys Glu Glu Val 50 55 60 Gly Leu Phe Asn Glu Leu Ala Ser Ser Phe Glu His Glu Phe Lys Lys 65 70 75 80 Ala Ser Asp Asp Asp Glu Lys Lys Gly Ala Ala Gly Pro Leu Asp Met 85 90 95 Ser Leu Pro Ser Thr Pro Asp Ile Lys Ile Lys Glu Glu Glu Pro Val 100 105 110 Glu Val Asp Ser Ser Pro Pro Asp Ser Pro Ala Ser Ser Pro Cys Ser 115 120 125 Pro Pro Leu Lys Glu Lys Glu Val Thr Thr Lys Pro Val Val Ile Ser 130 135 140 Thr Pro Thr Pro Thr Ile Val Arg Pro Gly Ser Leu Pro Leu His Leu 145 150 155 160 Gly Tyr Asp Pro Leu His Pro Thr Leu Pro Ser Pro Thr Ser Val Ile 165 170 175 Thr Gln Ala Pro Pro Ser Asn Arg Gln Ile Gly Ser Pro Thr Gly Ser 180 185 190 Leu Pro Leu Val Met His Leu Ala Asn Gly Gln Thr Met Pro Met Leu 195 200 205 Pro Gly Pro Pro Val Gln Met Pro Ser Val Ile Ser Leu Ala Arg Pro 210 215 220 Val Ser Met Val Pro Asn Ile Pro Gly Ile Pro Gly Pro Pro Val Asn 225 230 235 240 Asn Ser Gly Ser Ile Ser Pro Ser Gly His Pro Met Pro Ser Glu Ala 245 250 255 Lys Met Arg Leu Lys Ala Thr Leu Thr His Gln Val Ser Ser Ile Asn 260 265 270 Gly Gly Cys Gly Met Val Val Gly Thr Ala Ser Thr Met Val Thr Ala 275 280 285 Arg Pro Glu Gln Asn Gln Ile Leu Ile Gln His Pro Asp Ala Pro Ser 290 295 300 Pro Ala Gln Pro Gln Val Ser Pro Ala Gln Pro Thr Pro Ser Thr Gly 305 310 315 320 Gly Arg Arg Arg Arg Thr Val Asp Glu Asp Pro Asp Glu Arg Arg Gln 325 330 335 Arg Phe Leu Glu Arg Asn Arg Ala Ala Ala Ser Arg Cys Arg Gln Lys 340 345 350 Arg Lys Leu Trp Val Ser Ser Leu Glu Lys Lys Ala Glu Glu Leu Thr 355 360 365 Ser Gln Asn Ile Gln Leu Ser Asn Glu Val Thr Leu Leu Arg Asn Glu 370 375 380 Val Ala Gln Leu Lys Gln Leu Leu Leu Ala His Lys Asp Cys Pro Val 385 390 395 400 Thr Ala Leu Gln Lys Lys Thr Gln Gly Tyr Leu Gly Lys 405 410 86 58 PRT Mus musculus 86 Arg Ala Ala Ala Ser Arg Cys Arg Gln Lys Arg Lys Leu Trp Val Ser 1 5 10 15 Ser Leu Glu Lys Lys Ala Glu Glu Leu Thr Ser Gln Asn Ile Gln Leu 20 25 30 Ser Asn Lys Val Thr Leu Leu Arg Asn Glu Val Ala Gln Leu Lys Gln 35 40 45 Leu Leu Leu Ala His Lys Asp Cys Pro Gly 50 55 87 1031 PRT Mus musculus 87 Met Thr Asn Pro Lys Gly Lys Arg Arg Gly Thr Gln Ser Met Phe Ser 1 5 10 15 Arg Pro Phe Arg Lys His Gly Val Val Ser Leu Ala Thr Tyr Met Arg 20 25 30 Ile Tyr Lys Lys Arg Asp Ile Val Asp Ile Lys Gly Met Gly Thr Val 35 40 45 Gln Lys Gly Met Pro Cys Lys Cys Tyr His Gly Lys Thr Gly Arg Val 50 55 60 Tyr Asn Val Thr Gln His Ala Met Gly Ile Ile Val Asn Lys Gln Val 65 70 75 80 Lys Gly Lys Ile Leu Ala Lys Arg Ile Asn Val Gln Ile Glu His Ile 85 90 95 Lys His Ser Lys Ser Arg Asp Gly Phe Leu Lys Gln Gly Glu Ala Ala 100 105 110 His Phe Glu Tyr Leu Leu Tyr Pro Leu His Ser Ala Ser Ile Thr Gly 115 120 125 Leu Ala Thr Cys Ile Arg Lys Pro Leu Ile Ala Thr Cys Ser Leu Asp 130 135 140 Arg Ser Val Arg Ile Trp Asn Tyr Glu Ser Asn Ser Ser Cys Cys Lys 145 150 155 160 Ala Leu Arg Glu Asp Leu Trp Leu Leu Leu Leu Phe His Ile Thr Ala 165 170 175 Pro Ala Thr Leu Ser Ser Pro Pro Val Ile Phe Phe Cys Thr Leu Glu 180 185 190 Leu Tyr Lys Glu Tyr Gln Glu Glu Ala Tyr Thr Val Ser Leu His Pro 195 200 205 Ser Gly His Tyr Ile Val Val Gly Phe Ala Asp Lys Leu Arg Leu Met 210 215 220 Asn Leu Leu Ile Asp Asp Ile Arg Ser Phe Lys Glu Tyr Ser Val Arg 225 230 235 240 Gly Cys Lys Glu Cys Ala Phe Ser Asn Gly Gly His Leu Phe Ala Ala 245 250 255 Val Asn Gly Asn Val Ile His Ile Phe Thr Thr Thr Asn Leu Glu Asn 260 265 270 Ile Asn Asn Leu Lys Gly His Thr Gly Lys Arg Glu Thr Glu Cys Val 275 280 285 Leu Lys Val Cys Ser Tyr Asn Ser Val Thr Ile Ser Pro Asp Gly Lys 290 295 300 Val Ile Phe Ala Val Gly Ser Asp Gln Thr Leu Lys Glu Ile Ala Asp 305 310 315 320 Ser Leu Ile Leu Arg Glu Ile Pro Ala Phe Asp Val Val Tyr Thr Ala 325 330 335 Ile Thr Ile Ser His Ser Gly Arg Met Ile Phe Val Gly Thr Ser Val 340 345 350 Gly Thr Ile Arg Ala Met Lys Tyr Pro Leu Pro Leu Gln Arg Glu Phe 355 360 365 Asn Glu Tyr Gln Ala His Ala Gly Pro Val Thr Lys Ile Leu Leu Thr 370 375 380 Phe Asp Asp Gln Phe Leu Leu Thr Val Ser Glu Asp Gly Cys Leu Phe 385 390 395 400 Thr Trp Lys Val Phe Asp Lys Glu Gly Arg Gly Ile Lys Arg Glu Arg 405 410 415 Glu Val Gly Phe Ala Glu Glu Val Leu Val Thr Lys Thr Asp Met Glu 420 425 430 Glu Lys Ile Leu His Arg Asn Leu Ala Thr Glu Phe Arg Arg Pro Met 435 440 445 Ser Lys His Leu Glu Cys Pro Thr Ser Glu Thr Gly Pro Leu Thr Thr 450 455 460 Ile Asn Ile Ser Pro Val Gln Pro Arg Pro Trp Gly His Val Leu Thr 465 470 475 480 Cys Arg Thr Pro Val Ser Thr Asp Ser Ala Val Ala Ser Thr Arg Gly 485 490 495 Ser Val Asp Ser Ala Val Lys Pro Asp Arg Ser Thr Pro Thr Gln Glu 500 505 510 Val Arg Ile Pro Pro Lys Pro Ala Ser Gly Val His Thr Arg Cys Gln 515 520 525 Leu Gly Val Gln Lys Gln Met Glu His Val Ser Val Val Met Glu Val 530 535 540 Arg Glu Thr Asn Arg Gln Arg Gln Gly Gly Gly Ala Arg Asn Val Ile 545 550 555 560 Lys Ala Gln Ile Met Leu Glu Leu Lys Thr Arg Val Glu Glu Leu Lys 565 570 575 Met Glu Asn Glu Tyr Gln Leu Arg Leu Lys Asp Met Asn Tyr Ser Glu 580 585 590 Lys Ile Lys Glu Leu Thr Asp Lys Phe Ile Gln Glu Met Glu Ser Leu 595 600 605 Lys Thr Lys Asn Gln Val Leu Lys Thr Glu Lys Glu Lys Gln Asp Ile 610 615 620 Ser His Arg Glu His Leu Glu Asp Leu Ile Glu Arg Gln Ser Arg Glu 625 630 635 640 Leu Gln Asp Leu Glu Cys Cys Asn Asn Gln Lys Leu Leu Leu Glu Tyr 645 650 655 Glu Lys Tyr Gln Glu Leu Gln Leu Lys Ser Gln Arg Met Gln Glu Glu 660 665 670 Tyr Glu Lys Gln Leu Arg Asp Asn Asp Glu Thr Lys Ser Gln Ala Leu 675 680 685 Glu Glu Leu Thr Glu Phe Tyr Glu Ala Lys Leu Gln Glu Lys Thr Gly 690 695 700 Leu Leu Glu Glu Ala Leu Ser Thr Ala Ala Ser Pro Pro Leu Pro Ser 705 710 715 720 Ala His Val Leu Ser Pro Phe Pro Thr Leu Ser Gln Ala Gln Glu Asp 725 730 735 Val Arg Gln Gln Leu Arg Glu Phe Glu Glu Thr Lys Lys Gln Ile Glu 740 745 750 Glu Asp Glu Asp Arg Glu Ile Gln Asp Ile Lys Thr Lys Tyr Glu Arg 755 760 765 Lys Leu Arg Asp Glu Lys Glu Ser Asn Leu Arg Leu Lys Gly Glu Thr 770 775 780 Gly Ile Met Arg Lys Lys Phe Ser Ser Leu Gln Lys Glu Ile Glu Glu 785 790 795 800 Arg Thr Asn Asp Ile Glu Leu Leu Lys Thr Glu Gln Val Lys Leu Gln 805 810 815 Gly Val Ile Arg Ser Leu Glu Lys Asp Ile Gln Gly Leu Lys Arg Glu 820 825 830 Ile Gln Glu Arg Asp Glu Thr Ile Gln Asp Lys Glu Lys Arg Ile Tyr 835 840 845 Asp Leu Lys Lys Lys Asn Gln Glu Leu Glu Lys Phe Lys Phe Val Leu 850 855 860 Asp Tyr Lys Ile Lys Glu Leu Lys Lys Gln Ile Glu Pro Arg Glu Asn 865 870 875 880 Glu Ile Lys Val Met Lys Glu Gln Ile Gln Glu Asn Pro Val Asn His 885 890 895 Trp Leu Arg Ser Arg Glu Arg Glu Cys Val Thr Gln Pro Arg His Leu 900 905 910 Arg Leu Pro Ala Pro Gln Asn Lys Leu Asp Gly Asn Leu Ala Cys Gly 915 920 925 Pro Val Arg Gly Arg Leu Cys His Ser Asp Ala Thr Ser Gly Ala Leu 930 935 940 Asn Val Gln Gly Ile Leu Cys Leu Phe His Leu Pro Phe Pro Cys Asp 945 950 955 960 Arg Thr Pro Ser Phe Phe Pro Gly Glu Ala Cys Leu Leu Val Phe Ser 965 970 975 Leu Leu Ile Asp Val Leu Cys Arg Pro Thr Ser Asp Val Pro Val Ala 980 985 990 Ala Gly Asp Phe Leu Pro Cys Gly Gly Pro Leu His Leu Pro Pro Glu 995 1000 1005 Leu His His Leu Thr Val Ile Arg Thr Asn Ala Ser Pro Gln Lys 1010 1015 1020 Cys Tyr Pro Pro Thr Ser Pro Leu

