SEX SPECIFIC REGULATION OF TRANSCRIPTION IN DROSOPHILA

本项目将研究调节性别特异性的机制 果蝇的转录。 这将通过调查 遗传、性别分化途径和靶点之间的联系 由该通路调控的基因。 一个来自靶基因的增强子 在体内指导雌性和组织特异性转录。 它是 三个蛋白质结合位点，其中一个调节性别特异性 转录的方式，这完全取决于结合的两个 doublemex基因蛋白，DSX/F和DSX/M。 doublemex基因位于 性别分化途径的底部。 从这个DNA位点，DSX/F 在雌性中激活转录，而在雌性中DSX/M抑制转录。 男性。 这确立了两种doubletex蛋白及其结合位点 在增强子中作为通路和靶基因之间的连接。 该项目的主要目的是研究DSX 蛋白质调节该增强子的转录。 会来做这项工作 研究DSX/F和DSX/M的结构和功能。 首先， DSX蛋白的功能现在可以通过生物化学方法进行测定， 通过单细胞生物系统和一些生殖系转化 将进行实验研究。 在这项工作中， 四聚化结构域将被定位，然后氨基酸改变， 特异性阻断每个低聚步骤。 关于DSX蛋白寡聚化对DNA影响的假说 结合和DNA结合对DSX寡聚化的影响将是 通过使用野生型蛋白质和具有 阻断寡聚化的突变体。 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。 生殖系转化将用于 确定寡聚化的调节功能，并定位 DSX/F的转录激活结构域。 另外两种基因 从增强子起作用的调节蛋白将被分离， 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 DSX蛋白

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.