REGULATION OF EXDYSTEROID ACTIVATED GENES

类固醇激活基因的调控

• 批准号: 3292380
• 负责人: IAIN L CARTWRIGHT
• 金额: $ 13.08万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3292380
• 关键词: beta galactosidase; centrifugation; chromatin; cytogenetics; developmental genetics; ecdysone; gel electrophoresis; gene expression; genetic manipulation; hormone regulation /control mechanism; molecular cloning; molecular genetics; nucleic acid sequence; phase contrast microscopy; pleiotropism; regulatory gene; temperature sensitive mutant; tissue /cell culture

The steroid hormone ecdysterone is known to be intimately involved in the regulation of many developmental processes in Drosophila melanogaster. By attempting to identify newly induced interactions occurring between the DNA of responsive genes and trans acting factors activated in the presence of hormone, this proposal seeks to initiate a molecular description of how this hormonal regulation occurs, and at what level a differential stage and tissue specific response from various genes is established. Drosophila possess four small heat shock genes which, in addition to being heat shock inducible, are developmentally regulated by ecodysterone. A high resolution nuclear "footprinting" technique will be applied to hormonally responsive tissue culture cells to enable putative cis regulatory elements for each of these genes to be identified by virtue of their altered accessibility on hormonal induction. Ecdysterone mediated DNA-protein interactions at these elements will lead to a characteristic footprint. A direct functional test for ecdysterone regulation via these sequences will be provided by experiments that fuse synthetic copies of identified sequences to a suitable test gene (Beta-galactosidase), followed by assays in transfected cell lines or transformed flies. The availability of cell lines lacking hormone receptor, and fly strains conditional for ecdysterone responsiveness, will enable strong selection to be applied in testing of hormonally responsive elements. Once identified, elements can be assayed for their function as tissue specific enhancers. Attempts will be made to impose DNA or chromatin structural constraints that will impair the function of these elements, thereby revealing possible modes of action. As hormonally responsive elements are confirmed, fractionation of cellular extracts, using sensitive DNA-protein binding assays should lead to purification of specific trans acting components. With the identification of further genes that display differential responses to hormone, these approaches may help provide a molecular explanation of the pleiotropic regulatory pathways mediated by ecdysterone. The combined use of genetic and molecular approaches in the Drosophila system make it an attractive one for studies of hormonal regulation. This is likely to be of particular value given that steroid hormonal regulation is a fundamental physiological process in many cells of higher eukaryotes.

众所周知，类固醇激素ecdysterone与 调节果蝇中许多发育过程。 经过 试图识别DNA之间发生的新诱导的相互作用 在存在下激活的反应性基因和反式作用因子的 激素，该提议旨在启动有关如何的分子描述 这种荷尔蒙调节发生，以及差异阶段和 建立了各种基因的组织特异性反应。 果蝇 拥有四个小热冲击基因，除了热量冲击外 诱导性在发育中受环肽的调节。 高 分辨率核“足迹”技术将应用于荷尔蒙 反应性组织培养细胞以实现推定的顺式调节元件 每个基因都可以通过它们的改变来识别 激素诱导的可及性。 ecdystylone介导的DNA蛋白 这些元素的相互作用将导致特征性的足迹。 一个 通过这些序列调节ecdysterone的直接功能测试将 可以通过融合已鉴定的合成副本的实验提供 合适的测试基因（β-半乳糖苷酶）的序列，然后进行测定 在转染的细胞系或转化的苍蝇中。 细胞的可用性 缺乏激素受体的线和蝇菌的蝇毒 响应能力，将使强有力的选择适用于测试 荷尔蒙响应元素。 一旦确定，可以分析元素 它们作为组织特异性增强子的功能。 将尝试 施加会损害的DNA或染色质结构约束 这些元素的功能，从而揭示了可能的作用方式。 作为 荷尔蒙反应性元素已得到证实，细胞的分馏 提取物，使用敏感的DNA-蛋白结合测定应导致 纯化特定的反式作用成分。 与身份证明 在表现出对激素差异反应的其他基因中，这些 方法可能有助于提供对多效性的分子解释 ecdysterone介导的调节途径。 遗传的联合使用 果蝇系统中的分子方法使其成为吸引人的方法 用于荷尔蒙调节的研究。 这可能是特别的 鉴于类固醇激素调节是一种基本生理学 在许多较高真核生物的细胞中进行过程。