1025 1030 88 70 PRT Mus musculus 88 Lys Lys Phe Ser Ser Leu Gln Lys Glu Ile Glu Glu Arg Thr Asn Asp 1 5 10 15 Ile Glu Leu Leu Lys Ser Glu Arg Met Lys Leu Gln Gly Ile Ile Arg 20 25 30 Ser Leu Glu Lys Asp Ile Gln Gly Leu Lys Arg Glu Ile Gln Glu Arg 35 40 45 Asp Glu Thr Ile Gln Asp Met Glu Lys Leu Asp Tyr Lys Asp Asp Tyr 50 55 60 Asn Ser Asn Leu Glu Ile 65 70 89 56 PRT Mus musculus 89 Lys Lys Phe Ser Ser Leu Gln Lys Glu Ile Glu Glu Arg Thr Asn Asp 1 5 10 15 Ile Glu Leu Leu Lys Ser Glu Arg Met Lys Leu Gln Gly Ile Ile Arg 20 25 30 Ser Leu Glu Lys Asp Ile Gln Gly Leu Lys Arg Glu Ile Gln Glu Arg 35 40 45 Asp Glu Thr Ile Gln Asp Met Glu 50 55 90 217 PRT Mus musculus 90 Met Glu Val Glu Asn Glu Ala His Cys Cys Pro Gly Ser Ser Ser Gly 1 5 10 15 Gly Ser Arg Glu Tyr Lys Val Val Met Leu Gly Ala Gly Gly Val Gly 20 25 30 Lys Ser Ala Val Thr Met Gln Phe Ile Ser His Gln Phe Pro Asp Tyr 35 40 45 His Asp Pro Thr Ile Glu Asp Ala Tyr Lys Thr Gln Val Arg Ile Asp 50 55 60 Asn Glu Pro Ala Tyr Leu Asp Ile Leu Asp Thr Ala Gly Gln Ala Glu 65 70 75 80 Phe Thr Ala Met Arg Glu Gln Tyr Met Arg Gly Gly Glu Gly Phe Ile 85 90 95 Ile Cys Tyr Ser Val Thr Asp Arg Gln Ser Phe Gln Glu Ala Ala Lys 100 105 110 Phe Lys Glu Leu Ile Phe Gln Val Arg His Thr Tyr Glu Ile Pro Leu 115 120 125 Val Leu Val Gly Asn Lys Ile Asp Leu Glu Gln Phe Arg Gln Val Ser 130 135 140 Thr Glu Glu Gly Met Asn Leu Ala Arg Asp Tyr Asn Cys Ala Phe Phe 145 150 155 160 Glu Thr Ser Ala Ala Leu Arg Phe Gly Ile Asp Asp Ala Phe Gln Gly 165 170 175 Leu Val Arg Glu Ile Arg Arg Lys Glu Ser Met Leu Ser Leu Val Glu 180 185 190 Arg Lys Leu Lys Arg Lys Asp Ser Leu Trp Lys Lys Ile Lys Ala Ser 195 200 205 Leu Lys Lys Lys Arg Glu Asn Met Leu 210 215 91 50 PRT Mus musculus 91 Ala Ala Leu Arg Phe Gly Ile Asp Asp Ala Leu Gln Gly Leu Val Arg 1 5 10 15 Glu Ile Arg Arg Lys Glu Ser Met Leu Pro Leu Val Glu Arg Lys Leu 20 25 30 Lys Arg Lys Asp Ser Leu Trp Lys Lys Ile Lys Ala Ser Leu Lys Lys 35 40 45 Lys Arg 50 92 140 PRT Mus musculus 92 Gly Ala Thr Val Ile Thr Asn Leu Leu Ser Ala Ile Pro Tyr Ile Gly 1 5 10 15 Thr Thr Leu Val Glu Trp Ile Trp Gly Gly Phe Ser Val Asp Lys Ala 20 25 30 Thr Leu Thr Arg Phe Phe Ala Phe His Phe Ile Leu Pro Phe Ile Ile 35 40 45 Ala Ala Leu Ala Ile Val His Leu Leu Phe Leu His Glu Thr Gly Ser 50 55 60 Asn Asn Pro Thr Gly Leu Asn Ser Asp Ala Asp Lys Ile Pro Phe His 65 70 75 80 Pro Tyr Tyr Thr Ile Lys Asp Ile Leu Gly Ile Leu Ile Met Phe Leu 85 90 95 Ile Leu Met Thr Leu Val Leu Phe Phe Pro Asp Met Leu Gly Asp Pro 100 105 110 Asp Asn Tyr Met Pro Ala Asn Pro Leu Asn Thr Pro Pro His Ile Lys 115 120 125 Pro Glu Trp Tyr Phe Leu Phe Ala Tyr Ala Ile Leu 130 135 140 93 41 PRT Mus musculus 93 Ser Asp Ala Asp Lys Ile Pro Phe His Pro Tyr Tyr Thr Ile Lys Asn 1 5 10 15 Ile Leu Gly Ile Leu Ile Ile Phe Leu Ile Leu Ile Thr Leu Val Leu 20 25 30 Phe Phe Pro Asp Ile Leu Gly Asp Pro 35 40 94 311 PRT Mus musculus 94 Met Lys Ala Leu Trp Ala Val Leu Leu Val Thr Leu Leu Thr Gly Cys 1 5 10 15 Leu Ala Glu Gly Glu Pro Glu Val Thr Asp Gln Leu Glu Trp Gln Ser 20 25 30 Asn Gln Pro Trp Glu Gln Ala Leu Asn Arg Phe Trp Asp Tyr Leu Arg 35 40 45 Trp Val Gln Thr Leu Ser Asp Gln Val Gln Glu Glu Leu Gln Ser Ser 50 55 60 Gln Val Thr Gln Glu Leu Thr Ala Leu Met Glu Asp Thr Met Thr Glu 65 70 75 80 Val Lys Ala Tyr Lys Lys Glu Leu Glu Glu Gln Leu Gly Pro Val Ala 85 90 95 Glu Glu Thr Arg Ala Arg Leu Gly Lys Glu Val Gln Ala Ala Gln Ala 100 105 110 Arg Leu Gly Ala Asp Met Glu Asp Leu Arg Asn Arg Leu Gly Gln Tyr 115 120 125 Arg Asn Glu Val His Thr Met Leu Gly Gln Ser Thr Glu Glu Ile Arg 130 135 140 Ala Arg Leu Ser Thr His Leu Arg Lys Met Arg Lys Arg Leu Met Arg 145 150 155 160 Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala Val Tyr Lys Ala Gly Ala 165 170 175 Arg Glu Gly Ala Glu Arg Gly Val Ser Ala Ile Arg Glu Arg Leu Gly 180 185 190 Pro Leu Val Glu Gln Gly Arg Gln Arg Thr Ala Asn Leu Gly Ala Gly 195 200 205 Ala Ala Gln Pro Leu Arg Asp Arg Ala Gln Ala Phe Gly Asp Arg Ile 210 215 220 Arg Gly Arg Leu Glu Glu Val Gly Asn Gln Ala Arg Asp Arg Leu Glu 225 230 235 240 Glu Val Arg Glu His Met Glu Glu Val Arg Ser Lys Met Glu Glu Gln 245 250 255 Thr Gln Gln Ile Arg Leu Gln Ala Glu Ile Phe Gln Ala Arg Leu Lys 260 265 270 Gly Trp Phe Glu Pro Ile Val Glu Asp Met His Arg Gln Trp Ala Asn 275 280 285 Leu Met Glu Lys Ile Gln Ala Ser Val Ala Thr Asn Pro Ile Ile Thr 290 295 300 Pro Val Ala Gln Glu Asn Gln 305 310 95 31 PRT Mus musculus 95 Thr Glu Val Lys Ala Tyr Lys Lys Glu Leu Glu Glu Gln Leu Gly Pro 1 5 10 15 Val Ala Glu Glu Thr Arg Ala Arg Leu Gly Lys Glu Glu Gln Gly 20 25 30 96 695 PRT Mus musculus 96 Met Leu Pro Ser Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Val Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Lys Leu Asn Met His Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Glu Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Gly 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Thr His Ile Val 100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Ser Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Val Gly Ala Asp Thr Asp Tyr Ala Asp Gly Gly Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Asp Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Val Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Thr 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu 355 360 365 Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Asp Leu Glu Asn 385 390 395 400 Tyr Ile Ile Ala Leu Gln Ala Val Pro Pro Arg Pro His His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Thr Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln 530 535 540 Pro Trp His Pro Phe Gly Val Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala Glu Phe Gly His Asp Ser Gly Phe Glu Val 595 600 605 Arg His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu Gln Met Gln Asn 690 695 97 68 PRT Mus musculus 97 Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu 1 5 10 15 Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe 20 25 30 Ser Leu Asp Asp Leu Gln Pro Trp His Pro Ser Gly Val Asp Ser Val 35 40 45 Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala 50 55 60 Ala Asp Arg Gly 65 98 983 PRT Mus musculus 98 Met Lys Ala Gln Gln Ala Met Asp Lys Tyr Glu Gly Asp Ser Lys Ala 1 5 10 15 Arg Glu Thr Arg Ser Thr Ala Ala Met Val Gly Trp Arg Ser Asp Arg 20 25 30 Gly Leu Val Thr Cys Thr Arg Leu Arg Met Gln Asn Gly Ser Ser Leu 35 40 45 Lys Ala Phe Arg Ser Arg Val Gly Lys Trp Gly Glu Pro Ser Ser Arg 50 55 60 Ser His Lys Val Leu Lys Thr Ser Glu Thr Ser Gln Asp Ile Gln Lys 65 70 75 80 Val Ser Arg Glu Glu Ser Pro Ser Gln Leu Thr Ser Ala Val Pro Ala 85 90 95 Gln Arg Asn Cys Gln Pro Gly Ser Ala Ala Val Ile Asn Met Leu Arg 100 105 110 Gly Gly Gly Gly Val Arg Ser Pro Trp Thr Asp His His Ile Arg Gln 115 120 125 Arg Thr Asp His His Ile Arg Gln Pro Leu Phe Pro Ser Arg Arg Ser 130 135 140 Pro Gln Glu Asn Glu Asp Asp Asp Asp Asp Tyr Gln Met Phe Val Pro 145 150 155 160 Ser Phe Ser Ser Ser Asp Leu Asn Ser Thr Arg Leu Cys Glu Glu Asn 165 170 175 Ala Ser Ser Arg Pro Cys Ser Trp His Leu Gly Leu Ile Glu Pro Thr 180 185 190 Glu Ile Ser Ser Ser Gly His Arg Ile Val Arg Arg Ala Ser Ser Ala 195 200 205 Gly Glu Ser Asn Ala Cys Pro Pro Glu Val Arg Ile Arg Asp Cys Asp 210 215 220 Asp Ser Gln Tyr Cys Pro Gly Arg Gln Leu Gln Asn Ser Pro Arg Pro 225 230 235 240 Gly Gly Glu Arg Gly Met Thr Pro Tyr Gly Ser Ser Val Glu Leu Thr 245 250 255 Ile Asp Asp Ile Asp His Val Tyr Asp Asn Ile Ser Phe Glu Asp Leu 260 265 270 Lys Leu Met Val Ala Lys Arg Asp Glu Thr Glu Cys Ser Phe Ser Lys 275 280 285 Pro Ser Arg Asp Ser Val Arg Pro Lys Ser Thr Pro Glu Leu Ala Phe 290 295 300 Ser Lys Arg Gln Val Ser His Ser Thr Ser Ser Leu His Ser Arg Lys 305 310 315 320 Glu Ala Gly Leu Gly Gly Gln Glu Ala Ser Thr Gln Ser Val His Glu 325 330 335 His Gln Glu Val Glu Glu Asn Ile Tyr Asp Thr Ile Gly Leu Pro Asp 340 345 350 Pro Pro Ser Met Asn Leu Asn His Ser Ser Leu His Gln Pro Lys Arg 355 360 365 Ser Thr Phe Leu Gly Leu Glu Ala Asp Phe Ala Cys Cys Asp Ser Leu 370 375 380 Arg Pro Phe Val Ser Gln Asp Ser Leu Gln Phe Ser Glu Asp Asp Ile 385 390 395 400 Ser Tyr His Gln Gly Pro Ser Asp Thr Glu Tyr Leu Ser Leu Leu Tyr 405 410 415 Asp Ser Pro Arg Cys Asn Leu Pro Ile Ala Asp Lys Ala Leu Ser Asp 420 425 430 Lys Leu Ser Glu Glu Val Asp Glu Ile Trp Asn Asp Leu Glu Asn Tyr 435 440 445 Ile Lys Lys Asn Glu Asp Lys Ser Arg Asp Arg Leu Leu Ala Ala Phe 450 455 460 Pro Val Ser Lys Asp Asp Ala Pro Glu Arg Leu Tyr Val Asp Ser Thr 465 470 475 480 His Glu Leu Gly Arg Asp Thr Gly His Ala Thr Ser Met Leu Ala Leu 485 490 495 Pro Thr Ser Gln Thr Phe Leu Leu Pro Gly Lys Ser Arg Val Val Arg 500 505 510 Ala Ser Arg Ala Asn Cys Ser Leu Asp Asn Asp Ile Ile Ser Thr Glu 515 520 525 Gly Ser Phe Leu Ser Leu Asn Gln Leu Ser Leu Ala Ser Asp Gly Pro 530 535 540 Pro Val Asp Asn Pro Tyr Asp Leu Ala Asn Cys Ser Leu Pro Gln Thr 545 550 555 560 Asp Pro Glu Asn Pro Asp Pro Gly Met Glu Val Thr Asp Lys Thr Lys 565 570 575 Ser Arg Val Phe Met Met Ala Arg Gln Tyr Ser Gln Lys Ile Lys Lys 580 585 590 Val Asn Gln Ile Leu Lys Val Lys Ser Pro Glu Leu Glu Gln Pro Pro 595 600 605 Ser Ser Gln His Arg Pro Ser His Lys Asp Leu Val Ala Ile Leu Glu 610 615 620 Glu Lys Arg Gln Gly Gly Pro Ala Ile Gly Ala Arg Ile Ala Glu Tyr 625 630 635 640 Ser Gln Leu Tyr Asp Gln Ile Val Phe Arg Glu Thr Pro Leu Lys Ala 645 650 655 Gln Lys Asp Gly Trp Ala Ser Pro Gln Gly Pro Thr Leu His Arg Pro 660 665 670 Val Ser Pro Pro Gln Ala Gln Gly Ala Gly Glu Asp Trp Leu Trp His 675 680 685 Ser Pro Tyr Ser Asn Gly Glu Leu Ala Asp Phe Ser Pro Gln Thr Glu 690 695 700 Gln Asp Ser Lys Ser Lys Tyr Pro Ile Thr Leu Glu Ser Thr Thr Lys 705 710 715 720 Ile Arg Pro Arg Gln Leu Ser Gly Ala Cys Ser Val Pro Ser Leu Gln

