PREPUPAL ACTIVATION OF JH ESTERASE GENE

本研究的长期目标是确定棉铃虫保幼激素酯酶基因在变态前期精确调控表达的分子机制。这次竞争性续签是最初提出的5年研究项目的第二部分。在资助最初的部分时，研究科先前决定将最初的5年建议的具体目标分为两个连续拨款，第一个拨款是关于最初的特定目标1和2的即将到期的3年研究。我们对第一部分的研究进展顺利，意外发现JH酯酶基因显然是以更复杂的复合核心启动子的形式组织起来的，在鳞翅目昆虫和蚊子的C7-10细胞中都具有这种功能。我们在这里描述了第二部分剩余资金的研究，涉及最初提议的特定目标3(蛋白质的分离)和特定目标4(蛋白质的克隆)，以及这些分子的下一个初步特征，赋予核心启动子其作为复合核心启动子的特定属性。因此，本提案的具体目标是：特定目标1-使用蛋白质分级和配基结合方法(最初5年提案的特定目标3)分离与重要的转录活性启动子基序结合的蛋白(S)；特定目的2-克隆这些从蚊子中分离的转录因子，并使用这些克隆从蚊子cDNA库中分离相应的克隆(最初5年提案的特定目标4)特定目的3-从蚊子和蚊子各自的细胞系中克隆的因子的功能特征，以及为了发育，组织和激素的特异性NIH的热带医学和寄生虫学任务支持旨在研究和控制昆虫病媒的实验项目。拟议中的对精确控制尤为关键的基因复合启动子的研究将首次确定此类启动子的诊断成分和性质，用于在蚊子和其他医学上重要的昆虫中识别和破坏它们。

The long term objective of this study is to determine the molecular mechanisms by which the juvenile hormone esterase gene from Trichoplusia ni is precisely regulated to be expresssed during the prepupal stage of metamorphosis. This competitive renewal is for the second part of an originally proposed 5 year research project. In funding the initial part, the Study Section previously determined that the specific aims of the original 5 year proposal should be separated into two sequential grants, the first grant being the presently expiring 3 year study on the original specific aims 1 and 2. Our studies on the first part have progressed nicely on track, with the unexpected discovery that the JH esterase gene is apparently organized as a more complex composite core promoter, and functions so in both lepidopteran and mosquito C7-10 cells. We describe here the studies for the remaining funding of the second part, concerning the original specific aim 3 (isolation of the proteins) and specific aim 4 (cloning of the proteins) of the original proposal, as well as the next initial characterization of these molecules that confer the to the core promoter its specific attributes as composite core promoter. Thus, the specific aims of the present proposal are: Specific Aim 1 - Isolation of the protein(s) binding to the important, transcriptionally active promoter motifs, using protein fractionation and ligand-binding methods (specific aim 3 of original 5 year proposal) Specific Aim 2 - Cloning of these isolated transcription factors from T. ni and use of these clones to isolate the corresponding clones from mosquito cDNA libraries (specific aim 4 of original 5 year proposal) Specific Aim 3 - Functional characterization of the factors cloned from T. ni and mosquitos in their respective cell lines, and for developmental-, tissue and hormonal specificities The NIH mission in Tropical Medicine and Parasitology supports experimental projects designed toward study and control of insect disease vectors. The proposed research on much understudied composite promoters of genes whose precise control is especially crucial will identify for the first time the diagnostic component and properties of such promoters for their identification and disruption in mosquitoes and other medically important insects.