A Drosophila Juvenile Hormone Receptor

本研究拟通过研究模式昆虫黑腹果蝇幼体激素（JH）的分子作用，探讨激素对昆虫发育和繁殖的调控作用。JH在昆虫中的重要作用已经被确定，但这种激素的作用仍然是一个谜，因为还没有在任何昆虫中发现JH受体。一种遗传学方法鉴定了甲氧苯醚耐受性（Met）基因，并发现其编码的蛋白质（Met）可能作为JH受体的一个组成部分参与了这种昆虫的JH作用。在拟议的工作中，Met将作为理解该受体的重点。本研究主要包括两个目标：(1)MET的伴侣蛋白的鉴定。D. melanogaster中的Met+是bHLH-PAS转录调节因子家族的成员。PAS蛋白在异二聚体复合体中与伴侣蛋白一起起作用，没有理由相信MET是例外。对JH结合和接收的理解必须包括对伴侣蛋白的识别。一个有希望的候选基因是最近从md中克隆出来的同源基因，met - 1。腹。以Met+为诱饵的酵母双杂交技术将用于直接检测Met - l作为Met的伴侣，以及寻找与Met相互作用的其他蛋白。在这个成熟的技术中，候选基因在酵母细胞中一起表达。它们的基因产物之间的物理相互作用将驱动一个或多个报告基因的转录工程到酵母菌株，使这种相互作用的可视化。每个蛋白的相互作用将在体外使用MET抗体进行验证。任何新的基因（可能是通过功能）将从D. melanogaster序列数据库中识别出来，以进一步表征。(2) JH结合蛋白的鉴定与表征。先前的结合研究表明，jhiii在Met突变体的组织提取物中的结合亲和力要差得多，这解释了对甲氧二烯的抗性，并暗示Met是一种受体成分。为了鉴定JH结合蛋白并确定其结合特性，将测量JH III与单个蛋白和MET: PARTNER异二聚体复合物的结合。此外，JH III的光亲和类似物环氧法尼酯重氮乙酸酯将用于光亲和标记，并建立/确认哪个蛋白质是异二聚体复合体中的高亲和结合蛋白。这些研究的结果将定义假定的JH受体的成分和任何与MET物理相互作用的相关蛋白质。JH结合蛋白也将被鉴定，从而阐明JH作用的分子基础。这些结果将为进一步分析JH的转录调控提供必要的知识和分子工具。

The proposed work will investigate the hormonal control of development and reproduction in insects by probing the molecular action of juvenile hormone (JH) in the model insect Drosophila melanogaster. Important roles for JH in insects have been identified, but the action of this hormone remains an enigma because a JH receptor has not been identified in any insect. A genetic approach identified the Methoprene-tolerant (Met) gene, and found that the protein that it encodes (MET) is involved in the action of JH in this insect, probably as a component of a JH receptor. In the proposed work Met will serve as a focus for understanding this receptor. The proposed work consists of two objectives: (1) Identification of the partner protein of MET. Met+ in D. melanogaster is a member of the bHLH-PAS family of transcriptional regulators. PAS proteins function with a partner protein in a heterodimeric complex, and there is no reason to believe that MET is an exception. An understanding of JH binding and reception must include an identification of the partner protein. One promising candidate is a homologous gene, Met-like (Met-l), recently cloned fromD. melanogaster. Yeast two-hybrid technology using Met+ as bait will be employed to directly test MET-L as the partner of MET as well as search for other proteins that interact with MET. In this proven technology candidate genes are expressed together in yeast cells. Physical interaction between their gene products will drive transcription of one or more reporter genes engineered into the yeast strain, allowing ready visualization of this interaction. Interaction of each protein will be verified in vitro using antibody to MET. Any novel gene(s) will be identified, possibly by function, from the D. melanogaster sequence database, for further characterization. (2) Identification and characterization of the JH binding protein. Previous binding studies demonstrated that JH III binding affinity was much poorer in tissue extracts from Met mutants, accounting for the resistance to methoprene, and implicating MET as a receptor component. To identify the JH-binding protein and establish its binding characteristics, JH III binding to the individual proteins and to the MET: PARTNER heterodimeric complex will be measured. Additionally, a photoaffinity analog of JH III, epoxyfarnesyl diazoacetate, will be used to photoaffinity label and establish/confirm which protein is the high-affinity binding protein in the heterodimeric complex. Results from these studies will define the components of the putative JH receptor and any associated proteins that physically interact with MET. The JH-binding protein will also be identified, thus clarifying the poorly understood molecular basis for JH action. These results will provide the necessary knowledge and molecular tools for further analysis of transcriptional control by JH.