SEX SPECIFIC REGULATION OF TRANSCRIPTION IN DROSOPHILA

果蝇转录的性别特异性调控

• 批准号: 2444794
• 负责人: PIETER C WENSINK
• 金额: $ 26.79万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444794
• 关键词: DNA binding protein; Drosophilidae; chemical binding; conformation; crystallization; developmental genetics; dimer; gel mobility shift assay; gene induction /repression; genetic enhancer element; genetic promoter element; genetic regulation; genetic transcription; laboratory rat; molecular cloning; mutant; polymerization; protein isoforms; protein sequence; protein structure function; regulatory gene; sex differentiation; site directed mutagenesis; stoichiometry; transcription factor

This project will investigate mechanisms that regulate sex specific transcription in Drosophila. This will be done by investigating the connection between the genetic, sex differentiation pathway and a target gene that is regulated by the pathway. An enhancer from a target gene directs female and tissue specific transcription in vivo. It is composed of three protein binding sites, one of which regulates sex specific transcription in a way that depends solely on the binding of the two doublesex gene proteins, DSX/F and DSX/M. The doublesex gene is at the bottom of the sex differentiation pathway. From this DNA site, DSX/F activates transcription in females and DSX/M represses transcription in males. This establishes the two doublesex proteins and their binding site in the enhancer as the connection between the pathway and a target gene. The broad aim of the project is to investigate the mechanisms by which DSX proteins regulate transcription from this enhancer. This will be done by examining the structure and function of DSX/F and DSX/M. First those functions of DSX proteins that can now be assayed by biochemical methods, by unicellular biological systems and by a few germline transformation experiments will be studied. In this work the dimerization and tetramerization domains will be localized and then amino acid changes that specifically block each oligomerization step will be identified. Hypotheses concerning the effect of DSX protein oligomerization on DNA binding and the effect of DNA binding on DSX oligomerization will be tested by physical methods using wild type proteins and proteins with mutants blocking oligomerization. Similar methods will be used to test hypotheses that the sex-specific termini of the proteins affect oligomerization and DNA binding. Germline transformation will be used to determine the regulatory functions of oligomerization and to localize the transcriptional activation domain of DSX/F. Genes for the other two regulatory proteins that operate from the enhancer will be isolated and used to develop in vitro and unicellular assays for DSX protein function. These assays will then be used to examine the structure and function of the DSX proteins.

该项目将研究调节性别特异性的机制 果蝇中的转录。 这将通过调查来完成 遗传、性别分化途径和目标之间的联系 受该途径调控的基因。 来自靶基因的增强子 指导体内雌性和组织特异性转录。 它是由 三个蛋白质结合位点，其中之一调节性别特异性 转录方式仅取决于两者的结合 双性基因蛋白 DSX/F 和 DSX/M。 双性基因位于 性别分化途径的底部。 从该 DNA 位点，DSX/F 激活雌性中的转录，而 DSX/M 抑制雌性中的转录 男性。 这建立了两个双性蛋白及其结合位点 在增强子中作为途径和靶基因之间的连接。 该项目的主要目标是研究 DSX 的机制 蛋白质调节该增强子的转录。 这将由 检查 DSX/F 和 DSX/M 的结构和功能。 首先是那些 DSX 蛋白的功能现在可以通过生化方法进行测定， 通过单细胞生物系统和一些种系转化 将进行实验研究。 在这项工作中，二聚化和 四聚化结构域将被定位，然后氨基酸发生变化 将确定具体阻断每个寡聚步骤。 关于 DSX 蛋白寡聚化对 DNA 影响的假设 结合以及 DNA 结合对 DSX 寡聚化的影响将是 使用野生型蛋白质和具有以下性质的蛋白质通过物理方法进行测试 突变体阻断寡聚化。 类似的方法将用于测试 假设蛋白质的性别特异性末端影响 寡聚化和 DNA 结合。 种系转化将用于 确定寡聚化的调节功能并定位 DSX/F 的转录激活结构域。 另外两个的基因 由增强子起作用的调节蛋白将被分离并 用于开发 DSX 蛋白功能的体外和单细胞测定。 然后这些测定将用于检查结构和功能 DSX 蛋白。