Abstract:
The present invention relates to purified DNA sequences encoding all or a portion of an osteoclast-specific or -related gene products and a method for identifying such sequences. The invention also relates to antibodies directed against an osteoclast-specific or -related gene product. Also claimed are DNA constructs capable of replicating DNA encoding all or a portion of an osteoclast-specific or -related gene product, and DNA constructs capable of directing expression in a host cell of an osteoclast-specific or -related gene product.

Description:
RELATED APPLICATION 
     This application is a continuation of application Ser. No. 08/045,270 filed on Apr. 6, 1993 now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     Excessive bone resorption by osteoclasts contributes to the pathology of many human diseases including arthritis, osteoporosis, periodontitis, and hypercalcemia of malignancy. During resorption, osteoclasts remove both the mineral and organic components of bone (Blair, H. C., et al., J. Cell Biol. 102:1164 (1986)). The mineral phase is solubilized by acidification of the sub-osteoclastic lacuna, thus allowing dissolution of hydroxyapatite (Vaes, G., Clin. Orthop. Relat. 231:239 (1988)). However, the mechanism(s) by which type I collagen, the major structural protein of bone, is degraded remains controversial. In addition, the regulation of osteoclastic activity is only partly understood. The lack of information concerning osteoclast function is due in part to the fact that these cells are extremely difficult to isolate as pure populations in large numbers. Furthermore, there are no osteoclastic cell lines available. An approach to studying osteoclast function that permits the identification of heretofore unknown osteoclast-specific or -related genes and gene products would allow identification of genes and gene products that are involved in the resorption of bone and in the regulation of osteoclastic activity. Therefore, identification of osteclast-specific or -related genes or gene products would prove useful in developing therapeutic strategies for the treatment of disorders involving aberrant bone resorption. 
     SUMMARY OF THE INVENTION 
     The present invention relates to isolated DNA sequences encoding all or a portion of osteoclast-specific or -related gene products. The present invention further relates to DNA constructs capable of replicating DNA encoding osteoclast-specific or -related gene products. In another embodiment, the invention relates to a DNA construct capable of directing expression of all or a portion of the osteoclast-specific or -related gene product in a host cell. 
     Also encompassed by the present invention are prokaryotic or eukaryotic cells transformed or transfected with a DNA construct encoding all or a portion of an osteoclast-specific or -related gene product. According to a particular embodiment, these cells are capable of replicating the DNA construct comprising the DNA encoding the osteoclast-specific or -related gene product, and, optionally, are capable of expressing the osteoclast-specific or -related gene product. Also claimed are antibodies raised against osteoclast-specific or -related gene products, or portions of these gene products. 
     The present invention further embraces a method of identifying osteoclast-specific or -related DNA sequences and DNA sequences identified in this manner. In one embodiment, cDNA encoding osteoclast is identified as follows: First, human giant cell tumor of the bone was used to 1) construct a cDNA library; 2) produce  32  P-labelled cDNA to use as a stromal cell +  ; osteoclast +  probe, and 3) produce (by culturing) a stromal cell population lacking osteoclasts. The presence of osteoclasts in the giant cell tumor was confirmed by histological staining for the osteoclast marker, type 5 tartrate-resistant acid phosphatase (TRAP) and with the use of monoclonal antibody reagents. 
     The stromal cell population lacking osteoclasts was produced by dissociating cells of a giant cell tumor, then growing and passaging the cells in tissue culture until the cell population was homogeneous and appeared fibroblastic. The cultured stromal cell population did not contain osteoclasts. The cultured stromal cells were then used to produce a stromal cell + , osteoclast -   32  P-labelled cDNA probe. 
     The cDNA library produced from the giant cell tumor of the bone was then screened in duplicate for hybridization to the cDNA probes: one screen was performed with the giant cell tumor cDNA probe (stromal cell + , osteoclast + ), while a duplicate screen was performed using the cultured stromal cell cDNA probe (stromal cell + , osteoclast - ). Hybridization to a stromal + , osteoclast +  probe, accompanied by failure to hybridize to a stromal + , osteoclast -  probe indicated that a clone contained nucleic acid sequences specifically expressed by osteoclasts. 
     In another embodiment, genomic DNA encoding osteoclast -specific or -related gene products is identified through known hybridization techniques or amplification techniques. In one embodiment, the present invention relates to a method of identifying DNA encoding an osteoclast-specific or -related protein, or gene product, by screening a cDNA library or a genomic DNA library with a DNA probe comprising one or more sequences selected from the group consisting of the DNA sequences set out in Table I (SEQ ID NOs: 1-32). Finally, the present invention relates to an osteoclast-specific or related protein encoded by a nucleotide sequence comprising a DNA sequence selected from the group consisting of the sequences set out in Table I, or their complementary strands. 
    
    
     BRIEF DESCRIPTION OF FIG. 1 
     The FIG. 1 shows cDNA sequence (SEQ ID NO: 33) of human gelatinase B, and highlights those portions of the sequence represented by the osteoclast-specific or -related cDNA clones of the present invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     As described herein, Applicant has identified osteoclast-specific or osteoclast-related nucleic acid sequences. These sequences were identified as follows: Human giant cell tumor of the bone was used to 1) construct a cDNA library; 2) produce  32  P-labelled cDNA to use as a stromal cell + , osteoclast +  probe, and 3) produce (by culturing) a stromal cell population lacking osteoclasts. The presence of osteclasts in the giant cell tumor was confirmed by histological staining for the osteoclast marker, type 5 acid phosphatase (TRAP). In addition, monoclonal antibody reagents were used to characterize the multinucleated cells in the giant cell tumor, which cells were found to have a phenotype distinct from macrophages and consistent with osteoclasts. 
     The stromal cell population lacking osteoclasts was produced by dissociating cells of a giant cell tumor, then growing the cells in tissue culture for at least five passages. After five passages the cultured cell population was homogeneous and appeared fibroblastic. The cultured population contained no multinucleated cells at this point, tested negative for type 5 acid phosphatase, and tested variably alkaline phosphatase positive. That is, the cultured stromal cell population did not contain osteoclasts. The cultured stromal cells were then used to produce a stromal cell + , osteoclast -   32  P-labelled cDNA probe. 
     The cDNA library produced from the giant cell tumor of the bone was then screened in duplicate for hybridization to the cDNA probes: one screen was performed with the giant cell tumor cDNA probe (stromal cell + , osteroclast + ), while a duplicate screen was performed using the cultured stromal cell cDNA probe (stromal cell +  osteoclast - ) Clones that hybridized to the giant cell tumor cDNA probe (stromal + , osteoclast + ), but not to the stromal cell cDNA probe (stromal + , osteoclast - ), were assumed to contain nucleic acid sequences specifically expressed by osteoclasts. 
     As a result of the differential screen described herein, DNA specifically expressed in osteoclast cells characterized as described herein was identified. This DNA, and equivalent DNA sequences, is referred to herein as osteoclast-specific or osteoclast-related DNA. Osteoclast-specific or -related DNA of the present invention can be obtained from sources in which it occurs in nature, can be produced recombinantly or synthesized chemically; it can be cDNA, genomic DNA, recombinantly-produced DNA or chemically-produced DNA. An equivalent DNA sequence is one which hybridizes, under standard hybridization conditions, to an osteoclast-specific or -related DNA identified as described herein or to a complement thereof. 
     Differential screening of a human osteoclastoma cDNA library was performed to identify genes specifically expressed in osteoclasts. Of 12,000 clones screened, 195 clones were identified which are either uniquely expressed in osteoclasts, or are osteoclast-related. These clones were further identified as osteoclast-specific, as evidenced by failure to hybridize to mRNA derived from a variety of unrelated human cell types, including epithelium, fibroblasts, lymphocytes, myelomonocytic cells, osteoblasts, and neuroblastoma cells. Of these, 32 clones contain novel cDNA sequences which were not found in the GenBank database. 