725 730 735 Val Ser Asp Pro Leu Leu Gly Ser Val Gln Gln Arg Cys Ser Val Val 740 745 750 Val Ser Gln Pro His Lys Glu Asn Ser Gly Gln Ser Pro Leu Tyr Asn 755 760 765 Ser Leu Gly Arg Lys Ala Ile Ser Ala Lys Pro Gln Pro Tyr Ser Arg 770 775 780 Pro Gln Ser Ser Ser Ser Ile Leu Ile Asn Lys Ser Leu Asp Ser Ile 785 790 795 800 Asn Tyr Pro Ser Glu Thr Glu Thr Lys Gln Leu Leu Ser Ser Gln Lys 805 810 815 Ser Pro Arg Gly Ala Ser Gln Gln Asp Leu Pro Ser Gly Leu Ala Asn 820 825 830 Ser Cys Gln Gln Asp Arg Gly Lys Arg Ser Asp Leu Thr Leu Gln Asp 835 840 845 Ser Gln Lys Val Leu Val Val Asn Arg Asn Leu Pro Leu Ser Ala Gln 850 855 860 Ile Ala Thr Gln Asn Tyr Phe Cys Asn Phe Lys Asp Pro Glu Gly Asp 865 870 875 880 Glu Asp Asp Tyr Val Glu Ile Lys Ser Glu Glu Asp Glu Val Arg Leu 885 890 895 Asp Leu Ser Pro Arg Arg Gly Arg Lys Ser Asp Pro Gln Thr Pro Asp 900 905 910 Pro Asp Cys Ser Asp Ser Ile Cys Ser His Ser Thr Pro Tyr Ser Leu 915 920 925 Lys Glu Pro Val Ser Gly Arg Leu Gly Leu Pro Pro Tyr Leu Thr Ala 930 935 940 Cys Lys Asp Ser Asp Lys Leu Asn Asp Tyr Leu Trp Arg Gly Pro Ser 945 950 955 960 Pro Asn Gln Gln Asn Ile Val Gln Ser Leu Arg Glu Lys Phe Gln Cys 965 970 975 Leu Ser Ser Ser Ser Phe Ala 980 99 46 PRT Mus musculus 99 Phe Met Met Ala Arg Gln Tyr Ser Gln Lys Ile Lys Lys Val Asn Gln 1 5 10 15 Ile Leu Lys Val Lys Ser Pro Glu Leu Glu Gln Pro Pro Ser Ser Gln 20 25 30 His Arg Pro Ser His Lys Asp Leu Ala Ala Ile Leu Glu Lys 35 40 45 100 412 PRT Mus musculus 100 Met Ala Asn Val Ala Asp Thr Lys Leu Tyr Asp Ile Leu Gly Val Pro 1 5 10 15 Pro Gly Ala Ser Glu Asn Glu Leu Lys Lys Ala Tyr Arg Lys Leu Ala 20 25 30 Lys Glu Tyr His Pro Asp Lys Asn Pro Asn Ala Gly Asp Lys Phe Lys 35 40 45 Glu Ile Ser Phe Ala Tyr Glu Val Leu Ser Asn Pro Glu Lys Arg Glu 50 55 60 Leu Tyr Asp Arg Tyr Gly Glu Gln Gly Leu Arg Glu Gly Ser Gly Gly 65 70 75 80 Gly Gly Gly Met Asp Asp Ile Phe Ser His Ile Phe Gly Gly Gly Leu 85 90 95 Phe Gly Phe Met Gly Asn Gln Ser Arg Ser Arg Asn Gly Arg Arg Arg 100 105 110 Gly Glu Asp Met Met His Pro Leu Lys Val Ser Leu Glu Asp Leu Tyr 115 120 125 Asn Gly Lys Thr Thr Lys Leu Gln Leu Ser Lys Asn Val Leu Cys Ser 130 135 140 Ala Cys Ser Gly Gln Gly Gly Lys Ser Gly Ala Val Gln Lys Cys Ser 145 150 155 160 Ala Cys Arg Gly Arg Gly Val Arg Ile Met Ile Arg Gln Leu Ala Pro 165 170 175 Gly Met Val Gln Gln Met Gln Ser Val Cys Ser Asp Cys Asn Gly Glu 180 185 190 Gly Glu Val Ile Asn Glu Lys Asp Arg Cys Lys Lys Cys Glu Gly Lys 195 200 205 Lys Val Ile Lys Glu Val Lys Ile Leu Glu Val His Val Asp Lys Gly 210 215 220 Met Lys His Gly Gln Arg Ile Thr Phe Thr Gly Glu Ala Asp Gln Ala 225 230 235 240 Pro Gly Val Glu Pro Gly Asp Ile Val Leu Leu Leu Gln Glu Lys Glu 245 250 255 His Glu Val Phe Gln Arg Asp Gly Asn Asp Leu His Met Thr Tyr Lys 260 265 270 Ile Gly Leu Val Glu Ala Leu Cys Gly Phe Gln Phe Thr Phe Lys His 275 280 285 Leu Asp Ala Arg Gln Ile Val Val Lys Tyr Pro Pro Gly Lys Val Ile 290 295 300 Glu Pro Gly Cys Val Arg Val Val Arg Gly Glu Gly Met Pro Gln Tyr 305 310 315 320 Arg Asn Pro Phe Glu Lys Gly Asp Leu Tyr Ile Lys Phe Asp Val Gln 325 330 335 Phe Pro Glu Asn Asn Trp Ile Asn Pro Asp Lys Leu Ser Glu Leu Glu 340 345 350 Asp Leu Leu Pro Ser Arg Pro Glu Val Pro Asn Val Ile Gly Glu Thr 355 360 365 Glu Glu Val Glu Leu Gln Glu Phe Asp Ser Thr Arg Gly Ser Gly Gly 370 375 380 Gly Gln Arg Arg Glu Ala Tyr Asn Asp Ser Ser Asp Glu Glu Ser Ser 385 390 395 400 Ser His His Gly Pro Gly Val Gln Cys Ala His Gln 405 410 101 25 PRT Mus musculus 101 Leu Ser Asn Pro Glu Lys Arg Glu Leu Tyr Asp Arg Tyr Gly Glu Gln 1 5 10 15 Gly Leu Arg Glu Gly Ser Gly Gly Gly 20 25 102 29 PRT Mus musculus 102 Ala His Ser Phe Ser Val Phe Arg Leu Pro Ser Trp Trp Ile Val Gly 1 5 10 15 Trp Trp Ser Lys Gly Gly Val Gly Ser Asp Leu Glu Met 20 25 103 35 PRT Mus musculus 103 Pro Asp Ile Lys His Pro Gly Asn Leu Glu His Tyr Ile Lys Arg Val 1 5 10 15 Asn Leu Arg Ile Ile Ala Ile Glu Glu Gly Glu Lys Ser Gln Leu Lys 20 25 30 Gly Pro Lys 35 104 573 DNA Mus musculus 104 atgccattga ggcatctagc agacagattg gggcatctgg cagacagact gaggcatcta 60 acagacagat tgaggcatct agcagacaga ctgaggcatc taacagacag actgaggcat 120 ctagcagaca gactgaagca tctagcagac agactgaaac atctaacaga cagattgggg 180 catctaacag acagatcatg gcatctaaca gacagattgg ggcatctaac agacagattg 240 aggcatctaa cagacagatt ggggcatcta acagacagac agaggtatct agcagacaga 300 ttgaggcatc taacagacag attggggcat ctaacagaca aactgaggca tctaacagac 360 agattggggc atctaacaga cagactgagg catctaacag acagattggg gcatctaaca 420 gacagactga tgcatctaac agacagactg atgcatctaa cagacagact gaggcatcta 480 gcagacagac agaggcatct agcagacaga cagaggcatc tagcagacag actgaggcat 540 ctagcagaca aattgaggca tcagctgcag ctg 573 105 213 DNA Mus musculus 105 attgaggcat ctaacagaca gattggggca tctaacagac aaactgaggc atctaacaga 60 cagattgggc atctaacaga cagactgagg catctaacag acagattggg gcatctaaca 120 gacagactga tgcatctaac agacagactg atgcatctaa cagacagact gaggcatcta 180 gcagacagac agaggcatct agcagacaga ctg 213 106 165 DNA Mus musculus 106 gacaaactga ggcatctaac agacagattg gggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgatgcatc taacagacag actgatgcat 120 ctaacagaca gactgaggca tctagcagac agacagaggc acgac 165 107 135 DNA Mus musculus 107 gacagattgg ggcatctaac agacagactg aggcatctaa cagacagatt ggggcatcta 60 acagacagac tgatgcatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 108 135 DNA Mus musculus 108 gacagattgg ggcatctaac agacagactg aggcatctaa cagacagatt gggacatcta 60 acagacagac tgatgcatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 109 135 DNA Mus musculus 109 gacagattgg ggcatctaac agacagactg aggcatctaa cagacagatt ggggcatcta 60 acagacagac tgatgcatct aacagacaga ctgatgcatc taacagatag actgaggcat 120 ctagcagtca gacag 135 110 135 DNA Mus musculus 110 gacagattgg ggcatctaac agacagactg aggcatctaa cagacagatt ggggcatcta 60 acggacagac tgatgcatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 111 135 DNA Mus musculus 111 gacagattgg ggcatctaac agacagactg aggcatctaa cagacagatt ggggcatcta 60 acagacagac tgatgcatct aacagacaga ctgatgcatc taacagacag actgagacat 120 ctagcagtca gacag 135 112 135 DNA Mus musculus 112 gacagattgg ggcgtctaac agacagactg aggcatctaa cagacagatt ggggcatcta 60 acagacagac tgatgcatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 113 135 DNA Mus musculus 113 gacagattgg ggcgtctaac agacagactg aggcatctaa cagacagatt ggggcatcta 60 acagacaggc tgatgcatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 114 135 DNA Mus musculus 114 gacagattgg ggcatctaac agacagactg aggtatctaa cagacagatt ggggcatcta 60 acagacagac tgatgcatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 115 135 DNA Mus musculus 115 gacagattgg ggcatctaac agacagactg aggcatctaa cagacagatt ggggcatcta 60 acagacagac tgatgcatct aacagacaga ctgacgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 116 135 DNA Mus musculus 116 gacagattgg ggcatctaac agacagactg aagcatctaa cagacagatt ggggcatcta 60 acagacagac tgatccatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcagtca gacag 135 117 135 DNA Mus musculus 117 ggcagattgg ggcatctaac agacagactg aggcatctaa cagacagatt