A Drosophila Juvenile Hormone Receptor

果蝇保幼激素受体

• 批准号: 0109942
• 负责人: Thomas Wilson
• 金额: $ 21万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0109942&HistoricalAwards=false
• 关键词: Drosophila; Juvenile; Hormone; Receptor; Drosophila; Juvenile; Hormone; Receptor

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.

拟议的工作将通过探测模型昆虫果蝇果蝇中的少年激素（JH）的分子作用来研究昆虫发育和繁殖的激素控制。 已经确定了JH在昆虫中的重要作用，但是由于在任何昆虫中尚未鉴定出JH受体，这种激素的作用仍然是一种谜。 一种遗传方法鉴定出耐甲酯耐受剂（Met）基因，发现其编码（Met）的蛋白质参与了JH在该昆虫中的作用，这可能是JH受体的组成部分。 在拟议的工作中，MET将成为理解该受体的重点。 提出的工作由两个目标组成：（1）识别MET的伴侣蛋白。 Met+在D. Melanogaster中是转录调节剂BHLH-PAS家族的成员。 PAS蛋白质在异二聚体复合物中与伴侣蛋白一起发挥作用，没有理由相信MET是一个例外。 对JH结合和接收的理解必须包括对伴侣蛋白的识别。 一个有前途的候选人是一个同源基因，类似MET的（Met-L），最近被克隆了Fromd。 Melanogaster。 使用MET+作为诱饵的酵母两杂交技术将直接测试MET-L作为MET的合作伙伴以及搜索与MET相互作用的其他蛋白质。在这个经过验证的技术中，候选基因在酵母细胞中一起表达。 其基因产物之间的物理相互作用将驱动一个或多个在酵母菌菌株中设计的报告基因的转录，从而可以看到这种相互作用。 每种蛋白质的相互作用将在体外使用抗体进行验证。任何新型基因都可能通过功能从D. melanogaster序列数据库中鉴定出来，以进一步表征。 （2）JH结合蛋白的鉴定和表征。 先前的结合研究表明，在MET突变体的组织提取物中，JH III结合亲和力较差，考虑到对甲丁烯的抗性，并与MET作为受体成分。 为了鉴定JH结合蛋白并建立其结合特性，将测量JH III与单个蛋白质和MET：伴侣异二聚体复合物的结合。 此外，将使用JH III，环氧基重生乙酸的光亲和力类似物用于光接度标签，并建立/确认哪种蛋白是异二聚体复合物中的高亲和力结合蛋白。这些研究的结果将定义假定的JH受体的成分以及与MET物理相互作用的任何相关蛋白。 JH结合蛋白也将被鉴定，从而阐明了JH作用的分子基础知之甚少。 这些结果将提供必要的知识和分子工具，以进一步分析JH转录控制。