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。转录的 活跃的体细胞基因通常与DNA低甲基化有关 并对某些核酸酶具有DNA超敏反应。然而，它并不是 知道这些结构信号是否在 精子发生，一个细胞分化期，当基因 当鱼精蛋白取代DNA上的组蛋白时，转录最终停止 染色质浓缩。精子发生特异性的cDNA文库 基因将被产生，以定位任何低甲基化和 这些基因中的过敏点位置。因为这些基因是表达的 只有在精子发生过程中，才有可能出现低甲基化点 在生精DNA中会发现没有相应的位点 体细胞DNA中的敏感部位。染色质结构的变化 可以通过检查特定的阶段与基因活性直接相关 精子发生的过程。通过这种方法，DNA在之前、期间、 并在转录后可以确定每个基因的转录。特别是，它 很重要的一点是确定基因表达是否受到控制 然后，生精细胞中的不同信号在体细胞中被发现。 受精过程中低甲基化对基因表达的可能影响 将被调查，此外。基因的低甲基化 精子发生可能是传递这些基因转录的一种机制。 胚胎发育早期的基因。这些cDNA探针将被用来确定 在成熟精子中，任何基因在转录时都保持低甲基化 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 精子发生，并在整个分化过程中保持这种状态 在“标记”胚胎中的生殖细胞系方面可能很重要。这些 基因可以被跟踪进入胚胎以确定是否有任何差异 当生殖系和非生殖系的甲基化模式明显时 对组织进行分析。 这项提案中的实验将为 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.