Determination of transcription and translation of testis-specific histone RNA during spermatogenesis is essential for understanding the molecular mechanisms involved in the replacement of somatic histones during this differentiation process. During rat spermatogenesis, testis-specific histone variants are synthesized in primary spermatocytes and replace over half of somatic histones in whole testis, and there is an indication that somatic histones are nearly completely replaced by the variant histones in late stages of meiotic prophase. In contrast with somatic cells where synthesis of histones is coupled with DNA synthesis, the synthesis of testis-specific histones continues through meiotic prophage in which there is little DNA synthesis. The major objective of this application is to clone one of the testis-specific histone genes, the gene for variant H2B (TH2B-x) and use it as a hybridization probe to investigate transcription and translation of testis-specific histone RNA at different stages of spermatogenesis. 1. The cDNA for TH2B-x and H2B histone mRNA will be cloned from adult rat testis using standard molecular cloning methods. 2. The cDNAs will be sequenced, and the DNA and amino acid sequence of the TH2B-x and H2B histones will be compared. The regions which show extensive sequence variations will be subcloned into a bacterial plasmid and used as specific hybridization probes for TH2B-x and H2B RNA. 3. The presence of TH2B-x and H2B RNA will be determined by in situ cytohybridization using both Sertoli-spermatogenic co-cultures and whole-mount preparations of freshly-isolated, spermatogenic stage-specific seminiferous tubules. The cell types in which the histone genes are transcribed and the spermatogenic stages in which translation of the histones continue will be determined. 4. The possible storage of TH2B-x mRNA in translationally inactive cytoplasmic histone mRNA-protein complexes in pachytene spermatocytes will be examined. 5. The transcription of TH2B-x gene and synthesis of testis-specific histones during the transition of spermatogonia to spermatocytes will be studied using Sertoli-spermatogonia co-cultures.
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