MAMMALIAN SPERMATOGENESIS CONTROL OF GENE TRANSCRIPTION

DNA甲基化、DNA构象与基因活性的关系 将在哺乳动物精子发生过程中进行检查。 转录 活跃的体细胞基因通常与DNA低甲基化 以及对某些核酸酶的DNA超敏反应。 但不 已知这些结构性信号是否重要， 精子发生是细胞分化的一个阶段， 当鱼精蛋白取代DNA上的组蛋白时， 染色质浓缩。 精子发生特异性cDNA文库 将产生基因，以映射任何低甲基化和 这些基因中的超敏感位点。 由于这些基因的表达 只有在精子发生过程中，低甲基化点可能是预期的， 在生精DNA中会发现一些位点， 体细胞DNA中的超敏感位点。 染色质结构的变化 可以通过检查特定阶段直接与基因活性相关 精子发生的过程 通过这种方法，DNA的状态之前，期间， 以及每个基因的转录后。 特别是 这对于确定基因表达的控制是否受到 生精细胞中的不同信号然后在体细胞中被发现。 低甲基化对受精时基因表达的可能影响 此外，还将进行调查。 基因的低甲基化 精子发生可能是一种机制， 胚胎发育早期的基因 cDNA探针将用于确定 任何基因在成熟精子中保持低甲基化， 停止了。 如果是这样，这些基因在胚胎中的转录状态将 接受检查。 最后，早期低甲基化的基因， 并在整个分化过程中保持这种状态 可能对胚胎中生殖细胞系的“标记”很重要。 这些 基因可以被跟踪到胚胎中，以确定是否有任何差异， 当生殖系和非生殖系细胞 分析组织。 该提案中的实验将为 哺乳动物精子发生的长期分子检测。

The relationship among DNA methylation, DNA conformation and gene activity will be examined during mammalian spermatogenesis. Transcriptionally active somatic genes typically are associated both with DNA hypomethylation and with DNA hypersensitivity to certain nucleases. However, it is not known whether these structural signals are important during spermatogenesis, a period of cellular differentiation when gene transcription eventually ceases as protamines replace histones on the DNA and the chromatin condenses. cDNA libraries to spermatogenic-specific genes will be generated in order to map any hypomethylated and hypersensitive point sites in these genes. Since these genes are expressed only during spermatogenesis, it might be expected that hypomethylated point sites will be found in spermatogenic DNA without corresponding hypersensitive sites in somatic DNA. The changes in chromatin structure can be directly correlated with gene activity by examining specific stages of spermatogenesis. By this method the state of the DNA before, during, and after transcription of each gene can be ascertained. In particular, it would be important to determine if the control of gene expression is under different signals in spermatogenic cells then is found in somatic cells. The possible effects of hypomethylation on gene expression at fertilization will be investigated, in addition. Hypomethylation of genes during spermatogenesis may be a mechanism for signaling the transcription of these genes early in embryogenesis. The cDNA probes will be used to determine if any genes remain hypomethylated in the mature sperm when transcription has ceased. If so, the transcriptional state of these genes in the embryo will be examined. Finally, genes that are hypomethylated early in spermatogenesis and are retained in that state throughout differentiation may be important in "marking" the germ cell line in the embryo. These genes can be followed into the embryo to determine if any differences are apparent in the methylation pattern when the germ line and non-germ line tissues are analyzed. The experiments in this proposal will provide the foundation for a long-term molecular examination of mammalian spermatogenesis.