REGULATION OF EXDYSTEROID ACTIVATED GENES

类固醇激活基因的调控

基本信息

批准号：
3292381
负责人：
IAIN L CARTWRIGHT
金额：
$ 13.23万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
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

项目摘要

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.

已知类固醇激素蜕皮甾酮与果蝇许多发育过程的调节。经过试图识别 DNA 之间新诱导的相互作用在存在时激活的反应基因和反式作用因子激素，该提案旨在启动分子描述如何这种荷尔蒙调节发生，以及在什么水平上存在差异阶段和建立了来自各种基因的组织特异性反应。果蝇拥有四个小的热休克基因，除了热休克之外诱导型，在发育过程中受生态睾酮调节。一个高分辨率核“足迹”技术将应用于激素响应性组织培养细胞以实现假定的顺式调控元件这些基因中的每一个都可以通过它们的改变来识别激素诱导的可及性。蜕皮甾酮介导的DNA蛋白这些元素的相互作用将产生特征足迹。一个通过这些序列进行蜕皮甾酮调节的直接功能测试将由融合已识别的合成副本的实验提供对合适的测试基因（β-半乳糖苷酶）进行测序，然后进行测定在转染的细胞系或转化的果蝇中。小区的可用性缺乏激素受体的品系和需要蜕皮甾酮的果蝇品系响应能力，将使强有力的选择能够应用于测试荷尔蒙反应元素。一旦确定，就可以分析元素因其作为组织特异性增强剂的功能。将努力施加 DNA 或染色质结构限制，从而损害这些元素的功能，从而揭示可能的作用模式。作为荷尔蒙反应元件得到证实，细胞的分离提取物，使用敏感的 DNA-蛋白质结合测定应该会导致特定反式作用成分的纯化。凭身份证对激素表现出不同反应的其他基因，这些方法可能有助于提供多效性的分子解释由蜕皮甾酮介导的调节途径。遗传基因的综合运用果蝇系统中的分子方法使其成为一种有吸引力的系统用于荷尔蒙调节的研究。这很可能是特别的鉴于类固醇激素调节是基本的生理学价值高等真核生物的许多细胞中的过程。