REGULATION OF TRANSCRIPTION OF THE HISTONE H4T GENE

该项目的长期目标是确定机制 组蛋白基因在精子发生过程中的表达调控。这个 该项目的近期目标是确定捐款 由转录调控和转录后机制产生 例如在控制组蛋白的稳态水平中的mRNA稳定性 H4T基因在生发细胞发育的不同阶段。为了满足这些要求 目标方面，提出了以下三个具体目标。(1) 将对组蛋白H4T启动子进行分析，以确定 哪些转录调节组蛋白H4T基因的表达 精子发生。H4T基因转录的程度 在严格的控制下，在晚期粗线期下调 精母细胞和早期精子细胞中的基因以及该基因 在减数分裂粗线期转变过程中的组成性表达 精母细胞到减数分裂后的早期精子细胞将使用 体外转录检测。(2)H4T基因的稳定性将是 以确定信使核糖核酸的稳定程度 调节基因的表达。H4T mRNA的周转率将是 通过脉冲追逐实验测量。5‘-先导区的作用 突变和3‘-非编码区突变对H4T基因稳定性的影响 接受检查。具有编码区突变的H4T基因将用作 标记将置于可诱导启动子的控制下， 转化成特定类型的细胞。基因将会被诱导 突变的信使核糖核酸水平和周转率将被跟踪。(3) 我们将研究蛋白质与DNA的相互作用，以便了解 基因转录的机制也决定了组织 转录的特异性和转录水平。DNA结合 将对蛋白质进行鉴定、表征和纯化，以及DNA-蛋白质 启动子内部的相互作用将被建立。近端 将检查启动子和启动子的远端上游区域 使用体外和体内DNA足迹。近端启动子 该基因的区域以及上游和下游区域将被绘制成 核蛋白的体外结合模式。分析特定的 结合将通过电泳迁移率改变分析和 足迹分析。

The long term objective of this project is to determine mechanisms regulating expression of the histone genes during spermatogenesis. The immediate objective of this project is to determine the contributions made by transcriptional regulation and by posttranscriptional mechanisms such as mRNA stability in controlling the steady-state levels of histone H4t mRNA in various stages of germinal cell development. To meet these objectives, the following three specific aims are proposed. (1) The histone H4t promoter will be analyzed in order to establish the extent to which transcription regulates expression of the histone H4t gene during spermatogenesis. The degree to which transcription of the H4t gene is under stringent control and is downregulated in late pachytene spermatocytes and in early spermatids and the degree to which the gene is constitutively expressed during the transition from meiotic pachytene spermatocytes to postmeiotic early spermatids will be determined using in vitro transcription assays. (2) The stability of H4t mRNA will be measured in order to determine the degree to which mRNA stability regulates expression of the gene. H4t mRNA turnover rates will be measured by pulse chase experiments. The effects of 5'-leader region mutations and 3'-noncoding region mutations upon H4t mRNA stability will be examined. The H4t gene with a coding region mutation to be used as a marker will be placed under the control of an inducible promoter and transfected into specific cell types. The gene will be will be induced and the mutant mRNA levels and turnover rates will be followed. (3) Protein-DNA interactions will be examined in order to understand mechanisms by which the gene is transcribed also determine the tissue specificity of transcription and the level of transcription. DNA binding proteins will be identified, characterized, and purified and DNA-protein interactions within the promoter will be established. The proximal promoter and the distal upstream region of the promoter will be examined using in vitro and in vivo DNA footprinting. The proximal promoter region and upstream and downstream regions of the gene will be mapped for in vitro patterns of binding of nuclear proteins. Analysis of specific binding will be conducted by electrophoretic mobility shift assays and footprint analysis.