ggggcatcta 60 acagacagac tgatgcatct aacagacaga ctgatgcatc taacagacag actgaggcat 120 ctagcggtca gacag 135 118 114 DNA Mus musculus 118 gacagattga ggcatctagc agacagactg aggcatctaa ccgacagact gaggcatcta 60 gcagacagac tgaagcatct agcagacaga ctgaaacatc taacaaacag aaag 114 119 552 DNA Mus musculus 119 atgccattga ggcatctagc agacagattg gggcatctgg cagacagact gaggcatcta 60 acagacagat tgaggcatct agcagacaga ctgaggcatc taacagacag actgaggcat 120 ctagcagaca gactgaggca tctagcagac agactgaaac atctaacaga cagattgggg 180 catctaacag acagatcatg gcatctaaca gacagattgg ggcatctaac agacagattg 240 aggcatctaa cagacagatt ggggcatcta acagacagac agaggtatct agcagacaga 300 ttgaggcatc taacagacag attggggcat ctaacagaca gactgaggca tctaacagac 360 agattggggc atctaacaga cagactgagg catctaacag acagattggg gcatctaaca 420 gacagactga tgcatctaac agacagactg aggcatctag cagacagaca gaggcatcta 480 gcagacagac agaggcatct agcagacaga ctgaggcatc tagcagacaa attgaggcat 540 cagctgcagc tg 552 120 237 DNA Mus musculus 120 gaaaaagtga aaaccttgaa agcgcaaaac tccgagctgg catccacggc caacacgctc 60 agggaacagg tggcactgct taagcagaaa gtcatgaacc acgttaacag tgggtgccaa 120 ctcatgctaa cgcagcagtt gcaaacgttt tgggaacaga ctgtcagggc tgaggggcaa 180 tggaagaaaa aaaataacag agacaaactt gagaacttga ctggttgcga cagagaa 237 121 228 DNA Mus musculus 121 cggatcaagg cagagaggaa gcgcatgagg aaccgcattg ccgcctccga gtgccggaaa 60 aggaagctgg agcggatcgc tcggctagag gaaaaagtga aaaccttgaa agcgcaaaac 120 tccgagctgg catccacggc caacatgctc agggaacagg tggcacagct taagcagaaa 180 gtcatgaacc acgttaacag tgggtgccaa ctcatgctaa cacagcag 228 122 149 DNA Mus musculus 122 gggcatctgg cagacagact gaggcatcta acagacagat tgaggcatct agcagacaga 60 ctgaggcatc taacagacag actgaggcat ctagcagaca gactgaggca tctagcagac 120 agactgaaac atctaacaga cagatatgg 149 123 168 DNA Mus musculus 123 catctaacag acagactgag gcatctaaca gacagattgg ggcatctaac agacagactg 60 aggcatctaa cagacagatt gaggcatcta acagacagac tgatgcatct aacagacaga 120 ctggggcatc tagcagacag acagaggcat ctagcagaca gacagaga 168 124 132 DNA Mus musculus 124 gcagacagat tggggcatct ggcagacaga ctgaggcatc taacagacag attgaggcat 60 ctagcagaca gactgaggca tctaacagac agactgaggc atttagcaga cagactgagg 120 catctagcag ac 132 125 132 DNA Mus musculus 125 gcagacagat tggggcatct ggcagacaga ctgaggcatc taacagacag attgaggcat 60 ctagcagaca gactgagaca tctaacagac agactgaggc atttagcaga cagactgagg 120 catctagcag ac 132 126 81 DNA Mus musculus 126 gcagacagac tgaggcatct aacagacaga ttgaggcatc tagcagacag actgaggcat 60 ctaacagaca gactgaggca c 81 127 159 DNA Mus musculus 127 acagacagat tggggcatct aacagacaga ctgaggcatc taacagacag attggggcat 60 ctaacagaca gactgaggca tctaacagac agattggggc atctaacaga cagactgatg 120 catctaacag acagactgag gcatctagca gacagaccg 159 128 138 DNA Mus musculus 128 agacagaggc atctaacaga cagactgagg catctaacag acagattggg gcatctaaca 60 gacagactga ggcatctaac agacagattg gggcatctaa cagacagact gatgcatcta 120 acagacagac tgaggccc 138 129 117 DNA Mus musculus 129 gacagattga gccatctagc agacagactg aggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgaggcatc tagcagacag acagagg 117 130 117 DNA Mus musculus 130 gacagattga ggcatctagc agacagactg aggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgaggcatc tagcagacag acagagg 117 131 117 DNA Mus musculus 131 ggcagattga ggcatctagc agacagactg aggcatctaa cagacagact gaggcatcta 60 acagacagat tggggcatct aacagacaga ctgaggcatc tagcagacag acagagg 117 132 129 DNA Mus musculus 132 tatctagcag acagattgag gcatctaaca gacagattgg ggcatctgac agacaggctg 60 aggcatctaa cagacagatt ggggcatcta acagacagac tgaggcatct aacagacaga 120 ttggggcaa 129 133 129 DNA Mus musculus 133 tatctagcag acagattgag gcatctaaca gacagattga ggcatctaac agacaggctg 60 aggcatctaa cagacagatt ggggcatcta acagacagac tgaggcatct aacagacaga 120 ttggggcaa 129 134 228 DNA Mus musculus 134 cggatcaagg cagagaggaa gcgcatgagg aaccgcattg ccgcctccaa gtgccggaaa 60 aggaagctgg agcggatcgc tcggctagag gaaaaagtga aaaccttgaa agcgcaaaac 120 tccgagctgg catccacggc caacatgctc agggaacagg tggcacagct taagcagaaa 180 gtcatgaacc acgttaacag tgggtgccaa ctcatgctaa cacagcag 228 135 132 DNA Mus musculus 135 ttggggcatc taacagacag actgaggcat ctaacagaca gattggggca tctaacagac 60 agactgatgc atctaacaga cagactgagg catctagcag acagacagag gcatctagca 120 gacagacaga aa 132 136 108 DNA Mus musculus 136 ttggggcatc taacagacag actgaggcat ctaacagaca gattggggca tctaacagac 60 agactgatgc atctaacaga cagactgagg catctagcag acacccag 108 137 132 DNA Mus musculus 137 gggcatctaa cagacagact gaggcatcta acggacagat tggggcatct aacagacaga 60 ctgaggcatc taacagacag attggggcat ctaacagaca gactgatgca tctaacagac 120 agactgaggc ac 132 138 132 DNA Mus musculus 138 gggcatctaa cagacagact gaggcatcta acagacagat tggggcatct aacagacaga 60 ctgaggcatc taacagacag attggggcat ctaacagaca gactgatgca tctaacagac 120 agactgaggc ac 132 139 153 DNA Mus musculus 139 aggcatctaa cagacagatt ggggcatcta acagacagac tgaggcatct aacagacaga 60 ttggggcatc taacagacag actgatgcat ctaacagaca gactgaggca tctagcagac 120 agacagaggc atctagcaga caggcagagg cac 153 140 153 DNA Mus musculus 140 aggcatctaa cagacagatt ggggcatcta acagacagac tgaggcatct aacagacaga 60 ttggggcgtc taacagacag actgatgcat ctaacagaca gactgaggca tctagcagac 120 agacagaggc atctagcaga cagacagagg cac 153 141 1752 DNA Mus musculus 141 atgtcccatc aacctctgag ctgcctgact gagaaggggg acagcccttg tgagacccca 60 ggaaatggac cctccaatat ggttcacccc agcctggaca cattcacccc tgaggagctg 120 ctgcagcaaa tgaaggaact cctggttgag aaccaccagc tgaaagaagc catgaagcta 180 aataatcaag ctatgaaagg gcgatttgag gagctgtccg cctggacaga gaagcagaag 240 gaagagcgcc tgttgtttga gatgcaaagc aaagaggtta aggagcgcct taaggccctg 300 actcatgaaa atgagaggct gaaggaagag cttggaaaat tcaaagagaa atcagaaaag 360 ccattggaag acctcacagg tggctacagg tatcccagag ccttggagga ggaagtggag 420 aagctgaaga cccaggtgga gcaggaagtg gagcatctga agatccaggt gatgcgcctt 480 cgggctgaaa aggcagacct gctgggcatc gtctcagaac tgcagctcaa actcaactcc 540 ggcggctcct cggaagactc cttcgttgag atcaggatga ccgaaggaga gactgaaggg 600 gcaatgaagg agatgaagaa ctgccctaca cccacaagaa cagaccccat cagcttgagc 660 aactgtacag aggatgccag gagttgtgcg gagtttgaag aactgactgt gagccagctt 720 ctgctttgcc taagggaagg aaaccaaaag gtggagagac ttgaagtcgc cctcagagaa 780 gccaaagaaa gaatttcaga ttttgaaaag aaagcaaatg gccattcttc tactgagaag 840 cagacagcga ggagagcaga cagagagaag gaggacaaag gccaagagag tgttggaagc 900 gaagtggaaa cactgagcat tcaagtgacc tctctgttta aggagcttca agaggcacac 960 acaaaactca gtgaggctga gctgatgaag aagagacttc aagaaaagtg tcaggctctg 1020 gagaggaaga actctgcaac accatcagag ctgaatgaaa agcaagagct cgtttacagt 1080 aacaagaagt tagagctgca ggtggagagc atgcgctccg aaatcaagat ggagcaggcc 1140 aagacagagg aggagaagtc caggttagcc actctgcagg caactcacaa caagctcctt 1200 caagaacata ataaggcact gaaaacaatt gaagaactaa ccaagcaaca ggcagaaaag 1260 gtggacaaga tgttgctgca ggagctcagc gagaagctgg agctggcaga gcaggctctg 1320 gcatccaaac agctccagat ggatgagatg aagcagacgc tcgctaagca ggaggaagac 1380 ctggagacca tggccgtcct cagggctcag atggaggtgt actgctcaga ttttcacgct 1440 gagagagcag caagagagaa gattcatgaa gaaaaggagc agctggcctt gcagctcgcg 1500 attttgctga aagagaacaa tgacattgaa gagggaggca gtagacagtc

