TESTIS-SPECIFIC HISTONES AND MRNA DURING SPERMATOGENESIS

睾丸特异性组蛋白转录和翻译的测定 精子发生过程中的rna对于理解分子是必不可少的。 在此过程中涉及体细胞组蛋白替换的机制 分化过程。在大鼠精子发生过程中，睾丸特异 组蛋白变体在初级精母细胞中合成并取代 整个睾丸中有一半的体细胞组蛋白，而且有迹象表明 体细胞组蛋白几乎完全被变异的组蛋白所取代 减数分裂前期的晚期。与体细胞相比，在体细胞 组蛋白的合成与DNA合成相结合，组蛋白的合成 睾丸特异组蛋白通过减数分裂前驱体继续存在 是很少的DNA合成。此应用程序的主要目标是 克隆睾丸特异的组蛋白基因之一--H_2B变异体基因 (TH2B-x)，并将其用作杂交探针研究转录 以及睾丸特异性组蛋白RNA在不同阶段的翻译 精子发生。 1.从成年大鼠中克隆TH_2B-x和H_2B组蛋白基因的c DNA 使用标准的分子克隆方法克隆睾丸。 2.对cDNA进行测序，并测定其DNA和氨基酸序列 我们将比较TH2 B-x和H 2B组蛋白。显示广泛性的地区 序列变异将被亚克隆到细菌质粒中，并用作 TH_2B-x和H_2B RNA特异性杂交探针。 3.TH_2B-x和H_2B RNA的存在将通过原位测定 Sertoli-生精共培养和细胞杂交 新鲜分离的、特定于生精阶段的整装制剂 生精小管。组蛋白基因所在的细胞类型 转录和精子发生阶段的翻译 组蛋白的延续将被确定。 4.TH_2B-x mRNA在翻译非活动性中的可能储存 粗线期精母细胞胞浆组蛋白mRNA-蛋白复合体 接受检查。 5.TH_2B-x基因转录及睾丸特异性合成 精原细胞向精母细胞转变过程中的组蛋白 采用支持-精原细胞共培养的方法进行研究。

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.