     A large number of cDNA clones obtained by this procedure were found to represent 92 kDa type IV collagenase (gelatinase B; E.C. 3.4.24.35) as well as tartrate resistant acid phosphatase. In situ hybridization localized mRNA for gelatinase B to multinucleated giant cells in human osteoclastomas. Gelatinase B immunoreactivity was demonstrated in giant cells from 8/8 osteoclastomas, osteoclasts in normal bone, and in osteoclasts of Paget&#39;s disease by use of a polyclonal antisera raised against a synthetic gelatinase B peptide. In contrast, no immunoreactivity for 72 kDa type IV collagenase (gelatinase A; E.C. 3.4.24.24), which is the product of a separate gene, was detected in osteoclastomas or normal osteoclasts. 
     The present invention has utility for the production and identification of nucleic acid probes useful for identifying osteoclast-specific or -related DNA. Osteoclast-specific or -related DNA of the present invention can be used to produce osteoclast-specific or -related gene products useful in the therapeutic treatment of disorders involving aberrant bone resorption. The osteoclast-specific or -related sequences are also useful for generating peptides which can then be used to produce antibodies useful for identifying osteoclast-specific or -related gene products, or for altering the activity of osteoclast-specific or -related gene products. Such antibodies are referred to as osteoclast-specific antibodies. Osteoclast-specific antibodies are also useful for identifying osteoclasts. Finally, osteoclast -specific or -related DNA sequences of the present invention are useful in gene therapy. For example, they can be used to alter the expression in osteoclasts of an aberrant osteoclast -specific or -related gene product or to correct aberrant expression of an osteoclast-specific or -related gene product. The sequences described herein can further be used to cause osteoclast-specific or related gene expression in cells in which such expression does not ordinarily occur, i.e., in cells which are not osteoclasts. 
     Example 1--Osteoclast cDNA Libary Construction 
     Messenger RNA (mRNA) obtained from a human osteoclastoma (`giant cell tumor of bone`), was used to construct an osteoclastoma cDNA library. Osteoclastomas are actively bone resorptive tumors, but are usually non-metastatic. In cryostat sections, osteoclastomas consist of ˜30% multinucleated cells positive for tartrate resistant acid phosphatase (TRAP), a widely utilized phenotypic marker specific in vivo for osteoclasts (Minkin, Calcif. Tissue Int. 34:285-290 (1982)). The remaining cells are uncharacterized `stromal` cells, a mixture of cell types with fibroblastic/mesenchymal morphology. Although it has not yet been definitively shown, it is generally held that the osteoclasts in these tumors are non-transformed, and are activated to resorb bone in vivo by substance(s) produced by the stromal cell element. 
     Monoclonal antibody reagents were used to partially characterize the surface phenotype of the multinucleated cells in the giant cell tumors of long bone. In frozen sections, all multinucleated cells expressed CD68, which has previously been reported to define an antigen specific for both osteoclasts and macrophages (Horton, M. A. and M. H. Helfrich, In Biology and Physiology of the Osteoclast, B. R. Rifkin and C. V. Gay, editors, CRC Press, Inc. Boca Raton, Fla., 33-54 (1992)). In contrast, no staining of giant cells was observed for CD11b or CD14 surface antigens, which are present on monocyte/macrophages and granulocytes (Arnaout, M. A. et al. J. Cell. Physiol. 137:305 (1988); Haziot, A. et al. J. Immunol. 141:547 (1988)). Cytocentrifuge preparations of human peripheral blood monocytes were positive for CD68, CD11b, and CD14. These results demonstrate that the multinucleated giant cells of osteoclastomas have a phenotype which is distinct from that of macrophages, and which is consistent with that of osteoclasts. 
     Osteoclastoma tissue was snap frozen in liquid nitrogen and used to prepare poly A +  mRA according to standard methods. cDNA cloning into a pcDNAII vector was carried out using a commercially-available kit (Librarian, InVitrogen). Approximately 2.6×10 6  clones were obtained, &gt;95% of which contained inserts of an average length 0.6 kB. 
     Example 2--Stromal Cell mRNA Preparation 
     A portion of each osteoclastoma was snap frozen in liquid nitrogen for mRNA preparation. The remainder of the tumor was dissociated using brief trypsinization and mechanical disaggregation, and placed into tissue culture. These cells were expanded in Dulbecco&#39;s MEM (high glucose, Sigma) supplemented with 10% newborn calf serum (MA Bioproducts), gentamycin (0.5 mg/ml), 1-glutamine (2 mM) and non-essential amino acids (0.1 mM) (Gibco). The stromal cell population was passaged at least five times, after which it showed a homogenous, fibroblastic looking cell population that contained no multinucleated cells. The stromal cells were mononuclear, tested negative acid phosphatase, and tested variably alkaline phosphatase positive. These findings indicate that propagated stromal cells (i.e., stromal cells that are passaged in culture) are non-osteoclastic and non-activated. 
     Example 3--Identification of DNA Encoding Osteoclastoma-Specific or -Related Gene Products by Differential screening of an Osteoclastoma cDNA Library 
     A total of 12,000 clones drawn from the osteoclastoma cDNA library were screened by differential hybridization, using mixed  32  P labelled cDNA probes derived from (1) giant cell tumor mRNA (stromal cell + , OC + ), and (2) mRNA from stromal cells (stromal cell + , OC + ) cultivated from the same tumor. The probes were labelled with  32  [P]dCTP by random priming to an activity of ˜10 9  CPM/μg. Of these 12,000 clones, 195 gave a positive hybridization signal with giant cell (i.e., osteoclast and stromal cell) mRNA, but not with stromal cell mRNA. Additionally, these clones failed to hybridize to cDNA produced from mRNA derived from a variety of unrelated human cell types including epithelial cells, fibroblasts, lymphocytes, myelomonocytic cells, osteoblasts, and neuroblastoma cells. The failure of these clones to hybridize to cDNA produced from mRNA derived from other cell types supports the conclusion that these clones are either uniquely expressed in osteoclasts, or are osteoclast-related. 
     The osteoclast (OC) cDNA library was screened for differential hybridization to OC cDNA (stromal cell + , OC + ) and stromal cell cDNA (stromal cell + , OC - ) as follows: 
     NYTRAN filters (Schleicher &amp; Schuell) were placed on agar plates containing growth medium and ampicillin. Individual bacterial colonies from the OC library were randomly picked and transferred, in triplicate, onto filters with preruled grids and then onto a master agar plate. Up to 200 colonies were inoculated onto a single 90-mm filter/plate using these techniques. The plates were inverted and incubated at 37° C. until the bacterial inoculates had grown (on the filter) to a diameter of 0.5-1.0 mm. 
     The colonies were then lysed, and the DNA bound to the filters by first placing the filters on top of two pieces of Whatman 3 MM paper saturated with 0.5N NaOH for 5 minutes. The filters were neutralized by placing on two pieces of Whatman 3 MM paper saturated with 1M Tris-HCL, pH 8.0 for 3-5 minutes. Neutralization was followed by incubation on another set of Whatman 3 MM papers saturated with 1M Tris-HCL, pH 8.0/1.5M NaCl for 3-5 minutes. The filters were then washed briefly in 2×SSC. 
     DNA was immobilized on the filters by baking the filters at 80° C. for 30 minutes. Filters were best used immediately, but they could be stored for up to one week in a vacuum jar at room temperature. 
     Filters were prehybridized in 5-8 ml of hybridization solution per filter, for 2-4 hours in a heat sealable bag. An additional 2 ml of solution was added for each additional filter added to the hybridization bag. The hybridization buffer consisted of 5×SSC, 5×Denhardt&#39;s solution, 1% SDS and 100 μg/ml denatured heterologous DNA. 
