A Drosophila Juvenile Hormone Receptor

这项拟议的工作将通过探索保幼激素(JH)在模式昆虫黑腹果蝇中的分子作用来研究激素对昆虫发育和繁殖的控制。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。一种遗传方法鉴定了耐甲氧戊二烯(Met)基因，并发现它编码的蛋白质(MET)参与了这种昆虫体内JH的作用，可能是JH受体的一个组成部分。在这项拟议的工作中，Met将成为了解该受体的焦点。拟议的工作包括两个目标：(1)MET伙伴蛋白的鉴定。黑腹果蝇中的Met+是转录调节因子bHLH-PAS家族的成员。PAS蛋白与伴侣蛋白在异二聚体复合体中发挥作用，没有理由相信MET是一个例外。对JH结合和接受的理解必须包括对伙伴蛋白的鉴定。一个很有希望的候选基因是最近从D。黑猩猩。以Met+为诱饵的酵母双杂交技术将被用来直接检测Met-L作为MET的伴侣，并寻找与Met相互作用的其他蛋白质。在这项经过验证的技术中，候选基因在酵母细胞中一起表达。他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，这些基因被工程化为酵母菌株，使这种相互作用可以随时可视化。每种蛋白之间的相互作用将在体外用MET抗体进行验证。任何新的基因(S)都将被鉴定，可能是通过功能，从黑腹果蝇的序列数据库中进行进一步的鉴定。(2)JH结合蛋白的鉴定和鉴定。以往的结合研究表明，在Met突变体的组织提取物中，JH III结合亲和力较差，这是Met对甲基戊二烯产生抗性的原因，并暗示Met是一种受体成分。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：伙伴异二聚体复合体的结合。此外，JH III的光亲和类似物环氧法尼基重氮乙酸酯将被用于光亲和标记，并建立/确认哪个蛋白质是异二聚体复合体中的高亲和力结合蛋白。这些研究的结果将确定推定的JH受体的成分以及任何与MET物理上相互作用的相关蛋白质。JH结合蛋白也将被鉴定，从而澄清JH作用的鲜为人知的分子基础。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.