cctgatggaa 1560 atgcagtgcc gacacggggc aagaaccagt gactctgacc agcagactta cctgtttcaa 1620 agaggagccg aggacaggag ctggcagcac gggcagcagc ctcgcagtat tccgattcac 1680 tcctgcccca agtgcgggga ggtcctgccg gacatcgaca cgcttcagat ccatgtgatg 1740 gactgcatca tt 1752 142 324 DNA Mus musculus 142 ctgaagaccc aggtggagca ggaagtggag catctgaaga tccaggtgat gcgccttcgg 60 gctgaaaagg cagacctgct gggcatcgtc tcagaactgc agctcaaact caactccggc 120 ggctcctcgg aagactcctt cgttgagatc aggatgaccg aaggagagac tgaaggggca 180 atgaaggaga tgaagagctg ccctacaccc acaagaacag accccatcag cttgagcaac 240 tgtacagagg atgccaggag ttgtgcggag tttgaagaac tgactgtgag ccagcttctg 300 ctttgcctaa gggaaggaaa ccaa 324 143 186 DNA Mus musculus 143 catctgaaga tccaggtgat gcgccttcgg gctgaaaagg cagacctgct gggcatcgtc 60 tcagaactgc agctcaaact caactccggc ggctcctcgg aagactcctt cgttgagatc 120 aggatgaccg aaggagagac tgaaggggca atgaaggaga tgaagaactg ccctacaccc 180 acaaga 186 144 186 DNA Mus musculus 144 catctgaaga tccaggtgat gcgccttcgg gctgaaaagg cagacctgct gggcatcgtc 60 tcagaactgc agctcaaact caactccggc ggctcctcgg aagactcctt cgttgagatc 120 aggatgaccg aaggagagac tgaaggggca atgaaggaga tgaagaactg ccctgcaccc 180 acaaga 186 145 186 DNA Mus musculus 145 catctgaaga tccaggtgat gcgccttcgg gctgaaaagg cagacctgct gggcatcgtc 60 tcagaactgc ggctcaaact caactccggc ggctcctcgg aagactcctt cgttgagatc 120 aggatgaccg aaggagagac tgaaggggca atgaaggaga tgaagaactg ccctacaccc 180 acaaga 186 146 306 DNA Mus musculus 146 atgttgagtc gactgcagga gctccgcaag gaggaggaaa ccctgctgcg tctaaaggcg 60 gctctacacg accaactgaa ccgcctcaag gttgaagaat tagcccttca atccatgata 120 aattctcgag gaaggaccga gacactgtct tctcagcctg cacctgaaca gttatgtgat 180 atgtccctac atgtagacaa cgaagtgaca ataaatcaga ctacactgaa gctgagcaca 240 aggagcccta tggaagaaga ggaggaggaa gaggaggagg aagaggagga ggaagaatct 300 gattcg 306 147 249 DNA Mus musculus 147 gccctaagga agtggaaggg gatgttgagt cgactgcagg agctccgcaa ggaggtggaa 60 accctgctgc gtctaaaggc ggctctacac gaccaactga accgcctcaa ggttgaagaa 120 ttagcccttc aatccatgat aaattctcga ggaaggaccg agacactgtc ttctcagcct 180 gcacctgaac agttatgtga tatgtcccta catgtagaca acgaagtgac aataaatcag 240 actaggccg 249 148 237 DNA Mus musculus 148 aggaagtgga aggggacgtt gagtcgactg caggagctcc gcaaggaggt ggaaaccccg 60 ctgcgtctaa aggcggctct acacgaccaa ctgaaccgcc tcaaggttga agaattagcc 120 cttcaatcca tgataaattc tcgaggaagg accgagacac tgtcttctca gcctgcacct 180 gaacagttat gtgatatgtc cctacatgta gacaacgaag tgacaataaa tcagact 237 149 1239 DNA Mus musculus 149 atgggagacg acagaccgtt tgtgtgcagt gccccgggct gtggacagag atttacaaat 60 gaggaccacc tggcagttca taaacataag catgagatga cactgaaatt tggcccagcc 120 cgaacggact cagtcatcat tgcagatcaa acgcctactc caactagatt cctgaagaac 180 tgtgaggaag tggggctctt caatgaacta gctagctcct ttgaacatga atttaagaaa 240 gcttctgatg acgatgagaa aaagggtgct gctgggcctc ttgacatgtc tctgccttct 300 acaccagaca tcaaaatcaa ggaagaagag ccagtggaag tagactcatc gccccctgac 360 agtcctgctt ctagcccctg ttccccacca ctgaaggaga aggaagttac cacaaaaccg 420 gttgtgatct ctacccctac acctaccatt gtacgtcctg gctccctgcc tctccactta 480 ggttatgatc cacttcatcc aactcttcct tccccaacct ctgtcatcac acaggctcca 540 ccatccaaca ggcaaatagg atctcctact ggctccctcc ctctcgtcat gcatcttgct 600 aatggacaga ccatgcctat gttgccaggg cctccagtac agatgccttc tgttatttcg 660 ctggccagac ctgtgtccat ggtgcccaac attcctggta tacctggccc accggttaac 720 aacagtggct ccatttctcc ctctggccac cctatgccgt cagaagccaa aatgagacta 780 aaagccacgc tgacccatca agtttcttca atcaatggag gttgtggaat ggtggtgggt 840 actgcaagca ccatggtgac tgcccgtcca gagcaaaacc agatcctcat ccagcaccca 900 gatgccccat cccctgccca gccacaggtc tctccagctc agcccacccc tagcactggg 960 ggacggcgac ggcgtacagt ggatgaagat ccagatgagc ggcggcagcg gtttttagag 1020 cgaaacagag ctgcagcctc tcgatgccgg caaaagcgga aactgtgggt gtcctccctg 1080 gaaaagaagg cagaagaact tacttctcag aacattcagc tgagtaatga agtcacatta 1140 ctacgcaatg aggtggctca gctgaagcag ctactgttag ctcataaaga ttgcccagtc 1200 actgcactac agaaaaagac tcaaggctac ctaggtaag 1239 150 168 DNA Mus musculus 150 cgatgccggc aaaagcggaa actgtgggtg tcctccctgg aaaagaaggc agaagaactt 60 acttctcaga acattcagct gagtaatgaa gtcacattac tacgcaatga ggtggctcag 120 ctgaagcagc tactgttagc tcataaagat tgtccagtca ccgcacaa 168 151 3093 DNA Mus musculus 151 atgacaaacc caaaaggaaa gaggagaggt actcagtcta tgttctctag gccttttagg 60 aaacatggag ttgtttcttt ggccacatac atgcgaatct acaagaagcg tgatattgta 120 gacatcaagg gaatgggcac tgttcaaaaa ggaatgccct gtaagtgtta ccacggcaaa 180 accggaagag tctacaatgt cacccagcat gccatgggca tcattgtaaa caagcaagtt 240 aaaggcaaga ttctggccaa gaggatcaat gtgcagattg agcacatcaa gcactcgaag 300 agcagagacg gcttcctgaa gcagggagag gccgcccatt tcgaatacct gctgtaccca 360 cttcactcag cgtccatcac gggcctggcc acctgcatcc gaaaacccct cattgccacc 420 tgctccctgg atcgctccgt tcgcatctgg aattacgagt cgaattcctc ctgctgcaag 480 gctctcagag aagacctttg gctcctcctc cttttccaca tcactgcccc ggccaccctt 540 agctccccac cagtgatatt cttctgcaca ctggagctat ataaggaata ccaagaagag 600 gcctacacgg tcagccttca cccctccgga cactacattg tggtggggtt tgctgacaaa 660 cttcgcctta tgaacctgct cattgatgac atccgttctt tcaaagaata ttctgtcaga 720 ggatgcaaag agtgtgcctt tagcaatgga ggtcacctgt ttgctgccgt caatggtaat 780 gtgattcaca tcttcaccac cacgaacctg gagaatatca acaacctgaa aggccacaca 840 gggaagaggg agacagagtg tgtactcaag gtctgtagtt acaactcggt cactatctcc 900 cctgacggca aagttatctt cgctgttgga tcagaccaga ctcttaagga gatcgccgat 960 tctttgatcc ttcgagagat accagcattt gatgtcgtct acacggccat caccatctca 1020 cattccggac gcatgatatt cgtgggcact tcagtgggga ctatccgtgc catgaagtac 1080 ccgctgcctc tgcagagaga attcaatgag taccaggctc acgctggccc cgtcacgaag 1140 atactgctca ccttcgatga ccagttcctg ctgacggtct ctgaggatgg ctgcctgttc 1200 acctggaaag tctttgataa ggagggtcgg ggaatcaaac gagagaggga ggtgggcttt 1260 gctgaagagg tactcgtgac taagacagac atggaggaga agatactcca caggaactta 1320 gcaacggaat tcagaaggcc aatgagcaag caccttgagt gtcccacatc ggaaactggg 1380 ccactcacaa caataaatat ctccccggtc cagcccaggc cttggggcca tgtactcacc 1440 tgcagaacac ccgtcagcac tgacagtgct gttgcgtcta caagaggctc tgtggacagc 1500 gcagtgaagc cagataggtc aactccaacc caggaagtcc gcatcccacc aaagccagcc 1560 tcgggagtcc acaccaggtg ccagttagga gtacagaaac agatggaaca cgtttctgtt 1620 gtcatggagg tacgagaaac aaaccggcag agacagggtg ggggtgcgcg gaatgtaatc 1680 aaggctcaga tcatgctgga gctgaagacg cgtgtagagg aactgaaaat ggagaacgag 1740 tatcagctcc ggctgaagga catgaactac tcagagaaga tcaaggagct gacagacaag 1800 ttcatccagg agatggagtc cttgaagacg aagaaccagg ttttaaaaac agagaaagaa 1860 aaacaggaca tcagtcaccg agagcactta gaagacctca tagaaagaca gagccgggag 1920 ctgcaagacc tggaatgttg taacaaccag aagctgctcc ttgaatacga gaagtaccag 1980 gagctgcagc tcaagtccca gaggatgcag gaggagtacg aaaaacagct ccgagacaat 2040 gatgagacca agagccaagc actggaggag ctgaccgagt tctacgaggc caaactccag 2100 gagaaaaccg gccttctgga agaggccctc agcacagcag cctcaccacc ccttccctca 2160 gcacacgttc tctctccctt ccccactctg agccaggcac aggaagatgt ccgacagcag 2220 ctccgggaat tcgaggaaac caagaagcag attgaagaag atgaggacag ggagatccaa 2280 gacatcaaaa ccaagtatga gagaaagctt cgagatgaaa aggagtccaa ccttcggctt 2340 aagggagaaa caggaatcat gaggaagaag ttcagcagcc tgcagaagga gatcgaagag 2400 cgcaccaatg acatcgagct cctcaaaacg gagcaggtga agctgcaggg ggtcatcagg 2460 tccctagaga aggacatcca aggactcaag agagagatcc aggagaggga tgagaccatt 2520 caagacaagg agaagcgaat ttatgatctg aagaagaaga accaagagtt agagaaattc 2580 aaatttgtcc ttgactacaa aataaaggaa ctgaagaagc aaatagaacc aagggagaac 2640 gagatcaaag tgatgaagga gcagatccag gagaaccctg tcaatcactg gctcagaagc 2700 agggagagag aatgtgtcac acagccaagg catctgcggc ttccagctcc ccagaacaag 2760 ttagatggga atttagcttg tggaccggta agaggtcggt tgtgccactc agacgcgacc 2820 tcaggggccc tgaatgttca gggcatcctt tgtctcttcc acctgccatt tccctgtgat 2880 aggacgccat ctttcttccc cggagaagct tgtctcctgg ttttctctct tctgatagat 2940 gttctatgta gacccacctc tgacgtacca gtcgctgctg gcgattttct tccgtgtggc 3000 ggacctctgc acttgcctcc agagctgcac caccttacag tcatccggac caatgccagc 3060 ccacagaaat gctacccacc caccagtcct ctg 3093 152 210 DNA Mus musculus 152 aagaagttca gcagcctgca gaaggagatc gaagagcgca ccaacgacat cgagctcctc 60 aagtcggagc ggatgaagct gcagggcatc atcagatccc tggagaaaga catccaaggg 120 ctcaagagag agatccagga gagggacgag accattcaag acatggagaa gcttgactac 180 aaggacgatt ataattcaaa cctagagatc 210 153 168 DNA Mus musculus 153 aagaagttca gcagcctgca gaaggagatc gaagagcgca ccaacgacat cgagctcctc 60 aagtcggagc ggatgaagct gcagggcatc atcagatccc tggagaaaga catccaaggg 120 ctcaagagag agatccagga gagggacgag accattcaag acatggag 168 154 651 DNA Mus musculus 154 atggaagtag