TRANSCRIPTIONAL REGULATION DURING SPERMATOGENESIS

描述(改编自《调查者摘要》)：长期 该项目的目标是确定调控机制 睾丸特异基因的转录。这件事的一个直接目标是 项目是分离和鉴定一种睾丸特异的DNA结合蛋白 (TE结合蛋白)，并克隆编码这一推定的基因 组织特异性转录因子。最近，一个元素(TE) 发现具有高亲和力的结合TE结合蛋白位于 睾丸特异性组蛋白H1t基因的H1/AC盒和H1/CCAAT盒。这个 睾丸特异性TE结合蛋白首次在原代培养中被检测到 精母细胞与H1T发病之间存在时间相关性 基因转录和TE结合蛋白的表达。至 确定这种睾丸特异的DNA结合蛋白的生化性质 以及它在调节转录方面的作用，在特定的目标1中，蛋白质将 是纯净的。将对纯化多肽进行氨基酸序列分析 测定并与已知的多肽进行比较。为了进一步建立 蛋白质的性质，编码蛋白质的基因将被克隆和 在特定目标2中测序。在特定目标3中，多克隆抗体将 针对蛋白质和合成多肽做好准备 促进表达文库的筛选。的多肽序列 纯化的DNA结合蛋白将用于设计混合寡核苷酸 可用于筛选cdna和基因组文库的探针。要确定 TE结合蛋白在调节睾丸特异性中的作用 转录后，将在转录检测中检测其活性。

DESCRIPTION (Adapted from the Investigator's Abstract): A long term objective of this project is to determine mechanisms regulating transcription of testis-specific genes. An immediate objective of this project is to isolate and characterize a testis-specific DNA-binding protein (TE-binding protein) and clone the gene encoding this putative tissue-specific transcriptional factor. Recently, an element (TE) that binds TE-binding protein with high affinity was found located between the H1/AC box and H1/CCAAT box of the testis-specific histone H1t gene. The testis-specific TE-binding protein is first detectable in primary spermatocytes and a temporal correlation exists between the onset of H1t gene transcription and the expression of the TE-binding protein. To establish the biochemical nature of this testis-specific DNA-binding protein and its role in regulating transcription, in Specific Aim 1 the protein will be purified. Amino acid sequence analysis of purified polypeptides will be determined and compared to known polypeptides. To further establish the nature of the protein, the gene encoding the protein will be cloned and sequenced in Specific Aim 2. In Specific Aim 3, polyclonal antibodies will be prepared against the protein and against synthetic polypeptides to facilitate expression library screening. Sequences of peptides from the purified DNA-binding proteins will be used to design mixed oligonucleotide probes that can be used to screen cDNA and genomic libraries. To determine the role of the TE-binding protein in regulating testis-specific transcription, its activity will be examined in transcription assays.