     Prior to hybridization, labeled probe was denatured by heating in 1×SSC for 5 minutes at 100° C., then immediately chilled on ice. Denatured probe was added to the filters in hybridization solution, and the filters hybridized with continuous agitation for 12-20 hours at 65° C. 
     After hybridization, the filters were washed in 2×SSC/0.2% SDS at 50°-60° C. for 30 minutes, followed by washing in 0.2×SSC/0.2% SDS at 60° C. for 60 minutes. 
     The filters were then air dried and autoradiographed using an intensifying screen at -70° C. overnight. 
     Example 4--DNA Sequencing of Selected Clones 
     Clones reactive with the mixed tumor probe, but unreactive with the stromal cell probe, are expected to contain either osteoclast-related, or in vivo `activated` stromal-cell-related gene products. One hundred and forty-four cDNA clones that hybridized to tumor cell cDNA, but not to stromal cell cDNA, were sequenced by the dideoxy chain termination method of Sanger et al. (Sanger F., et al. Proc. Natl. Acad. Sci. USA 74:5463 (1977)) using sequenase (US Biochemical). The DNASIS (Hitatchi) program was used to carry out sequence analysis and a homology search in the GenBank/EMBL database. 
     Fourteen of the 195 tumor +  stromal -  clones were identified as containing inserts with a sequence identical to the osteoclast marker, type 5 tartrate-resistant acid phosphatase (TRAP) (GenBank accession number J04430 M19534). The high representation of TRAP positive clones also indicates the effectiveness of the screening procedure in enriching for clones which contain osteoclast-specific or related cDNA sequences. 
     Interestingly, an even larger proportion of the tumor +  stromal -  clones (77/195; 39.5%) were identified as human gelatinase B (macrophage-derived gelatinase) (Wilhelm, S. M. J. Biol. Chem. 264:17213 (1989)), again indicating high expression of this enzyme by osteoclasts. Twenty-five of the gelatinase B clones were identified by dideoxy sequence analysis; all 25 showed 100% sequence homology to the published gelatinase B sequence (Genbank accession number J05070). The portions of the gelatinase B cDNA sequence covered by these clones is shown in the FIGURE (SEQ ID NO: 33). An additional 52 gelatinase B clones were identified by reactivity with a  32  P-labelled probe for gelatinase B. 
     Thirteen of the sequenced clones yielded no readable sequence. A DNASIS search of GenBank/EMBL databases revealed that, of the remaining 91 clones, 32 clones contain novel sequences which have not yet been reported in the databases or in the literature. These partial sequences are presented in Table I. Note that three of these sequences were repeats, indicating fairly frequent representation of mRNA related to this sequence. The repeat sequences are indicated by a , b  superscripts (Clones 198B, 223B and 32C of Table I). 
     
                                           TABLE I__________________________________________________________________________PARTIAL SEQUENCES OF 32 NOVEL OC-SPECIFIC OR -RELATEDEXPRESSED GENES (cDNA CLONES)34A (SEQ ID NO: 1)1  GCAAATATCT        AAGTTTATTG                  CTTGGATTTC                            TAGTGAGAGC                                      TGTTGAATTT                                                GGTGATGTCA61 AATGTTTCTA        GGGTTTTTTT                  AGTTTGTTTT                            TATTGAAAAA                                      TTTAATTATT                                                TATGCTATAG121   GTGATATTCT        CTTTGAATAA                  ACCTATAATA                            GAAAATAGCA                                      GCAGACAACA4B (SEQ ID NO: 2)1  GTGTCAACCT        GCATATCCTA                  AAAATGTCAA                            AATGCTGCAT                                      CTGGTTAATG                                                TCGGGGTAGG61 GGG12B (SEQ ID NO: 3)1  CTTCCCTCTC        TTGCTTCCCT                  TTCCCAAGCA                            GAGGTGCTCA                                      CTCCATGGCC                                                ACCGCCACCA61 CAGGCCCACA        GGGAGTACTG                  CCAGACTACT                            GCTGATGTTC                                      TCTTAAGGCC                                                CAGGGAGTCT121   CAACCAGCTG        GTGGTGAATG                  CTGCCTGGCA                            CGGGACCCCC                                      CCC28B (SEQ ID NO: 4)1  TTTTATTTGT        AAATATATGT                  ATTACATCCC                            TAGAAAAAGA                                      ATCCCAGGAT                                                TTTCCCTCCT61 GTGTGTTTTC        GTCTTGCTTC                  TTCATGGTCC                            ATGATGCCAG                                      CTGAGGTTGT                                                CAGTACAATG121   AAACCAAACT        GGCGGGATGG                  AAGCAGATTA                            TTCTGCCATT                                      TTTCCAGGTC                                                TTT37B (SEQ ID NO: 5)1  GGCTGGACAT        GGGTGCCCTC                  CACGTCCCTC                            ATATCCCCAG                                      GCACACTCTG                                                GCCTCAGGTT61 TTGCCCTGGC        CATGTCATCT                  ACCTGGAGTG                            GGCCCTCCCCC                                      TTCTTCAGCC                                                TTGAATCAAA121   AGCCACTTTG        TTAGGCGAGG                  ATTTCCCAGA                            CCACTCATCA                                      CATTAAAAAA                                                TATTTTGAAA181   ACAAAAAAAA        AAAAAAA55B (SEQ ID NO: 6)1  TTGACAAAGC        TGTTTATTTC                  CACCAATAAA                            TAGTATATGG                                      TGATTGGGGT                                                TTCTATTTAT61 AAGAGTAGTG        GCTATTATAT                  GGGGTATCAT                            GTTGATGCTC                                      ATAAATAGTT                                                CATATCTACT121   TAATTTGCCT        TC60B (SEQ ID NO: 7)1  GAAGAGAGTT        GTATGTACAA                  CCCCAACAGG                            CAAGGCAGCT                                      AAATGCAGAG                                                GGTACAGAGA61 GATCCCGAGG        GAATT86B (SEQ ID NO: 8)1  GGATGGAAAC        ATGTAGAAGT                  CCAGAGAAAA                            ACAATTTTAA                                      AAAAAGGTGG                                                AAAAGTTACG61 GCAAACCTGA        GATTTCAGCA                  TAAAATCTTT                            AGTTAGAAGT                                      GAGAGAAAGA                                                AGAGGGAGGC121   TGGTTGCTGT        TGCACGTATC                  AATAGGTTAT                            C87B (SEQ ID NO: 9)1  TTCTTGATCT        TTAGAACACT                  ATGAATAGGG                            AAAAAAGAAA                                      AAACTGTTCA                                                AAATAAAATG61 TAGGAGCCGT        GCTTTTGGAA                  TGCTTGAGTG                            AGGAGCTCAA                                      CAAGTCCTCT                                                CCCAAGAAAG181   CAATGATAAA        ACTTGACAAA                  A98B (SEQ ID NO: 10)1  ACCCATTTCT        AACAATTTTT                  ACTGTAAAAT                            TTTTGGTCAA                                      AGTTCTAAGC                                                TTAATCACAT61 CTCAAAGAAT        AGAGGCAATA                  TATAGCCCAT                            CTTACTAGAC                                      ATACAGTATT                                                AAACTGGACT121   GAATATGAGG        ACAAGCTCTA                  GTGGTCATTA                            AACCCCTCAG                                      AA110B (SEQ ID NO: 11)1  ACATATATTA        ACAGCATTCA                  TTTGGCCAAA                            ATCTACACGT                                      TTGTAGAATC                                                CTACTGTATA61 TAAAGTGGGA        ATGTATCAAG                  TATAGACTAT                            GAAAGTGCAA                                      ATAACAAGTC                                                AAGGTTAGAT121   TAACTTTTTT        TTTTTACATT                  ATAAAATTAA                            CTTGTTT118B (SEQ ID NO: 12)1  CCAAATTTCT        CTGGAATCCA                  TCCTCCCTCC                            CATCACCATA                                      GCCTCGAGAC                                                GTCATTTCTG61 TTTGACTACT        CCAGC133B (SEQ ID NO: 13)1  AACTAACCTC        CTCGGACCCC                  TGCCTCACTC                            