aaaacgaagc ccactgctgc cctggcagct catcaggcgg gtccagagag 60 tacaaggtgg taatgctggg cgcagggggc gttggtaaaa gcgcagtcac aatgcagttt 120 ataagccacc agttcccgga ctatcacgac cccacaatcg aagatgctta taaaacccag 180 gtgaggattg ataatgagcc tgcttactta gacatcttgg acactgctgg tcaggcagag 240 ttcacggcca tgcgggagca gtacatgcgt gggggagagg gcttcatcat ctgctattct 300 gtcactgacc gccagtcatt ccaggaggct gccaagttca aggagcttat tttccaggtc 360 cgtcacacct atgaaattcc ccttgtgcta gtgggtaaca aaattgactt ggagcagttc 420 cgtcaggtat ctacagaaga aggcatgaat cttgctcgag actacaactg tgccttcttt 480 gagacatctg cagccctgcg attcggtatc gatgatgctt ttcaaggctt agtgagagaa 540 attcgcagga aggaatccat gctgtccttg gtggaaagga aattgaagag gaaggacagc 600 ctgtggaaga agataaaagc ctccctgaag aagaagagag aaaacatgtt g 651 155 150 DNA Mus musculus 155 gcagccctgc gattcggtat cgatgatgct cttcaaggct tagtgagaga aattcgcagg 60 aaggaatcca tgctgccctt ggtggaaagg aaattgaaga ggaaggacag cctgtggaag 120 aagataaaag cctccctgaa gaagaagagg 150 156 420 DNA Mus musculus 156 ggtgccacag ttattacaaa cctcctatca gccatcccat atattggaac aaccctagtc 60 gaatgaattt gagggggctt ctcagtagac aaagccacct tgacccgatt cttcgctttc 120 cacttcatct taccatttat tatcgcggcc ctagcaatcg ttcacctcct cttcctccac 180 gaaacaggat caaacaaccc aacaggatta aactcagatg cagataaaat tccatttcac 240 ccctactata caatcaaaga tatcctaggt atcctaatca tattcttaat tctcataacc 300 ctagtattat ttttcccaga catactagga gacccagaca actacatacc agctaatcca 360 ctaaacaccc caccccatat taaacccgaa tgatatttcc tatttgcata cgccattcta 420 157 123 DNA Mus musculus 157 tcagatgcag ataaaattcc atttcacccc tactatacaa tcaaaaatat cctaggtatc 60 ctaatcatat tcttaattct cataacccta gtattatttt tcccagacat actaggagac 120 cca 123 158 933 DNA Mus musculus 158 atgaaggctc tgtgggccgt gctgttggtc acattgctga caggatgcct agccgaggga 60 gagccggagg tgacagatca gctcgagtgg caaagcaacc aaccctggga gcaggccctg 120 aaccgcttct gggattacct gcgctgggtg cagacgctgt ctgaccaggt ccaggaagag 180 ctgcagagct cccaagtcac acaagaactg acggcactga tggaggacac tatgacggaa 240 gtaaaggctt acaaaaagga gctggaggaa cagctgggtc cagtggcgga ggagacacgg 300 gccaggctgg gcaaagaggt gcaggcggca caggcccgac tcggagccga catggaggat 360 ctacgcaacc gactcgggca gtaccgcaac gaggtgcaca ccatgctggg ccagagcaca 420 gaggagatac gggcgcggct ctccacacac ctgcgcaaga tgcgcaagcg cttgatgcgg 480 gatgccgatg atctgcagaa gcgcctagct gtgtacaagg caggggcacg cgagggcgcc 540 gagcgcggtg tgagtgccat ccgtgagcgc ctggggcctc tggtggagca aggtcgccag 600 cgcactgcca acctaggcgc tggggccgcc cagcctctgc gcgatcgcgc ccaggctttt 660 ggtgaccgca tccgagggcg gctggaggaa gtgggcaacc aggcccgtga ccgcctagag 720 gaggtgcgtg agcacatgga ggaggtgcgc tccaagatgg aggaacagac ccagcaaata 780 cgcctgcagg cggagatctt ccaggcccgc ctcaagggct ggttcgagcc aatagtggaa 840 gacatgcatc gccagtgggc aaacctgatg gagaagatac aggcctctgt ggctaccaac 900 cccatcatca ccccagtggc ccaggagaat caa 933 159 90 DNA Mus musculus 159 acggaagtaa aggcttacaa aaaggagctg gaggaacagc tgggtccagt ggcggaggag 60 acacgggcca ggctgggcaa agaggagcag 90 160 2085 DNA Mus musculus 160 atgctgccca gcttggcact gctcctgctg gccgcctgga cggttcgggc tctggaggta 60 cccactgatg gcaacgccgg gctgctggca gaaccccaga tcgccatgtt ctgtggtaaa 120 ctcaacatgc acatgaatgt gcagaatgga aagtgggagt cagacccgtc agggaccaaa 180 acctgcattg gcaccaagga gggcatcttg cagtactgcc aagaggtcta ccctgaactg 240 cagatcacaa acgtggtgga agccaaccag ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac acacacccac atcgtgattc cttaccgttg cctagttggt 360 gagtttgtga gcgacgccct tctcgtgccc gacaagtgca agttcctaca ccaggagcgg 420 atggatgttt gtgagaccca tcttcactgg cacaccgtcg ccaaagagac atgcagcgag 480 aagagcacta acttgcacga ctatggcatg ctgctgccct gcggcatcga caagttccga 540 ggggtagagt ttgtatgctg cccgttggcc gaggaaagcg acagcgtgga ttctgcggat 600 gcagaggagg atgactctga tgtctggtgg gttggagcgg acacagacta cgctgatggc 660 ggtgaagaca aagtagtaga agtcgccgaa gaggaggaag tggctgatgt tgaggaagag 720 gaagctgatg atgatgagga tgtggaggat ggggacgagg tggaggagga ggccgaggag 780 ccctacgaag aggccaccga gagaacaacc agcactgcca ccaccaccac aaccaccact 840 gagtccgtgg aggaggtggt ccgagttccc acgacagcag ccagcacccc cgacgccgtc 900 gacaagtacc tggagacacc cggggacgag aacgagcatg cccatttcca gaaagccaaa 960 gagaggctgg aagccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagagcgtc aagccaagaa cttgcccaaa gctgacaaga aggccgttat ccagcatttc 1080 caggagaaag tggaatctct ggaacaggaa gcagccaatg agagacagca gcttgtagag 1140 acacacatgg ccagagttga agccatgctc aatgaccgcc gccgcctgga cctcgagaat 1200 tacatcatcg cactgcaggc ggtgccccca aggcctcatc atgtgttcaa catgctgaag 1260 aagtacgtcc gtgcggagca gaaagacaga cagcacaccc taaagcattt tgaacatgtg 1320 cgcatggtgg accccaagaa agctactcag atccggtccc aggttatgac acacctccgt 1380 gtgatctacg agcgcatgaa ccagtctctg tccctgctct acaatgtccc tgcggtggct 1440 gaggagattc aagatgaagt cgatgagctg cttcagaagg agcagaacta ctccgacgat 1500 gtcttggcca acatgatcag tgagcccaga atcagctacg gaaacgacgc tctcatgcct 1560 tcgctgacgg aaaccaagac caccgtggag ctccttcccg tgaatgggga attcagcctg 1620 gatgacctcc agccgtggca cccttttggg gtggactctg tgccagccaa taccgaaaat 1680 gaagtcgagc ctgttgacgc ccgccccgct gctgaccgag gactgaccac tcgaccaggt 1740 tctgggctga caaacatcaa gacggaagag atctcggaag tgaagatgga tgcagaattc 1800 ggacatgatt caggatttga agtccgccat caaaaactgg tgttctttgc tgaagatgtg 1860 ggttcgaaca aaggcgccat catcggactc atggtgggcg gcgttgtcat agcaaccgtg 1920 attgtcatca ccctggtgat gttgaagaag aaacagtaca catccatcca tcatggcgtg 1980 gtggaggtcg acgccgccgt gaccccagag gagcgccatc tctccaagat gcagcagaac 2040 ggatatgaga atccaactta caagttcttt gagcaaatgc agaac 2085 161 201 DNA Mus musculus 161 agtgagccca gaatcagcta cggaaacgac gctctcatgc cttcgctgac ggaaaccaag 60 accaccgtgg agctccttcc cgtgaatggg gaattcagcc tggatgacct ccagccgtgg 120 cacccttctg gggtggactc tgtgccagcc aataccgaaa atgaggtcga gcctgttgac 180 gcccgccccg ctgctgaccg a 201 162 1236 DNA Mus musculus 162 atggcgaacg tggccgacac gaagctgtac gacatcctgg gcgtccctcc cggcgctagc 60 gagaacgagc tgaagaaggc ataccgaaag ttagccaaag aataccaccc tgataagaat 120 ccaaatgctg gagacaaatt taaagaaata agttttgcat atgaagtatt gtcaaatcca 180 gagaagcgag agctgtatga cagatatgga gaacaaggcc tacgggaagg cagcggcgga 240 ggcggtggaa tggatgatat cttctcacat atttttggtg gaggattgtt tggctttatg 300 ggcaatcaga gtagaagtcg aaatggcaga agaagaggcg aggacatgat gcatccacta 360 aaagtatctt tagaagacct gtacaatggc aagacaacca aactacaact tagcaagaat 420 gtgctctgta gtgcatgcag tggccaaggt gggaagtctg gagctgttca gaaatgcagc 480 gcttgtcggg gtcgaggtgt gcgcattatg atcagacagc tggctccagg aatggtgcag 540 cagatgcagt ccgtgtgctc cgactgtaat ggagaagggg aggtcatcaa tgaaaaagac 600 cgctgtaaaa aatgtgaagg gaagaaggta atcaaagaag tcaagattct ggaagtccat 660 gtagacaaag gcatgaaaca tggacagagg attacgttca ctggggaagc agaccaggct 720 ccaggagtgg aacctggaga tattgttctt ttgctacagg aaaaagaaca tgaggtgttc 780 cagagagatg ggaatgattt gcatatgaca tataagatag gacttgttga agctttatgt 840 ggatttcagt tcacatttaa acatcttgat gctcgtcaga ttgtggtgaa atacccccct 900 ggcaaagtaa ttgaaccagg atgtgttcgt gttgttcgag gtgaaggaat gccacagtat 960 cgtaatccct ttgaaaaggg tgatctttac ataaagtttg atgtacagtt tcctgagaat 1020 aactggatca acccagacaa actttctgaa ttagaagatc tcctgccatc tagaccagaa 1080 gttcctaatg ttattggaga gacagaagaa gtggagcttc aggaatttga tagcactcga 1140 ggctctggcg gtggtcagag acgtgaagcc tataatgata gctctgatga agaaagtagc 1200 agccatcatg gacctggagt gcagtgtgcc catcag 1236 163 75 DNA Mus musculus 163 ttgtcaaatc cagagaagcg agagctgtat gacagatatg gagaacaagg cctacgggaa 60 ggcagcggag gaggc 75 164 2949 DNA Mus musculus 164 atgaaggctc agcaggccat ggacaaatat gaaggagata gcaaggcgag ggagacccgg 60 agcacagcgg ccatggtagg ctggagaagt gacagaggcc tggtgacttg caccaggctt 120 aggatgcaga atggtagctc actaaaagct tttcggagca gggtggggaa gtggggagaa 180 ccttcctcga gatcacacaa agtgttgaag accagcgaaa cttcacaaga tatccagaag 240 gtttctagag aggaaagccc ttcccagctg acttctgccg tgcctgccca gaggaactgc 300 cagcccggca gcgctgccgt tatcaatatg cttcgcgggg gaggcggtgt tagaagccca 360 tggacagatc accacatcag gcagcggacg gatcaccaca tccggcagcc cttgtttcca 420 agccgccggt ctccacagga gaatgaggac gatgacgacg attaccagat gttcgtcccg 480 tccttttctt cctcggatct caactccacc aggttgtgtg aagagaacgc ttcaagtcgc 540 ccttgcagct ggcatctggg cctcatcgaa cccacagaga tctccagctc tggtcacagg 600 atagtccgac gggccagtag tgcaggtgaa agcaacgcat gccctcctga agtaagaatt 660 agagactgtg atgactctca gtactgtccc gggagacagc tgcagaacag tcctcgacca 720 ggaggagaga gggggatgac tccctatggg tcgtctgtag agttgaccat tgatgatata 780 gaccatgtct atgataacat aagttttgaa gacttaaagt taatggttgc taagcgggat 840 gaaactgaat gttctttctc caaaccatcc agagactctg ttcgccccaa gagtacccca 900 gagttagcct tctcaaagag

