Rsb66 And The Hsd13 Encoded Protein Function Study

Mammalian spermatogenesis provides a unique model to address the underlying mechanism governing cell cycle regulation and differentiation. In this progress, stem cells undergoing mitotic proliferation proceed into meiosis, followed by a remodeling of the haploid spermatids to form mature spermatozoa, associated with the expression of multiple genes to form essential proteins under stringent temporal and spatial regulation. Laser Capture Microdissection(LCM) was a novel technique recently developed to isolate pure population of specific cells from miscellaneous tissues. The captured cell population can be used in the subsequent assay for DNA, RNA and protein. In our laboratory, tens of thousands of rat spermatocytes and round spermatids had been isolated by LCM and total RNA of these two cells had been extracted. The Suppression Subtracted Hybridization (SSH) library of round spermatid-specific cDNAs against those of primary spermatocyte (RSB) had been constructed.RSB-66 was obtained from RSB subtracted library. Its full-length cDNA composed of 564bp and was assigned the GenBank accession number AY121839. The reading frame encodes a polypeptide consisting of 168 amino acid residues. Northern blot showed that RSB66 gene was specifically expressed in rat testis. RSB66 human homolog was also cloned, the coding sequences of RSB66 and RSB66 human homolog showed 88.2% homology on the nucleotide and 92.3% identity on the amino acid level. Such highly homolog between human and rodents suggests that this gene is conserved in the evolution.Yeast two-hybrid system had been used to screen the partners of RSB66 protein in human testis cDNA library. Two positive clone which was confirmed to interact with pAS2-l-RSB66, but not to interact with the control pAS2-l and pLAM5'-l was isolated and designated as HSD45 (AY604176) and HSD46 (AY604177). The full-length cDNA of HSD45 composed of 985bp.The reading frame encodes a polypeptide consisting of 221 amino acid residues. Blast analysis showed that HSD45 was identical to INCA1 (Inhibitor of CDK interacting with Cyclin A1), a novel cyclin A1/CDK2 interaction partner in a yeast-triple hybrid approach. The full-length cDNA of HSD46 composed of 1163bp.The reading frame encodes a polypeptide consisting of 317 amino acid residues. The HSD46 homologs had been found in the genome of Pan troglodytes, Canis familiaris and Bos Taurus and show highly conserved in the evolution. No functional study of this gene is available so far.In vitro and in vivo system had been used to comfirm the interaction between RSB66 and HSD45. Firstly, His-RSB66 and GST-HSD45 fusion proteins had been expressed in E coli, and purified by affinity chromatography. GST pull-down assay between these two recombinant proteins was performed. The result showed that His-RSB66 can co-elute with GST-HSD45, but not with GST alone. To further examine...