ATTTACACCA                                      ACCACCCAAC                                                TATCTATAAA61 CCTGAGCCAT        GGCCATCCCT                  TATGAGCGGC                            GCAGTGATTA                                      TAGGCTTTCG                                                CTCTAAGATA121   AAAT140B (SEQ ID NO: 14)1  ATTATTATTC        TTTTTTTATG                  TTAGCTTAGC                            CATGCAAAAT                                      TTACTGGTGA                                                AGCAGTTAAT61 AAAACACACA        TCCCATTGAA                  GGGTTTTGTA                            CATTTCAGTC                                      CTTACAAATA                                                ACAAAGCAAT121   GATAAACCCG        GCACGTCCTG                  ATAGGAAATT                            C144B (SEQ ID NO: 15)1  CGTGACACAA        ACATGCATTC                  GTTTTATTCA                            TAAAACAGCC                                      TGGTTTCCTA                                                AAACAATACA61 AACAGCATGT        TCATCAGCAG                  GAAGCTGGCC                            GTGGGCAGGG                                      GGGCC198B.sup.a (SEQ ID NO: 16)1  ATAGGTTAGA        TTCTCATTCA                  CGGGACTAGT                            TAGCTTTAAG                                      CACCCTAGAG                                                GACTAGGGTA61 ATCTGACTTC        TCACTTCCTA                  AGTTCCCTCT                            TATATCCTCA                                      AGGTAGAAAT                                                GTCTATGTTT121   TCTACTCCAA        TTCATAAATC                  TATTCATAAG                            TCTTTGGTAC                                      AAGTTACATG                                                ATAAAAAGAA181   ATGTGATTTG        TCTTCCCTTC                  TTTGCACTTT                            TRAAATAAAG                                      TATTTATCTC                                                CTGTCTACAG241   TTTAAT212B (SEQ ID NO: 17)1  GTCCAGTATA        AAGGAAAGCG                  TTAAGTCGGT                            AAGCTAGAGG                                      ATTGTAAATA                                                TCTTTTATGT61 CCTCTAGATA        AAACACCCGA                  TTAACAGATG                            TTAACCTTTT                                      ATGTTTTGAT                                                TTGCTTTAAA121   AATGGCCTTC        TACACATTAG                  CTCCAGCTAA                            AAAGACACAT                                      TGAGAGCTTA                                                GAGGATAGTC181   TCTGGAGC223B.sup.b (SEQ ID NO: 18)1  GCACTTGGAA        GGGAGTTGGT                  GTGCTATTTT                            TGAAGCAGAT                                      GTGGTGATAC                                                TGAGATTGTC61 TGTTCAGTTT        CCCCATTTGT                  TTGTGCTTCA                            AATGATCCTT                                      CCTACTTTGC                                                TTCTCTCCAC121   CCATGACCTT        TTTCACTGTG                  GCCATCAAGG                            ACTTTCCTGA                                      CAGCTTGTGT                                                ACTCTTAGGC181   TAAGAGATGT        GACTACAGCC                  TGCCCCTGAC                            TG241B (SEQ ID NO: 19)1  TGTTAGTTTT        TAGGAAGGCC                  TGTCTTCTGG                            GAGTGAGGTT                                      TATTAGTCCA                                                CTTCTTGGAG61 CTAGACGTCC        TATAGTTAGT                  CACTGGGGAT                            GGTGAAAGAG                                      GGAGAAGAGG                                                AAGGGCGAAG121   GGAAGGGCTC        TTTGCTAGTA                  TCTCCATTTC                            TAGAAGATGG                                      TTTAGATGAT                                                AACCACAGGT181   CTATATGAGC        ATAGTAAGGC                  TGT32C.sup.b (SEQ ID NO: 20)1  CCTATTTCTG        ATCCTGACTT                  TGGACAAGGC                            CCTTCAGCCA                                      GAAGACTGAC                                                AAAGTCATCC121   TCCGTCTACC        AGAGCGTGCA                  CTTGTGATCC                            TAAAATAAGC                                      TTCATCTCCG                                                GCTGTGCCTT161   GGGTGGAAGG        GGCAGGATTC                  TGCAGCTGCT                            TTTGCATTTC                                      TCTTCCTAAA                                                TTTCATT34C (SEQ ID NO: 21)1  CGGAGCGTAG        GTGTGTTTAT                  TCCTGTACAA                            ATCATTACAA                                      AACCAAGTCT                                                GGGGCAGTCA61 CCGCCCCCAC        CCATCACCCC                  AGTGCAATGG                            CTAGCTGCTG                                      GCCTTT47C (SEQ ID NO: 22)1  TTAGTTCAGT        CAAAGCAGGC                  AACCCCCTTT                            GGCACTGCTG                                      CCACTGGGGT                                                CATGGCGGTT61 GTGGCAGCTG        GGGAGGTTTC                  CCCAACACCC                            TCCTCTGCTT                                      CCCTGTGTGT                                                CGGGGTCTCA121   GGAGCTGACC        CAGAGTGGA65C (SEQ ID NO: 23)1  GCTGAATGTT        TAAGAGAGAT                  TTTGGTCTTA                            AAGGCTTCAT                                      CATGAAAGTG                                                TACATGCATA61 TGCAAGTGTG        AATTACGTGG                  TATGGATGGT                            TGCTTGTTTA                                      TTAACTAAAG                                                ATGTACAGCA121   AACTGCCCGT        TTAGAGTCCT                  CTTAATATTG                            ATGTCCTAAC                                      ACTGGGTCTG                                                CTTATGC79C (SEQ ID NO: 24)1  GGCAGTGGGA        TATGGAATCC                  AGAAGGGAAA                            CAAGCACTGG                                      ATAATTAAAA                                                ACAGCTGGGG61 AGAAAACTGG        GGAAACAAAG                  GATATATCCT                            CATGGCTCGA                                      AATAAGAACA                                                ACGCCTGTGG121   CATTGCCAAC        CTGGCCAGCT                  TCCCCAAGAT                            GTGACTCCAG                                      CCAGAAA84C (SEQ ID NO: 25)1  GCCAGGGCGG        ACCGTCTTTA                  TTCCTCTCCT                            GCCTCAGAGG                                      TCAGGAAGGA                                                GGTCTGGCAG61 GACCTGCAGT        GGGCCCTAGT                  CATCTGTGGC                            AGCGAAGGTG                                      AAGGGACTCA                                                CCTTGTCGCC121   CGTGCCTGAG        TAGAACTTGT                  TCTGGAATTC                            C86C (SEQ ID NO: 26)1  AACTCTTTCA        CACTCTGGTA                  TTTTTAGTTT                            AACAATATAT                                      GTGTTGTGTC                                                TTGGAAATTA61 GTTCATATCA        ATTCATATTG                  AGCTGTCTCA                            TTCTTTTTTT                                      AATGGTCATA                                                TACAGTAGTA121   TTCAATTATA        AGAATATATC                  CTAATACTTT                            TTAAAA87C (SEQ ID NO: 27)1  GGATAAGAAA        GAAGGCCTGA                  GGCCTAGGGG                            CCGRGGCTGG                                      CCTGCGTCTC                                                AGTCCTGGGA61 CGCAGCAGCC        CGCACAGGTT                  GAGAGGGGCA                            CTTCCTCTTG                                      CTTAGGTTGG                                                TGAGGATCTG121   GTCCTGGTTG        GCCGGTGGAG                  AGCCACAAAA88C (SEQ ID NO: 28)1  CTGACCTTCG        AGAGTTTGAC                  CTGGAGCCGG                            ATACCTACTG                                      CCGCTATGAC                                                TCGGTCAGCG61 TGTTCAACGG        AGCCGTGAGC                  GACGACTCCG                            GTGGGGAAGT                                      TCTGCGGCGA                                                T89C (SEQ ID NO: 29)1  ATCCCTGGCT        GTGGATAGTG                  CTTTTGTGTA                            GCAAATGCTC                                      