acaggttagc cacagtacaa gctctctcca ctcaaggaaa 960 gaggcaggcc tcggtggtca agaggcatcc acccaaagcg tacatgaaca ccaggaagtg 1020 gaagaaaaca tctatgacac catagggctt ccagacccac catcgatgaa cttgaaccac 1080 agcagccttc atcagcccaa aaggagcacc ttcctgggtc tggaagctga ttttgcatgc 1140 tgtgacagcc tgagaccatt tgtctcccag gatagcctcc agttcagtga ggatgacata 1200 tcttaccacc agggaccctc cgatactgaa tatttgagtt tgttatatga ctctccccgc 1260 tgtaatctgc ctattgcaga taaggctctg tccgacaaat tgtctgaaga agtagatgaa 1320 atctggaatg atctggaaaa ttacatcaag aaaaatgaag acaaatcaag agaccgcctc 1380 cttgcggcct ttcctgtgag caaagacgat gcaccagaga ggctatatgt tgacagcacc 1440 catgagctgg gcagggatac aggacatgcc acatctatgc tggcccttcc tacaagccag 1500 actttcctcc tgcctgggaa gagcagagtt gtcagagcta gcagggccaa ctgctccctg 1560 gataatgaca ttatttcaac agaaggttcc ttcctgagtc ttaaccaact ctctctggcc 1620 agtgatgggc ctcctgtgga caatccctat gacctggcca actgcagcct gccccagaca 1680 gacccagaaa accctgaccc cgggatggag gtcacagaca agactaagag cagggtcttt 1740 atgatggcca ggcagtatag tcaaaagatc aagaaggtaa atcagatttt gaaagtgaaa 1800 agcccagagt tggagcaacc accgtccagt cagcataggc ccagtcacaa agacctggtg 1860 gccatcttgg aagagaagag gcaaggaggg cccgccattg gtgccaggat cgctgaatat 1920 tcccaactgt atgaccagat tgtgttcaga gagacacccc ttaaagctca gaaggatggc 1980 tgggccagcc cccaaggacc caccctccac aggcctgtgt cacctcccca ggcccagggc 2040 gctggtgagg actggctctg gcattcgccc tacagtaacg gagagttggc agatttctct 2100 ccccagacag aacaagactc aaaatcaaaa taccccatca cattagagag caccacaaaa 2160 attaggccca ggcagttgtc gggtgcttgt tcggtgccgt ctctccaagt gtcggaccct 2220 ctgctgggct ctgtgcagca gagatgcagc gtggtggtca gccagcccca caaagagaac 2280 tcaggtcaga gtcctcttta caactcgctg ggtcgcaaag caatcagcgc taaaccccag 2340 ccttatagca ggcctcagtc atcttcctca atcttgataa acaaatctct ggactctatc 2400 aactacccca gtgagacaga gacgaagcaa ctactctctt cacagaaaag tcccagaggc 2460 gcgagccagc aggatttgcc gtcagggcta gcaaactcat gtcaacagga caggggcaaa 2520 cggtccgatc tcacgctcca agactcgcag aaggttctcg tggtaaatag aaatttaccc 2580 ttaagcgctc aaatagcgac gcagaactac ttttgtaatt tcaaagatcc cgagggagat 2640 gaagatgact atgtggaaat caagtcagaa gaggacgaag tgcgtctgga tctctctcca 2700 aggcggggca ggaagtctga cccacagacc ccggaccctg actgttcgga tagcatctgt 2760 agccacagca caccttactc tttgaaggag ccagtgagtg gcaggcttgg gcttcctcct 2820 tacctgacag catgtaagga ctctgataaa ctgaatgatt atctgtggag ggggccctca 2880 cccaatcagc aaaacattgt ccaatctctg agggagaaat ttcagtgtct tagctcaagc 2940 agctttgcc 2949 165 138 DNA Mus musculus 165 tttatgatgg ccaggcagta tagtcaaaag atcaagaagg taaatcagat tttgaaagtg 60 aaaagcccag agttggagca accaccgtcc agtcagcata ggcccagtca caaagacctg 120 gcggccatct tggaaaag 138 166 87 DNA Mus musculus 166 gcacactcat tctccgtttt cagactcccg agttggtgga ttgtgggctg gtggagcaaa 60 ggtggagtag gctctgattt agaaatg 87 167 105 DNA Mus musculus 167 cctgatataa aacatccagg aaatctggaa cactatataa aaagagtaaa cctaagaata 60 atagcaatag aagaaggaga aaaatcccag ctcaaaggcc cgaaa 105 168 5172 DNA Artificial Sequence vector CMV-FosCBPzz 168 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 60 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 120 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 180 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 240 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 300 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 360 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc 420 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 480 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 540 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta 600 gcgattacgc caagctcgaa attaaccctc actaaaggga acaaaagctg gagctccacc 660 gcggtggcgg ccgctctagc ccgggcggat cacgatcccg cgaaattaat acgactcact 720 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag 780 aaggagatat accatggcta gcatgactgg tggacagcaa atgggtcgcg gatccggcag 840 agcgcagagc atcggcagaa ggggcaaagt agagcagcta tctcctgaag aggaagagaa 900 acggagaatc cgaagggaac ggaataagat ggctgcagcc aagtgccgga atcggaggag 960 ggagctgaca gatacactcc aagcggagac agatcaactt gaagatgaga agtctgcgtt 1020 gcagactgag attgccaatc tgctgaaaga gaaggaaaaa ctggagttta ttttggcagc 1080 ccaccgacct gcctgcaaga tccccgatga ccttggcctc gagctcaaga gaagatggaa 1140 aaagaatttc atagccgtct cagcagccaa ccgctttaag aaaatctcat cctccggggc 1200 acttggatca gattatgata ttccaactac tgctagcgag aatttgtatt ttcagggtgg 1260 taccaaaacc gcggctcttg cgcaacacga tgaagccgta gacaacaaat tcaacaaaga 1320 acaacaaaac gcgttctatg agatcttaca tttacctaac ttaaacgaag aacaacgaaa 1380 cgccttcatc caaagtttaa aagatgaccc aagccaaagc gctaaccttt tagcagaagc 1440 taaaaagcta aatgatgctc aggcgccgaa agtagacaac aaattcaaca aagaacaaca 1500 aaacgcgttc tatgagatct tacatttacc taacttaaac gaagaacaac gaaacgcctt 1560 catccaaagt ttaaaagatg acccaagcca aagcgctaac cttttagcag aagctaaaaa 1620 gctaaatgat gctcaggcgc cgaaagtaga cgcgaattct agctctgtac cccatcacca 1680 tcaccatcac taagtcgact tcgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 1740 atggagatcc aatttttaag tgtataatgt gttaaactac tgattctaat tgtttgtgta 1800 ttttagattc acagtcccaa ggctcatttc aggcccctca gtcctcacag tctgttcatg 1860 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac 1920 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 1980 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 2040 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt 2100 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 2160 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt 2220 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa 2280 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag 2340 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt 2400 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg 2460 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc 2520 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg 2580 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 2640 accctgataa atgcttcaat aatattgaaa aaggaagaat cctgaggcgg aaagaaccag 2700 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 2760 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 2820 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 2880 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 2940 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 3000 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg 3060 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt 3120 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt 3180 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc 3240 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc 3300 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga 3360 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat 3420 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca 3480 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga 3540 tgatctggac gaagaacatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc 3600 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat 3660 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga 3720 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagaacttg gcggcgaatg 3780 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt 3840 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa 3900 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg 3960 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 4020 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg 4080 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg 4140 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga 4200 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg 4260 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt 4320 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 4380 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 4440 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 4500 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 4560 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 4620 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 4680 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 4740 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 4800 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 4860 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 4920 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 4980 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 5040 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 5100 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 5160 cgtattaccg cc 5172 169 70 DNA Artificial Sequence PCR primer 5'SP6(O29)T7-FosCBPzz 169 gaatttaggt gacactatag aacaacaaca acaacaaaca acaacaaaat ggctagcatg 60 actggtggac 70 170 20 DNA Artificial Sequence PCR primer 3'FosCBPzz 170 ggatctccat tcgccattca 20 171 89 DNA Artificial Sequence DNA beit template DNA-Fos/Jun 171 cgactctgac ggcagtttac gtgactcatg agtcatgact catgagtcat gactcatgag 60 tcacgttaga acgcggctac aattaatac 89 172 21 DNA Artificial Sequence PCR primer 5'DNA 172 cgactctgac ggcagtttac g 21 173 26 DNA Artificial Sequence PCR primer 3'DNA 173 gtattaattg tagccgcgtt ctaacg 26 174 67 DNA Artificial Sequence main chain of adaptor (O29) 174 gaacaacaac aacaacaaac aacaacaaaa tgactggtgg acagcaaatg ggtgcggccg 60 cgaattc 67 175 68 DNA Artificial Sequence main chain of adaptor (O29-2) 175 gaacaacaac aacaacaaac aacaacaaaa tggctagcat gactggtgga cagcaaatgg 60 cgaattcc 68 176 32 DNA Artificial Sequence random primer for reverse transcription 176 tcatcgtcct tgtagtcaag cttnnnnnnn nn 32 177 58 DNA Artificial Sequence PCR 5' primer (O29) 177 ggaagatcta tttaggtgac actatagaac aacaacaaca acaaacaaca acaaaatg 58 178 36 DNA Artificial Sequence PCR 3' primer 178 ttttttttct tgtcgtcatc gtccttgtag tcaagc 36 179 3851 DNA Artificial Sequence pDrive vector 179 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcta 240 atacgactca ctatagggaa agctcggtac cacgcatgct gcagacgcgt tacgtatcgg 300 atccagaatt cgtgatatct gaattcgtcg acaagcttct cgagcctagg ctagctctag 360 accacacgtg tgggggcccg agctcgcggc cgctgtattc tatagtgtca cctaaatggc 420 cgcacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 480 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 540 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt 600 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 660 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 720 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 780 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 840 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 900 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 960 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 1020 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 1080 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 1140 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 1200 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 1260 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 1320 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 1380 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 1440 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 1500 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 1560 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 1620 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 1680 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 1740 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 1800 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 1860 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 1920 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 1980 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 2040 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 2100 gatctaggtg aagatccttt ttgataatct catgaacaat aaaactgtct gcttacataa 2160 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc 2220 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg 2280 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc 2340 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact 2400 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg 2460 catggttact caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc 2520 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga 2580 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat 2640 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc 2700 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg 2760 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt 2820 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga 2880 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg 2940 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat 3000 taattcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 3060 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 3120 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 3180 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 3240 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 3300 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 3360 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 3420 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 3480 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 3540 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 3600 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 3660 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 3720 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 3780 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 3840 gaagcggaag a 3851 180 58 DNA Artificial Sequence primer 5'F3 180 ggaagatcta tttaggtgac actatagaac aacaacaaca acaaacaaca acaaaatg 58 181 27 DNA Artificial Sequence primer 3'R3 181 ttttttttct cgagcttgtc gtcatcg 27 182 18 DNA Artificial Sequence primer Optn_F 182 tgggcatcgt ctcagaac 18 183 18 DNA Artificial Sequence primer Optn_R 183 tgtgggtgta gggcagtt 18 184 18 DNA Artificial Sequence primer SNAP19_F 184 aaaccctgct gcgtctaa 18 185 19 DNA Artificial Sequence primer SNAP19_R 185 atcatggatt gaagggcta 19 186 17 DNA Artificial Sequence primer C130020M04RIK_F 186 ggtgtcctcc ctggaaa 17 187 20 DNA Artificial Sequence primer C130020M04RIK_R 187 tgggcaatct ttatgagcta 20 188 18 DNA Artificial Sequence primer Rattus FLJ32000_F 188 aagagcgcac caatgaca 18 189 20 DNA Artificial Sequence primer Rattus FLJ32000_R 189 tcttgaatgg tctcatccct 20 190 21 DNA Artificial Sequence primer 5'M13_F 190 gttttcccag tcacgacgtt g 21 191 24 DNA Artificial Sequence primer 3'M13_R 191 gaaacagcta tgaccatgat tacg 24

