MOLECLAR BASIS OF ECDYSTEROID ACTION IN THE MOSQUITO

该提案的目标是描述监管中的因素， 介导蚊子中20-羟基蜕皮激素（20 E）作用的级联。 我们克隆了一个编码蜕皮激素受体（AaEcR）的cDNA， 其在埃及伊蚊卵黄发生过程中的表达。 最近，我们还克隆了一个编码蚊子超气门的cDNA， 同源物（AaUSP），其是蜕皮类固醇受体的异二聚体伴侣。 我们进一步证明了AaEcR和AaUSP，从克隆的cDNA表达， 通过体外转录/翻译，能够将激素和DNA 作为功能性异二聚体结合。20 E应答基因编码 还克隆了蚊卵黄生成羧肽酶（VCP）基因。然而，在这方面， 存在于两个等位基因VCP基因中的潜在激素反应元件是 与蜕皮激素反应的共有序列不同 元素此外，利用放线菌酮，抑制剂， 结果表明，20 E对蚊卵黄蛋白的作用 卵黄蛋白原和VCP的基因是间接的，可能是由一种 类似于果蝇的调节级联。这些研究为 阐明基因调控的分子机制的阶段 20 E在蚊子中的表达。 在这项提案中，我们计划通过克隆亚型特异性AaEcR来分析AaEcR。 cDNA和基因并研究它们的表达。可能的亚型 还将分析AaUSP及其基因。AaEcR和AaUSP都将 在杆状病毒系统中表达，并对其纯化蛋白进行研究 关于生物化学性质和配体（20 E）结合。我们将 研究AaEcR/USP复合物对荧光响应结合特性 蚊子和果蝇的20 E调控基因的元件。最后， 我们将确定转录因子介导的20 E反应， 蚊子 寻找替代的灭蚊策略是基于 确定控制发育的过程和基因， 繁殖能力或矢量能力。阐明关键监管 蜕皮激素受体等分子控制着基因的表达 对蚊子的发育和繁殖至关重要， 重视这些努力。

The goal of this proposal is to characterize factors in the regulatory cascade that mediate 20-hydroxyecdysone (20E) action in the mosquito. We cloned a cDNA encoding ecdysteroid receptor (AaEcR) and characterized its expression during vitellogenesis in the mosquito Adedes aegypti. Recently, we also cloned a cDNA encoding for the mosquito Ultraspiracle homolog (AaUSP) which is a heterodimeric partner of ecdysteroid receptor. We further demonstrated that AaEcR and AaUSP, expressed from cloned cDNAs by in vitro transcription/translation, are capable of hormone and DNA binding as a functional heterodimer. The 20E-responsive gene coding for mosquito vitellogenic carboxypeptidase (VCP) was also cloned. However, potential hormone response elements present in two allelic VCP genes are different from that of the consensus sequence for the ecdysone response element. Furthermore, experiments utilizing cycloheximide, an inhibitor of protein synthesis, showed that 20E action on the mosquito yolk protein genes for vitellogenin and VCP is indirect and likely mediated by a regulatory cascade similar to that in Drosophila. These studies have set the stage for elucidating the molecular mechanism of regulation of gene expression by 20E in the mosquito. In this proposal we plan to analyze AaEcR by cloning isoform-specific cDNAs and gene(s) and studying their expression. Possible isoforms of AaUSP and its gene(s) will also be analyzed. Both AaEcR and AaUSP will be expressed in a baculovirus system and their purified proteins studied with respect to biochemical properties and ligand (20E) binding. We will investigate binding properties of AaEcR/USP complexes to hormone-response elements of 20E-regulated genes from mosquito and Drosophila. Finally, we will identify transcription factors mediating 20E response in the mosquito. The search for alternative strategies of mosquito control is based on the identification of processes and genes that govern development, reproduction, or vectorial capacity. Elucidation of how key regulatory molecules such as ecdysteroid receptor govern the expression of genes essential to mosquito development and reproduction is of fundamental importance to these efforts.