CCTCCTTAAG                                                GTTATAGGGC61 TCCCTGAGTT        TGGGAGTGTG                  GAAGTACTAC                            TTAACTGTCT                                      GTCCTGCTTG                                                GCTGTOGTTA121   TCGTTTTCTG        GTGATGTTGT                  GCTAACAATA                            AGAATAC101C (SEQ ID NO: 30)1  GGCTGGGCAT        CCCTCTCCTC                  CTCCATCCCC                            ATACATCACC                                      AGGTCTAATG                                                TTTACAAACG61 GTGCCAGCCC        GGCTCTGAAG                  CCAAGGGCCG                            TCCGTGCCAC                                      GGTGGCTGTG                                                AGTATTCCTC121   CGTTAGCTTT        CCCATAAGGT                  TGGAGTATCT                            GC112C (SEQ ID NO: 31)1  CCAACTCCTA        CCGCGATACA                  GACCCACAGA                            GTGCCATCCC                                      TGAGAGACCA                                                GACCGCTCCC161   CAATACTCTC        CTAAAATAAA                  CATGAAGCAC114C (SEQ ID NO: 32)1  CATGGATGAA        TGTCTCATGG                  TGGGAAGGAA                            CATGGTACAT                                      TTC__________________________________________________________________________ .sup.a Repeated 3 times .sup.b Repeated 2 times 
    
     Sequence analysis of the OC +  stromal cell -  cloned DNA sequences revealed, in addition to the novel sequences, a number of previously-described genes. The known genes identified (including type 5 acid phosphatase, gelatinase B, cystatin C (13 clones), Alu repeat sequences (11 clones), creatnine kinase (6 clones) and others) are summarized in Table II. In situ hybridization (described below) directly demonstrated that gelatinase B mRNA is expressed in multinucleated osteoclasts and not in stromal cells. Although gelatinase B is a well-characterized protease, its expression at high levels in osteoclasts has not been previously described. The expression in osteoclasts of cystatin C, a cysteine protease inhibitor, is also unexpected. This finding has not yet been confirmed by in situ hybridization. Taken together, these results demonstrate that most of these identified genes are osteoclast-expressed, thereby confirming the effectiveness of the differential screening strategy for identifying DNA encoding osteoclast-specific or -related gene products. Therefore, novel genes identified by this method have a high probability of being OC-specific or related. 
     In addition, a minority of the genes identified by this screen are probably not expressed by OCs (Table II). For example, type III collagen (6 clones), collagen type I (1 clone), dermatansulfate (1 clone), and type VI collagen (1 clone) are more likely to originate from the stromal cells or from osteoblastic cells which are present in the tumor. These cDNA sequences survive the differential screening process either because the cells which produce them in the tumor in vivo die out during the stromal cell propagation phase, or because they stop producing their product in vitro. These clones do not constitute more than 5-10% of the all sequences selected by differential hybridization. 
     
                       TABLE II______________________________________SEQUENCE ANALYSIS OF CLONES ENCODING KNOWNSEQUENCES FROM AN OSTEOCLASTOMA cDNALIBRARYClones with Sequence Homology                        25 totalto Collagenase Type IVClones with Sequence Homology to                        14 totalType 5 Tartrate Resistant Acid PhosphataseClones with Sequence Homology to                        13 totalCystatin C:Clones with Sequence Homology to                        11 totalAlu-repeat SequencesClones with Sequence Homology to                         6 totalCreatnine KinaseClones with Sequence Homology to                         6 totalType III CollagenClones with Sequence Homology to                         5 totalMHC Class I γ Invariant ChainClones with Sequence Homology to                         3 totalMHC Class II β ChainOne or Two Clone(s) with Sequence Homology to Each                        10 totalof the Following:αI collagen type Iγ interferon inducible proteinosteopontinHuman chondroitin/dermatansulfateα globinβ glucosidase/sphingolipid activatorHuman CAPL protein (Ca binding)Human EST 01024Type VI collagenHuman EST 00553______________________________________ 
    
     Example 5--In situ Hybridiation of OC-Expressed Genes 
     In situ hybridization was performed using probes derived from novel cloned sequences in order to determine whether the novel putative OC-specific or -related genes are differentially expressed in osteoclasts (and not expressed in the stromal cells) of human giant cell tumors. Initially, in situ hybridization was performed using antisense (positive) and sense (negative control) cRNA probes against human type IV collagenase/gelatinase B labelled with  35  S-UTP. 
     A thin section of human giant cell tumor reacted with the antisense probe resulted in intense labelling of all OCs, as indicated by the deposition of silver grains over these cells, but failed to label the stromal cell elements. In contrast, only minimal background labelling was observed with the sense (negative control) probe. This result confirmed that gelatinase B is expressed in human OCs. 
     In situ hybridization was then carried out using cRNA probes derived from 11/32 novel genes, labelled with digoxigenin UTP according to known methods. 
     The results of this analysis are summarized in Table III. Clones 28B, 118B, 140B, 198B, and 212B all gave positive reactions with OCs in frozen sections of a giant cell tumor, as did the positive control gelatinase B. These novel clones therefore are expressed in OCs and fulfill all criteria for OC-relatedness. 198B is repeated three times, indicating relatively high expression. Clones 4B, 37B, 88C and 98B produced positive reactions with the tumor tissue; however the signal was not well-localized to OCs. These clones are therefore not likely to be useful and are eliminated from further consideration. Clones 86B and 87B failed to give a positive reaction with any cell type, possibly indicating very low level expression. This group of clones could still be useful but may be difficult to study further. The results of this analysis show that 5/11 novel genes are expressed in OCs, indicating that ˜50% of novel sequences likely to be OC-related. 
     To generate probes for the in situ hybridizations, cDNA derived from novel cloned osteoclast-specific or -related cDNA was subcloned into a BlueScript II SK(-) vector. The orientation of cloned inserts was determined by restriction analysis of subclones. The T7 and T3 promoters in the BlueScriptII vector was used to generate  35  S-labelled ( 35  S-UTP 850 Ci/mmol, Amersham, Arlington Heights, Ill.), or UTP digoxygenin labelled cRNA probes. 
     
                       TABLE III______________________________________In Situ HYBRIDIZATION USING PROBESDERIVED FROM NOVEL SEQUENCES        Reactivity with:Clone          Osteoclasts                    Stromal Cells______________________________________4B             +         +28B*           +         -37B            +         +86B            -         -87B            -         -88C            +         +98B            +         +118B*          +         -140B*          +         -198B*          +         -212B*          +         -Gelatinase B*  +         -______________________________________ *OC-expressed, as indicated by reactivity with antisense probe and lack o reactivity with sense probe on OCs only. 
    
     In situ hybridization was carried out on 7 micron cryostat sections of a human osteoclastoma as described previously (Chang, L.-C. et al. Cancer Res. 49:6700 (1989)). Briefly, tissue was fixed in 4% paraformaldehyde and embedded in OCT (Miles Inc., Kankakee, Ill.). The sections were rehydrated, postfixed in 4% paraformaldehyde, washed, and pretreated with 10 mM DTT, 10 mM iodoacetamide, 10 mM N-ethylmaleimide and 0.1 triethanolamine-HCL. Prehybridization was done with 50% deionized formamide, 10 mM Tris-HCl, pH 7.0, 1× Denhardt&#39;s, 500 mg/ml tRNA, 80 mg/ml salmon sperm DNA, 0.3M NaCl, mM EDTA, and 100 mM DTT at 45° C. for 2 hours. Fresh hybridization solution containing 10% dextran sulfate and 1.5 ng/ml  35  S-labelled or digoxygenin labelled RNA probe was applied after heat denaturation. Sections were coverslipped and then incubated in a moistened chamber at 45°-50° C. overnight. Hybridized sections were washed four times with 50% formamide, 2× SSC, containing 10 mM DTT and 0.5% Triton X-100 at 45° C. Sections were treated with RNase A and RNase T1 to digest single-stranded RNA, washed four times in 2× SSC/10 mM DTT. 