Claims

1. A protein of the following (a) or (b): (a) a protein comprising any one of the amino acid sequences of SEQ ID NOS: 1 to 14, SEQ ID NOS: 15 to 19, SEQ ID NOS: 20 to 22, SEQ ID NOS: 47 to 56, SEQ ID NOS: 57 to 76, SEQ ID NOS: 77 to 81, SEQ ID NOS: 82 to 84, SEQ ID NOS: 85 to 86, SEQ ID NOS: 87 to 89, SEQ ID NOS: 90 to 91, SEQ ID NOS: 92 to 93, SEQ ID NOS: 94 to 95, SEQ ID NOS: 96 to 97, SEQ ID NOS: 98 to 99, SEQ ID NOS: 100 to 101, SEQ ID NO: 102 and SEQ ID NO: 103, (b) a protein that comprises any one of the amino acid sequences of SEQ ID NOS:1 to 14, SEQ ID NOS: 15 to 19, SEQ ID NOS: 20 to 22, SEQ ID NOS: 47 to 56, SEQ ID NOS: 57 to 76, SEQ ID NOS: 77 to 81, SEQ ID NOS: 82 to 84, SEQ ID NOS: 85 to 86, SEQ ID NOS: 87 to 89, SEQ ID NOS: 90 to 91, SEQ ID NOS: 92 to 93, SEQ ID NOS: 94 to 95, SEQ ID NOS: 96 to 97, SEQ ID NOS: 98 to 99, SEQ ID NOS: 100 to 101, SEQ ID NO: 102 and SEQ ID NO: 103, including deletion, substitution or addition of one or several amino acid residues and interacts with a c-Fos protein.

2. A protein according to claim 1, which comprises any one of the amino acid sequences of SEQ ID NOS: 1 to 14, SEQ ID NOS: 15 to 19, SEQ ID NOS: 20 to 22, SEQ ID NOS: 47 to 56, SEQ ID NOS: 57 to 76, SEQ ID NOS: 77 to 81, SEQ ID NOS: 82 to 84, SEQ ID NOS: 85 to 86, SEQ ID NOS: 87 to 89, SEQ ID NOS: 90 to 91, SEQ ID NOS: 92 to 93, SEQ ID NOS: 94 to 95, SEQ ID NOS: 96 to 97, SEQ ID NOS: 98 to 99, SEQ ID NOS: 100 to 101, SEQ ID NO: 102 and SEQ ID NO: 103.

3. A nucleic acid encoding the protein according to claim 1.

4. A nucleic acid of the following (a) or (b): (a) a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 23 to 38, SEQ ID NOS: 39 to 43, SEQ ID NOS: 44 to 46, SEQ ID NOS: 104 to 118, SEQ ID NOS: 119 to 140, SEQ ID NOS: 141 to 145, SEQ ID NOS: 146 to 148, SEQ ID NOS: 149 to 150, SEQ ID NOS: 151 to 153, SEQ ID NOS: 154 to 155, SEQ ID NOS: 156 to 157, SEQ ID NOS: 158 to 159, SEQ ID NOS: 160 to 161, SEQ ID NOS: 162 to 163, SEQ ID NOS: 164 to 165, SEQ ID NO: 166, and SEQ ID NO: 167, (b) a nucleic acid that hybridizes with a nucleic acid comprising any one of the nucleotide sequences of SEQ ID NOS: 23 to 38, SEQ ID NOS: 39 to 43, SEQ ID NOS: 44 to 46, SEQ ID NOS: 104 to 118, SEQ ID NOS: 119 to 140, SEQ ID NOS: 141 to 145, SEQ ID NOS: 146 to 148, SEQ ID NOS: 149 to 150, SEQ ID NOS: 151 to 153, SEQ ID NOS: 154 to 155, SEQ ID NOS: 156 to 157, SEQ ID NOS: 158 to 159, SEQ ID NOS: 160 to 161, SEQ ID NOS: 162 to 163, SEQ ID NOS: 164 to 165, SEQ ID NO: 166, and SEQ ID NO: 167, under a stringent condition and encodes a protein that interacts with a c-Fos protein.

5. A nucleic acid according to claim 4, which comprises any one of the nucleotide sequences of SEQ ID NOS: 23 to 38, SEQ ID NOS: 39 to 43, SEQ ID NOS: 44 to 46, SEQ ID NOS: 104 to 118, SEQ ID NOS: 119 to 140, SEQ ID NOS: 141 to 145, SEQ ID NOS: 146 to 148, SEQ ID NOS: 149 to 150, SEQ ID NOS: 151 to 153, SEQ ID NOS: 154 to 155, SEQ ID NOS: 156 to 157, SEQ ID NOS: 158 to 159, SEQ ID NOS: 160 to 161, SEQ ID NOS: 162 to 163, SEQ ID NOS: 164 to 165, SEQ ID NO: 166, and SEQ ID NO: 167.

6. An inhibitor for an interaction between a protein that interacts with a c-Fos protein and the c-Fos protein, which comprises the protein according to claim 1 or 2 or a protein translated from the nucleic acid according to any one of claims 3 to 5 or 133 as an active ingredient.

7. A method for detecting an interaction between a bait and a prey, which comprises bringing the bait and the prey into contact and detecting a complex formed by the contact, wherein the bait is the protein according to claim 1 or 2 or a protein translated from the nucleic acid according to any one of claims 3 to 5 or 133.

8. A method for screening for a prey that interacts with a bait, which comprises the step of detecting an interaction between the bait and a prey by the method according to claim 7 and the step of selecting a prey for which an interaction was detected.

9-132. (canceled)

133. A nucleic acid encoding the protein according to claim 2.