REGULATION OF TRANSCRIPTION OF THE HISTONE H4T GENE

该项目的长期目标是确定机制 调节精子发生过程中组蛋白基因的表达。 的 该项目的直接目标是确定 由转录调节和转录后机制产生 如mRNA稳定性控制组蛋白的稳态水平 H4 tmRNA在生殖细胞发育的各个阶段。 满足这些 为了实现这些目标，提出了以下三个具体目标。(1)的 将分析组蛋白H4 t启动子，以确定 其转录调节组蛋白H4 t基因的表达 精子发生 H4 t基因转录的程度是 在严格控制下，在粗线期晚期下调 精母细胞和早期精子细胞中，以及该基因被 在减数分裂粗线期过渡期间组成型表达 精母细胞到减数分裂后早期精子细胞将使用 体外转录测定。(2)H4 t mRNA的稳定性将是 为了确定mRNA的稳定性 调节基因的表达。 H4 t mRNA周转率将是 通过脉冲追踪实验测量。 5 '-前导区的作用 突变和3 '-非编码区突变对H4 t mRNA稳定性的影响将 接受检查。 具有编码区突变的H4 t基因被用作 标记将置于诱导型启动子的控制下， 转染到特定的细胞类型中。 该基因将被诱导 并跟踪突变体mRNA水平和周转率。（三） 将检查蛋白质与DNA的相互作用以了解 基因转录的机制也决定了 转录的特异性和转录水平。 DNA结合 蛋白质将被鉴定、表征和纯化， 将建立启动子内的相互作用。 近侧 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 基因的上游和下游区域将被定位， 核蛋白结合的体外模式。 分析具体 结合将通过电泳迁移率变动分析进行， 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.