     In order to detect  35  S-labelling by autoradiography, slides were dehydrated, dried, and coated with Kodak NTB-2 emulsion. The duplicate slides were split, and each set was placed in a black box with desiccant, sealed, and incubated at 4° C. for 2 days. The slides were developed (4 minutes) and fixed (5 minutes) using Kodak developer D19 and Kodak fixer. Hematoxylin and eosin were used as counter-stains. 
     In order to detect digoxygenin-labelled probes, a Nucleic Acid Detection Kit (Boehringer-Mannheim, Cat. #1175041) was used. Slides were washed in Buffer 1 consisting of 100 mM Tris/150 mM NaCl, pH7.5, for 1 minute. 100 μl Buffer 2 was added (made by adding 2 mg/ml blocking reagent as provided by the manufacturer) in Buffer 1 to each slide. The slides were placed on a shaker and gently swirled at 20° C. 
     Antibody solutions were diluted 1:100 with Buffer 2 (as provided by the manufacturer). 100 μl of diluted antibody solution was applied to the slides and the slides were then incubated in a chamber for 1 hour at room temperature. The slides were monitored to avoid drying. After incubation with antibody solution, slides were washed in Buffer 1 for 10 minutes, then washed in Buffer 3 containing 2 mM levamisole for 2 minutes. 
     After washing, 100 μl color solution was added to the slides. Color solution consisted of nitroblue/tetrazolium salt (NBT) (1:225 dilution) 4.5 μl, 5-bromo-4-chloro-3-indolyl phosphate (1:285 dilution) 3.5 μl, levamisole 0.2 mg in Buffer 3 (as provided by the manufacturer) in a total volume of 1 ml. Color solution was prepared immediately before use. 
     After adding the color solution, the slides were placed in a dark, humidified chamber at 20° C. for 2-5 hours and monitored for color development. The color reaction was stopped by rinsing slides in TE Buffer. 
     The slides were stained for 60 seconds in 0.25% methyl green, washed with tap water, then mounted with water-based Permount (Fisher). 
     Example 6--Immunohistochemistry 
     Immunohistochemical staining was performed on frozen and paraffin embedded tissues as well as on cytospin preparations (see Table IV). The following antibodies were used: polyclonal rabbit anti-human gelatinase antibodies; Ab110 for gelatinase B; monoclonal mouse anti-human CD68 antibody (clone KP1) (DAKO, Denmark); Mol (anti-CD11b) and Mo2 (anti-CD14) derived from ATCC cell lines HB CRL 8026 and TIB 228/HB44. The anti-human gelatinase B antibody Ab110 was raised against a synthetic peptide with the amino acid sequence EALMYPMYRFTEGPPLHK (SEQ ID NO: 34), which is specific for human gelatinase B (Corcoran, M. L. et al. J. Biol. Chem, 267:515 (1992)). 
     Detection of the immunohistochemical staining was achieved by using a goat anti-rabbit glucose oxidase kit (Vector Laboratories, Burlingame Calif.) according to the manufacturer&#39;s directions. Briefly, the sections were rehydrated and pretested with either acetone or 0.1% trypsin. Normal goat serum was used to block nonspecific binding. Incubation with the primary antibody for 2 hours or overnight (Abl10:1/500 dilution) was followed by either a glucose oxidase labeled secondary anti-rabbit serum, or, in the case of the mouse monoclonal antibodies, were reacted with purified rabbit anti-mouse Ig before incubation with the secondary antibody. 
     Paraffin embedded and frozen sections from osteoclastomas (GCT) were reacted with a rabbit antiserum against gelatinase B (antibody 110) (Corcoran, M. L. et al. J. Biol. chem. 267:515 (1992)), followed by color development with glucose oxidase linked reagents. The osteoclasts of a giant cell tumor were uniformly strongly positive for gelatinase B, whereas the stromal cells were unreactive. Control sections reacted with rabbit preimmune serum were negative. Identical findings were obtained for all 8 long bone giant cell tumors tested (Table IV). The osteoclasts present in three out of four central giant cell granulomas (GCG) of the mandible were also positive for gelatinase B expression. These neoplasms are similar but not identical to the long bone giant cell tumors, apart from their location in the jaws (Shafer, W. G. et al., Textbook of Oral Pathology, W. B. Saunders Company, Philadelphia, pp. 144-149 (1983)). In contrast, the multinucleated cells from a peripheral giant cell tumor, which is a generally non-resorptive tumor of oral soft tissue, were unreactive with antibody (Shafer, W. G. et. al., Textbook of Oral Pathology, W. B. Saunders Company, Philadelphia, pp. 144-149 (1983)). 
     Antibody 110 was also utilized to assess the presence of gelatinase B in normal bone (n=3) and in Paget&#39;s disease, in which there is elevated bone remodeling and increased osteoclastic activity. Strong staining for gelatinase B was observed in osteoclasts both in normal bone (mandible of a 2 year old), and in Paget&#39;s disease. Staining was again absent in controls incubated with preimmune serum. Osteoblasts did not stain in any of the tissue sections, indicating that gelatinase B expression is limited to osteoclasts in bone. Finally, peripheral blood monocytes were also reactive with antibody 110 (Table IV). 
     
                       TABLE IV______________________________________DISTRIBUTION OF GELATINASE B IN VARIOUSTISSUES          Antibodies tested          Ab 110Samples        gelatinase B______________________________________GCT frozen(n = 2)giant cells    +stromal cells  -GCT paraffin(n = 6)giant cells    +stromal cells  -central GCG(n = 4)giant cells      +(3/4)stromal cells  -peripheral GCT(n - 4)giant cells    -stromal cells  -Paget&#39;s disease(n = 1)osteoclasts    +osteoblasts    -normal bone(n = 3)osteoclasts    +osteoblasts    -monocytes      +(cytospin)______________________________________ Distribution of gelatinase B in multinucleated giant cells, osteoclasts, osteoblasts and stromal cells in various tissues. In general, paraffin embedded tissues were used for these experiments; exceptions are indicated. 
    
     Equivalents 
     Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. 
