|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,400,000 (Direct Cost : ¥1,400,000)
We had produced several differentiation stage specific monoclonal antibodies against mouse testicular germ cells, and we isolated the several cDNAs for those antigen genes from a mouse testis cDNA library using these antibodies. In these genes, calmegin had 611 amino acids, highly homology against calreticulin, and Ca binding activity. And next, we cloned the 5' up-stream region of calmegin genomic DNA, and analyzed its sequences and promoter activity. As the results, the testis specific promoter activity was present within about 200 bp of the 5' up-stream region from it's transcription start site, and this region had a GC box and high GC contents. Furthermore, gel shift assay revealed that there were several testis specific retarded bands using testicular nuclear extracts. Moreover, calmegin genomic DNA was introduced into a targeting vector, and the several ES cell lines which were happened to homologous recombinations were isolated. Now, we tried to make a chimera mouse using these ES cell lines.
In addition, we made germ cell specific polyclonal antibodies, analyzed those antigen molecules by western blotting. We identified more than ten antigen molecules which were presented in differentiation specific manner. And next, we isolated 11 cDNA clones against these antigens using the testis expression cDNA library, it revealed that 6 clones were new genes and 5 clones had homology for other genes. By northern blotting analysis, these genes were specifically expressed in testis. In these genes, GAG12 had highly homology for rat SSeCKS, which was tumor suppressor gene. GAG12 was expressed at meiosis, and was seemed that it may be related to the cell cycle of germ cells. GAG5 had highly homology for rat SCP-1, which was composed of synaptonemal complex at meiosis. And then, we isolated a human homologue gene for SCP-1, and decided its sequences.