A Drosophila Juvenile Hormone Receptor

果蝇保幼激素受体

• 批准号: 0322136
• 负责人: Thomas Wilson
• 金额: --
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-31 至 2004-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0322136&HistoricalAwards=false
• 关键词: 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.

本研究以果蝇为模型，探讨保幼激素（JH）对昆虫发育和生殖的分子调控作用。 JH在昆虫中的重要作用已被确定，但这种激素的作用仍然是一个谜，因为JH受体尚未在任何昆虫中被确定。 一种遗传学方法鉴定了耐甲氧普仑（Met）基因，并发现它编码的蛋白质（MET）参与了JH在这种昆虫中的作用，可能是JH受体的一个组成部分。 在拟议的工作中，Met将作为了解这种受体的焦点。 拟议的工作包括两个目标： (1)MET伴侣蛋白的鉴定。 Met+在D.黑腹菌是转录调节子bHLH-PAS家族的成员。 PAS蛋白在异二聚体复合物中与伴侣蛋白一起起作用，没有理由相信MET是例外。 对JH结合和接受的理解必须包括对伴侣蛋白的鉴定。 一个有希望的候选者是最近从D.黑腹菌 以Met+为诱饵的酵母双杂交技术将被用于直接检测MET-L作为MET的伴侣以及寻找与MET相互作用的其他蛋白。在这种成熟的技术中，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工程化到酵母菌株中的报告基因的转录，从而允许这种相互作用的现成可视化。 将使用MET抗体在体外验证每种蛋白质的相互作用。任何新的基因将被鉴定，可能是通过功能，从D。melanogaster序列数据库，用于进一步表征。(2)JH结合蛋白的鉴定和表征。 以前的结合研究表明，JH III的结合亲和力差得多的组织提取物从Met突变体，占耐甲氧普烯，并牵连MET作为受体成分。 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，环氧法呢基重氮乙酸酯，将用于光亲和标记和建立/确认哪种蛋白质是异二聚体复合物中的高亲和力结合蛋白。这些研究的结果将确定推定的JH受体的组分和与MET物理相互作用的任何相关蛋白质。JH结合蛋白也将被鉴定，从而澄清了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。