     
         __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 34(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 170 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GCAAATATCTAAGTTTATTGCTTGGATTTCTAGTGAGAGCTGTTGAATTTGGTGATGTCA60AATGTTTCTAGGGTTTTTTTAGTTTGTTTTTATTGAAAAATTTAATTATTTATGCTATAG120GTGATATTCTCTTTGAATAAACCTATAATAGAAAATAGCAGCAGACAACA170(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 63 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GTGTCAACCTGCATATCCTAAAAATGTCAAAATGCTGCATCTGGTTAATGTCGGGGTAGG60GGG63(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 163 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:CTTCCCTCTCTTGCTTCCCTTTCCCAAGCAGAGGTGCTCACTCCATGGCCACCGCCACCA60CAGGCCCACAGGGAGTACTGCCAGACTACTGCTGATGTTCTCTTAAGGCCCAGGGAGTCT120CAACCAGCTGGTGGTGAATGCTGCCTGGCACGGGACCCCCCCC163(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 173 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:TTTTATTTGTAAATATATGTATTACATCCCTAGAAAAAGAATCCCAGGATTTTCCCTCCT60GTGTGTTTTCGTCTTGCTTCTTCATGGTCCATGATGCCAGCTGAGGTTGTCAGTACAATG120AAACCAAACTGGCGGGATGGAAGCAGATTATTCTGCCATTTTTCCAGGTCTTT173(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 197 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:GGCTGGACATGGGTGCCCTCCACGTCCCTCATATCCCCAGGCACACTCTGGCCTCAGGTT60TTGCCCTGGCCATGTCATCTACCTGGAGTGGGCCCTCCCCTTCTTCAGCCTTGAATCAAA120AGCCACTTTGTTAGGCGAGGATTTCCCAGACCACTCATCACATTAAAAAATATTTTGAAA180ACAAAAAAAAAAAAAAA197(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 132 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:TTGACAAAGCTGTTTATTTCCACCAATAAATAGTATATGGTGATTGGGGTTTCTATTTAT60AAGAGTAGTGGCTATTATATGGGGTATCATGTTGATGCTCATAAATAGTTCATATCTACT120TAATTTGCCTTC132(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 75 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:GAAGAGAGTTGTATGTACAACCCCAACAGGCAAGGCAGCTAAATGCAGAGGGTACAGAGA60GATCCCGAGGGAATT75(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 151 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:GGATGGAAACATGTAGAAGTCCAGAGAAAAACAATTTTAAAAAAAGGTGGAAAAGTTACG60GCAAACCTGAGATTTCAGCATAAAATCTTTAGTTAGAAGTGAGAGAAAGAAGAGGGAGGC120TGGTTGCTGTTGCACGTATCAATAGGTTATC151(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 141 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:TTCTTGATCTTTAGAACACTATGAATAGGGAAAAAAGAAAAAACTGTTCAAAATAAAATG60TAGGAGCCGTGCTTTTGGAATGCTTGAGTGAGGAGCTCAACAAGTCCTCTCCCAAGAAAG120CAATGATAAAACTTGACAAAA141(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 162 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:ACCCATTTCTAACAATTTTTACTGTAAAATTTTTGGTCAAAGTTCTAAGCTTAATCACAT60CTCAAAGAATAGAGGCAATATATAGCCCATCTTACTAGACATACAGTATTAAACTGGACT120GAATATGAGGACAAGCTCTAGTGGTCATTAAACCCCTCAGAA162(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 157 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:ACATATATTAACAGCATTCATTTGGCCAAAATCTACACGTTTGTAGAATCCTACTGTATA60TAAAGTGGGAATGTATCAAGTATAGACTATGAAAGTGCAAATAACAAGTCAAGGTTAGAT120TAACTTTTTTTTTTTACATTATAAAATTAACTTGTTT157(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 75 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:CCAAATTTCTCTGGAATCCATCCTCCCTCCCATCACCATAGCCTCGAGACGTCATTTCTG60TTTGACTACTCCAGC75(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 124 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:AACTAACCTCCTCGGACCCCTGCCTCACTCATTTACACCAACCACCCAACTATCTATAAA60CCTGAGCCATGGCCATCCCTTATGAGCGGCGCAGTGATTATAGGCTTTCGCTCTAAGATA120AAAT124(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 151 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:ATTATTATTCTTTTTTTATGTTAGCTTAGCCATGCAAAATTTACTGGTGAAGCAGTTAAT60AAAACACACATCCCATTGAAGGGTTTTGTACATTTCAGTCCTTACAAATAACAAAGCAAT120GATAAACCCGGCACGTCCTGATAGGAAATTC151(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 105 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:CGTGACACAAACATGCATTCGTTTTATTCATAAAACAGCCTGGTTTCCTAAAACAATACA60AACAGCATGTTCATCAGCAGGAAGCTGGCCGTGGGCAGGGGGGCC105(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 246 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:ATAGGTTAGATTCTCATTCACGGGACTAGTTAGCTTTAAGCACCCTAGAGGACTAGGGTA60ATCTGACTTCTCACTTCCTAAGTTCCCTCTTATATCCTCAAGGTAGAAATGTCTATGTTT120TCTACTCCAATTCATAAATCTATTCATAAGTCTTTGGTACAAGTTACATGATAAAAAGAA180ATGTGATTTGTCTTCCCTTCTTTGCACTTTTGAAATAAAGTATTTATCTCCTGTCTACAG240TTTAAT246(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 188 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:GTCCAGTATAAAGGAAAGCGTTAAGTCGGTAAGCTAGAGGATTGTAAATATCTTTTATGT60CCTCTAGATAAAACACCCGATTAACAGATGTTAACCTTTTATGTTTTGATTTGCTTTAAA120AATGGCCTTCTACACATTAGCTCCAGCTAAAAAGACACATTGAGAGCTTAGAGGATAGTC180TCTGGAGC188(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 212 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:GCACTTGGAAGGGAGTTGGTGTGCTATTTTTGAAGCAGATGTGGTGATACTGAGATTGTC60TGTTCAGTTTCCCCATTTGTTTGTGCTTCAAATGATCCTTCCTACTTTGCTTCTCTCCAC120CCATGACCTTTTTCACTGTGGCCATCAAGGACTTTCCTGACAGCTTGTGTACTCTTAGGC180TAAGAGATGTGACTACAGCCTGCCCCTGACTG212(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 203 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:TGTTAGTTTTTAGGAAGGCCTGTCTTCTGGGAGTGAGGTTTATTAGTCCACTTCTTGGAG60CTAGACGTCCTATAGTTAGTCACTGGGGATGGTGAAAGAGGGAGAAGAGGAAGGGCGAAG120GGAAGGGCTCTTTGCTAGTATCTCCATTTCTAGAAGATGGTTTAGATGATAACCACAGGT180CTATATGAGCATAGTAAGGCTGT203(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 177 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:CCTATTTCTGATCCTGACTTTGGACAAGGCCCTTCAGCCAGAAGACTGACAAAGTCATCC60TCCGTCTACCAGAGCGTGCACTTGTGATCCTAAAATAAGCTTCATCTCCGGCTGTGCCTT120GGGTGGAAGGGGCAGGATTCTGCAGCTGCTTTTGCATTTCTCTTCCTAAATTTCATT177(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 106 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:CGGAGCGTAGGTGTGTTTATTCCTGTACAAATCATTACAAAACCAAGTCTGGGGCAGTCA60CCGCCCCCACCCATCACCCCAGTGCAATGGCTAGCTGCTGGCCTTT106(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 