SEX-SPECIFIC REGULATION OF TRANSCRIPTION IN DROSOPHILA

果蝇性别特异性转录调控

• 批准号: 3305631
• 负责人: PIETER C WENSINK
• 金额: $ 11.23万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305631
• 关键词: DNA binding protein; DNA footprinting; Drosophilidae; affinity chromatography; chemical binding; developmental genetics; gel filtration chromatography; gene expression; genetic enhancer element; genetic mapping; genetic promoter element; genetic regulatory element; genetic transcription; ion exchange chromatography; molecular genetics; nucleic acid sequence; polymerase chain reaction; protein purification; protein structure function; regulatory gene; sex determination; stoichiometry; transcription factor

This project will investigate the molecular mechanism by which a major, well-characterized developmental regulatory hierarchy of Drosophila melanogaster directs the sex-specificity of target gene transcription. The significance of the project lies in its advanced analysis of the structure and function of a cellular enhancer from a higher organism and in its study of the direct molecular connection between a developmental regulatory hierarchy and target genes. Understanding developmental regulation and cellular enhancer function is crucial to understanding defects in gene expression that lead to human disease and human developmental abnormalities. The project will investigate a particular enhancer that conveys the male- specific repression signal from the hierarchy to a pair of target genes. We will determine how this repression signal is received by the enhancer and then sent to the promoters. A gene at the bottom of the regulatory hierarchy encodes a male-specific repressor of female genes and a female- specific repressor of male genes. In vitro both male and female repressors bind to the male-specific regulatory elements of the enhancer. We will examine the mechanism operating in vivo to determine whether the male repressor is sufficient to cause repression in males and whether the female repressor does not bind these elements, or does bind them but does not repress. This will be done by using in vitro mutagenesis/germline transformation methods to identify the functional elements within the enhancer and to determine whether the bound repressor acts on other enhancer elements or directly on the promoter. These studies will show whether this repressor action occurs by steric hindrance or DNA looping mechanisms. We will also investigate the biochemistry of the interactions between the DNA sites and the purified, expressed, repressors. This biochemical portion of the project aims at an explanation for the male and female repressor activities at this male-specific enhancer. We will then use a transcription system derived from an appropriate purified tissue in order to reproduce this sex-specific repression in vitro and investigate the function of proteins that operate in the repression mechanism.

该项目将研究一种主要的， 果蝇发育调控系统 黑腹果蝇指导靶基因转录的性别特异性。 的 该项目的意义在于其先进的结构分析 和功能的细胞增强剂从一个更高的有机体和在其研究 发育调节基因和细胞周期之间 层次和靶基因。 了解发育调节和 细胞增强子功能对于理解基因缺陷至关重要， 表达导致人类疾病和人类发育 异常 该项目将研究一种特殊的增强剂，它可以传达男性的信息- 特异性阻遏信号从该层级传递到一对靶基因。 我们将确定这种抑制信号是如何被增强子接收的 然后发给发起人 一个位于调节基因底部的基因 等级编码女性基因的男性特异性阻遏物和女性- 雄性基因的特异性抑制物。 体外雄性和雌性阻遏物 与增强子的雄性特异性调节元件结合。 我们将 检查在体内运作的机制，以确定男性是否 抑制因子足以引起男性的抑制，女性是否 阻遏物不结合这些元件，或结合它们但不 压抑 这将通过使用体外诱变/种系进行 转换方法，以识别 增强子，并确定结合的阻遏物是否作用于其他 增强子元件或直接在启动子上。 这些研究将显示 这种阻遏物的作用是通过空间位阻还是DNA成环发生的 机制等 我们还将研究相互作用的生物化学 在DNA位点和纯化的表达的阻遏物之间。 这 该项目的生物化学部分旨在解释男性和 女性阻遏物在这个男性特异性增强子的活动。 然后我们将 使用源自适当纯化组织的转录系统， 为了在体外重现这种性别特异性抑制， 在抑制机制中发挥作用的蛋白质的功能。