TRANSCRIPTIONAL REGULATION DURING SPERMATOGENESIS

描述（改编自研究者摘要）：长期 该项目的目标是确定监管机制， 睾丸特异性基因的转录。 一个直接的目标， 一个项目是分离和鉴定睾丸特异性DNA结合蛋白 （TE结合蛋白）并克隆编码这种推定的 组织特异性转录因子。 最近，一种元素（TE）， 与TE结合蛋白的高亲和力结合被发现位于 睾丸特异性组蛋白H1t基因的H1/AC盒和H1/CCAAT盒。 的 睾丸特异性TE结合蛋白是第一次检测到的原发性 精母细胞和时间相关性之间存在的发病H1t 基因转录和TE结合蛋白的表达。 到 确定这种睾丸特异性DNA结合蛋白的生物化学性质 及其在调节转录中的作用，在特异性目的1中，蛋白质将 被净化 将对纯化的多肽进行氨基酸序列分析。 测定并与已知多肽比较。 进一步树立 蛋白质的性质，将克隆编码蛋白质的基因， 具体目标2。 在特定目标3中，多克隆抗体将 针对蛋白质和针对合成多肽制备， 促进表达文库筛选。 肽的序列， 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。 以确定 TE结合蛋白在调节睾丸特异性 转录，其活性将在转录测定中检查。

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.