REGULATION OF EXDYSTEROID ACTIVATED GENES

类固醇激素蜕皮激素已知与 果蝇多种发育过程的调控。通过 试图确定DNA之间发生的新的诱导相互作用 反应基因和反式作用因子的激活 荷尔蒙，这项提议试图启动一个分子描述如何 这种荷尔蒙调节发生在什么水平上，处于不同的阶段和 建立了来自不同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 可诱导的，是由生态酮发育调节的。一次高潮 解析核“足迹”技术将应用于荷尔蒙 反应性组织培养细胞启动可能的顺式调控元件 对于这些基因中的每一个都要通过它们的改变来识别 荷尔蒙诱导的可及性。蜕皮酮介导的DNA蛋白 这些元素之间的相互作用将导致一种独特的足迹。一个 通过这些序列进行蜕皮酮调节的直接功能测试将 是由融合了已识别出的 对合适的测试基因(β-半乳糖苷酶)进行测序，然后进行分析 在转基因的细胞系或转化的果蝇中。信元的可用性 缺乏激素受体的品系和对蜕皮激素有条件的苍蝇品系 响应性，将使强大的选择应用于测试 荷尔蒙反应元素。一旦确定，就可以对元素进行分析 它们作为组织特异性增强剂的功能。我们将尝试 施加DNA或染色质结构限制，这将损害 这些要素的作用，从而揭示了可能的行动模式。AS 激素反应元件被证实，细胞的分级 提取物，使用敏感的DNA-蛋白质结合分析应该导致 特定反式作用组分的纯化。带着身份证明 对激素表现出不同反应的其他基因，这些 这些方法可能有助于提供多效性的分子解释。 蜕皮激素介导的调节通路。基因的组合使用 果蝇系统中的分子方法使其成为一种有吸引力的方法 用于荷尔蒙调节的研究。这很可能是特别的 考虑到类固醇激素调节是一种基本的生理学 在高等真核生物的许多细胞中都有这种过程。

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.