TESTIS-SPECIFIC HISTONES AND MRNA DURING SPERMATOGENESIS

睾丸特异性组蛋白转录和翻译的测定 RNA在精子发生过程中的作用对于理解精子发生的分子机制至关重要。 在此过程中，参与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， 组蛋白变体在初级精母细胞中合成， 整个睾丸中有一半的体细胞组蛋白，有迹象表明， 体细胞组蛋白几乎完全被变异组蛋白所取代， 减数分裂前期的晚期阶段。 与体细胞相反， 组蛋白的合成与DNA合成偶联， 睾丸特异性组蛋白继续通过减数分裂前噬菌体，其中有 是一个小的DNA合成。 本申请的主要目的是 克隆一个睾丸特异性组蛋白基因，即变异H2 B的基因 （TH 2B-x），并将其用作杂交探针来研究转录 睾丸特异性组蛋白RNA在不同发育阶段的表达和翻译 精子发生 1.将从成年大鼠中克隆TH 2B-x和H2 B组蛋白mRNA的cDNA 用标准的分子克隆方法。 2.将对cDNA进行测序，并将DNA和氨基酸序列的DNA和氨基酸序列的DNA和氨基酸序列的DNA和氨基酸序列的氨基酸序列的DNA和氨基酸序列的氨基酸序列的DNA和氨基酸序列的DNA和氨基酸序列的DNA和氨基酸序列的氨基酸序列的DNA和氨基酸序列的氨基酸序列的DNA和氨基酸序列的DNA和氨基酸序列的DNA和氨基酸序列的DNA和氨基酸序列的 将比较TH 2B-x和H2 B组蛋白。 显示广泛的区域 将序列变异亚克隆到细菌质粒中，并用作 TH 2B-x和H2 B RNA的特异性杂交探针。 3. TH 2B-x和H2 B RNA的存在将通过原位杂交测定。 使用支持-生精共培养物和 新鲜分离的生精阶段特异性 生精小管 其中组蛋白基因被 转录和精子发生阶段，其中翻译的 将继续测定组蛋白。 4. TH 2B-x mRNA可能储存在免疫失活细胞中， 粗线期精母细胞的细胞质组蛋白mRNA-蛋白复合物将 接受检查。 5. TH 2B-x基因的转录和睾丸特异性抗原的合成 组蛋白在精原细胞向精母细胞的转变过程中， 研究使用Sertoli精原细胞共培养。

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.