STRUCTURE AND REGULATION OF ECDYSONE-RESPONSIVE GENES

最近使用脊椎动物系统的研究表明，类固醇激素， 与它们的受体蛋白结合到 特定的基因，调节这些基因的活动。 蜕皮激素 昆虫的类固醇激素，已经研究了很多年，但 行动细节尚不清楚。 我们建议澄清 通过鉴定那些DNA序列， 赋予蜕皮激素诱导的特定基因（ERE），并通过研究 与这些序列结合的分子。 该提案的重点是 果蝇基因，以促进最终的遗传分析， 这些调控途径。 Eip 28/29和Eip 40基因是已充分表征的果蝇基因， 是由蜕皮激素诱导的。 间接测试表明， 它们参与这些细胞对类固醇的初级反应 激素. 在Eip 40位点存在一种新的情况： DNA被转录，两种转录物都是在早期诱导的。 对蜕皮激素的反应 Eip 40位点的剩余结构问题将通过以下方法解决： 标准程序 这些实验应该表明RNA是否 互补性在该基因座起调节作用。 所有3个的ERE 将绘制转录单位。 一旦绘制完成，DNA元素将被 作为探针来识别与它们相互作用的蛋白质， 包括蜕皮激素受体。 确定这些关键 响应的组成部分应允许设计体内测定， 探索其结构。 这些测定可能包括DNA酶和Sl的测试 超敏反应和体内足迹。 发展模式 EIP基因在果蝇中的表达将被确定，并将在 根据刚刚描述的关键结构的分析。 最后， 将努力揭示Eip基因的功能作用 产品. 这些实验应该加强我们对蜕皮激素的理解， 真核生物中的转录调控，以及 类固醇激素和其他发育调节因子 设备.

Recent studies using vertebrate systems have shown that steroid hormones, in concert with their receptor proteins, bind to DNA sequences near particular genes, modulating the activities of those gene. Ecdysone, the steroid hormone of insects, has been studied for many years, but the details of its action are as yet unknown. We propose to elucidate the mechanism of ecdysone action by identifying those DNA sequences which confer ecdysoneinducibility on particular genes (EREs) and by studying the molecules that bind to those sequences. This proposal focuses on Drosophila genes in order to facilitate the eventual genetic analysis of these regulatory pathways. The Eip28/29 and Eip40 genes are well-characterized Drosophila genes which are induced by ecdysonein several cell lines. Indirect tests suggest that they participate in the primary response of these cells to the steroid hormone. A novel situation exists at the Eip40 locus: both strands of the DNA are transcribed and both transcripts are induced during the early response to ecdysone. Remaining structural questions at the Eip40 locus will be resolved by standard procedures. These experiments should suggest whether RNA complementarity plays a regulatory role at this locus. EREs for all 3 transcription units will be mapped. Once mapped, the DNA elements will be used as probes to identify the proteins with which they interact, perhaps including the ecdysone receptor. Identification of these critical components of the response should permit the design of in vivo assays to probe their structure. Such assays might include tests of DNase and Sl hypersensitivity and in vivo footprinting. The developmental pattern of Eip gene expression in flies will be determined and will be interpreted in the light of the assays of critical structures just described. Finally, efforts will be made to uncover the functional roles of the Eip gene products. These experiments should enhance our understanding of ecdysone, of transcriptional regulation in eukaryotes, and of the relation between steroid hormones and other elements of the developmental regulatory apparatus.