139 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:TTAGTTCAGTCAAAGCAGGCAACCCCCTTTGGCACTGCTGCCACTGGGGTCATGGCGGTT60GTGGCAGCTGGGGAGGTTTCCCCAACACCCTCCTCTGCTTCCCTGTGTGTCGGGGTCTCA120GGAGCTGACCCAGAGTGGA139(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 177 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:GCTGAATGTTTAAGAGAGATTTTGGTCTTAAAGGCTTCATCATGAAAGTGTACATGCATA60TGCAAGTGTGAATTACGTGGTATGGATGGTTGCTTGTTTATTAACTAAAGATGTACAGCA120AACTGCCCGTTTAGAGTCCTCTTAATATTGATGTCCTAACACTGGGTCTGCTTATGC177(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 167 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:GGCAGTGGGATATGGAATCCAGAAGGGAAACAAGCACTGGATAATTAAAAACAGCTGGGG60AGAAAACTGGGGAAACAAAGGATATATCCTCATGGCTCGAAATAAGAACAACGCCTGTGG120CATTGCCAACCTGGCCAGCTTCCCCAAGATGTGACTCCAGCCAGAAA167(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 151 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:GCCAGGGCGGACCGTCTTTATTCCTCTCCTGCCTCAGAGGTCAGGAAGGAGGTCTGGCAG60GACCTGCAGTGGGCCCTAGTCATCTGTGGCAGCGAAGGTGAAGGGACTCACCTTGTCGCC120CGTGCCTGAGTAGAACTTGTTCTGGAATTCC151(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 156 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:AACTCTTTCACACTCTGGTATTTTTAGTTTAACAATATATGTGTTGTGTCTTGGAAATTA60GTTCATATCAATTCATATTGAGCTGTCTCATTCTTTTTTTAATGGTCATATACAGTAGTA120TTCAATTATAAGAATATATCCTAATACTTTTTAAAA156(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 150 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:GGATAAGAAAGAAGGCCTGAGGGCTAGGGGCCGGGGCTGGCCTGCGTCTCAGTCCTGGGA60CGCAGCAGCCCGCACAGGTTGAGAGGGGCACTTCCTCTTGCTTAGGTTGGTGAGGATCTG120GTCCTGGTTGGCCGGTGGAGAGCCACAAAA150(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 212 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:GCACTTGGAAGGGAGTTGGTGTGCTATTTTTGAAGCAGATGTGGTGATACTGAGATTGTC60TGTTCAGTTTCCCCATTTGTTTGTGCTTCAAATGATCCTTCCTACTTTGCTTCTCTCCAC120CCATGACCTTTTTCACTGTGGCCATCAAGGACTTTCCTGACAGCTTGTGTACTCTTAGGC180TAAGAGATGTGACTACAGCCTGCCCCTGACTG212(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 157 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:ATCCCTGGCTGTGGATAGTGCTTTTGTGTAGCAAATGCTCCCTCCTTAAGGTTATAGGGC60TCCCTGAGTTTGGGAGTGTGGAAGTACTACTTAACTGTCTGTCCTGCTTGGCTGTCGTTA120TCGTTTTCTGGTGATGTTGTGCTAACAATAAGAATAC157(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 152 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:GGCTGGGCATCCCTCTCCTCCTCCATCCCCATACATCACCAGGTCTAATGTTTACAAACG60GTGCCAGCCCGGCTCTGAAGCCAAGGGCCGTCCGTGCCACGGTGGCTGTGAGTATTCCTC120CGTTAGCTTTCCCATAAGGTTGGAGTATCTGC152(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 90 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:CCAACTCCTACCGCGATACAGACCCACAGAGTGCCATCCCTGAGAGACCAGACCGCTCCC60CAATACTCTCCTAAAATAAACATGAAGCAC90(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 43 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:CATGGATGAATGTCTCATGGTGGGAAGGAACATGGTACATTTC43(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2333 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:AGACACCTCTGCCCTCACCATGAGCCTCTGGCAGCCCCTGGTCCTGGTGCTCCTGGTGCT60GGGCTGCTGCTTTGCTGCCCCCAGACAGCGCCAGTCCACCCTTGTGCTCTTCCCTGGAGA120CCTGAGAACCAATCTCACCGACAGGCAGCTGGCAGAGGAATACCTGTACCGCTATGGTTA180CACTCGGGTGGCAGAGATGCGTGGAGAGTCGAAATCTCTGGGGCCTGCGCTGCTGCTTCT240CCAGAAGCAACTGTCCCTGCCCGAGACCGGTGAGCTGGATAGCGCCACGCTGAAGGCCAT300GCGAACCCCACGGTGCGGGGTCCCAGACCTGGGCAGATTCCAAACCTTTGAGGGCGACCT360CAAGTGGCACCACCACAACATCACCTATTGGATCCAAAACTACTCGGAAGACTTGCCGCG420GGCGGTGATTGACGACGCCTTTGCCCGCGCCTTCGCACTGTGGAGCGCGGTGACGCCGCT480CACCTTCACTCGCGTGTACAGCCGGGACGCAGACATCGTCATCCAGTTTGGTGTCGCGGA540GCACGGAGACGGGTATCCCTTCGACGGGAAGGACGGGCTCCTGGCACACGCCTTTCCTCC600TGGCCCCGGCATTCAGGGAGACGCCCATTTCGACGATGACGAGTTGTGGTCCCTGGGCAA660GGGCGTCGTGGTTCCAACTCGGTTTGGAAACGCAGATGGCGCGGCCTGCCACTTCCCCTT720CATCTTCGAGGGCCGCTCCTACTCTGCCTGCACCACCGACGGTCGCTCCGACGGGTTGCC780CTGGTGCAGTACCACGGCCAACTACGACACCGACGACCGGTTTGGCTTCTGCCCCAGCGA840GAGACTCTACACCCGGGACGGCAATGCTGATGGGAAACCCTGCCAGTTTCCATTCATCTT900CCAAGGCCAATCCTACTCCGCCTGCACCACGGACGGTCGCTCCGACGGCTACCGCTGGTG960CGCCACCACCGCCAACTACGACCGGGACAAGCTCTTCGGCTTCTGCCCGACCCGAGCTGA1020CTCGACGGTGATGGGGGGCAACTCGGCGGGGGAGCTGTGCGTCTTCCCCTTCACTTTCCT1080GGGTAAGGAGTACTCGACCTGTACCAGCGAGGGCCGCGGAGATGGGCGCCTCTGGTGCGC1140TACCACCTCGAACTTTGACAGCGACAAGAAGTGGGGCTTCTGCCCGGACCAAGGATACAG1200TTTGTTCCTCGTGGCGGCGCATGAGTTCGGCCACGCGCTGGGCTTAGATCATTCCTCAGT1260GCCGGAGGCGCTCATGTACCCTATGTACCGCTTCACTGAGGGGCCCCCCTTGCATAAGGA1320CGACGTGAATGGCATCCGGCACCTCTATGGTCCTCGCCCTGAACCTGAGCCACGGCCTCC1380AACCACCACCACACCGCAGCCCACGGCTCCCCCGACGGTCTGCCCCACCGGACCCCCCAC1440TGTCCACCCCTCAGAGCGCCCCACAGCTGGCCCCACAGGTCCCCCCTCAGCTGGCCCCAC1500AGGTCCCCCCACTGCTGGCCCTTCTACGGCCACTACTGTGCCTTTGAGTCCGGTGGACGA1560TGCCTGCAACGTGAACATCTTCGACGCCATCGCGGAGATTGGGAACCAGCTGTATTTGTT1620CAAGGATGGGAAGTACTGGCGATTCTCTGAGGGCAGGGGGAGCCGGCCGCAGGGCCCCTT1680CCTTATCGCCGACAAGTGGCCCGCGCTGCCCCGCAAGCTGGACTCGGTCTTTGAGGAGCC1740GCTCTCCAAGAAGCTTTTCTTCTTCTCTGGGCGCCAGGTGTGGGTGTACACAGGCGCGTC1800GGTGCTGGGCCCGAGGCGTCTGGACAAGCTGGGCCTGGGAGCCGACGTGGCCCAGGTGAC1860CGGGGCCCTCCGGAGTGGCAGGGGGAAGATGCTGCTGTTCAGCGGGCGGCGCCTCTGGAG1920GTTCGACGTGAAGGCGCAGATGGTGGATCCCCGGAGCGCCAGCGAGGTGGACCGGATGTT1980CCCCGGGGTGCCTTTGGACACGCACGACGTCTTCCAGTACCGAGAGAAAGCCTATTTCTG2040CCAGGACCGCTTCTACTGGCGCGTGAGTTCCCGGAGTGAGTTGAACCAGGTGGACCAAGT2100GGGCTACGTGACCTATGACATCCTGCAGTGCCCTGAGGACTAGGGCTCCCGTCCTGCTTT2160GCAGTGCCATGTAAATCCCCACTGGGACCAACCCTGGGGAAGGAGCCAGTTTGCCGGATA2220CAAACTGGTATTCTGTTCTGGAGGAAAGGGAGGAGTGGAGGTGGGCTGGGCCCTCTCTTC2280TCACCTTTGTTTTTTGTTGGAGTGTTTCTAATAAACTTGGATTCTCTAACCTTT2334(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:GluAlaLeuMetTyrProMetTyrArgPheThrGluGlyProProLeu151015HisLys__________________________________________________________________________