REGULATION OF EXDYSTEROID ACTIVATED GENES

已知类固醇激素蜕皮甾酮密切参与 调节果蝇的许多发育过程。 通过 试图识别DNA之间发生的新诱导的相互作用 反应基因和反式作用因子的激活， 激素，这项建议旨在启动一个分子描述如何 这种激素调节发生，在什么水平上的差异阶段， 建立了来自各种基因的组织特异性应答。 果蝇 拥有四个小的热休克基因，除了热休克， 可诱导的，在发育上受蜕皮甾酮调节。 高 分辨率核“足迹”技术将应用于 使推定的顺式调节元件 对于这些基因中的每一个， 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导致特征足迹。 一 通过这些序列进行蜕皮甾酮调节的直接功能测试将 通过实验提供， 序列与合适的测试基因（β-半乳糖苷酶），然后进行测定 在转染的细胞系或转化的果蝇中。 细胞的可用性 缺乏激素受体的品系，以及蜕皮甾酮条件的蝇品系 响应性，将使强选择适用于测试 反应灵敏的元素。 一旦确定，可以分析元素 因为它们作为组织特异性增强剂的功能。 将试图 施加DNA或染色质结构限制， 这些元素的功能，从而揭示可能的行动模式。 作为 细胞反应元件得到确认，细胞的分级分离 提取物，使用敏感的DNA-蛋白质结合试验应导致 纯化特定的反式作用组分。 与识别 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， 蜕皮甾酮介导的调节途径。 基因的联合使用 果蝇系统的分子方法使其成为一个有吸引力的系统 用于研究荷尔蒙调节 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 在高等真核生物的许多细胞中的过程。

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.