PREPUPAL ACTIVATION OF JH ESTERASE GENE

本研究的长期目标是确定粉纹夜蛾保幼激素酯酶基因在变态的预蛹期精确调控表达的分子机制。 这次竞争性更新是最初提出的5年研究项目的第二部分。 在资助初始部分时，研究科先前确定，原始5年提案的具体目标应分为两个连续的赠款，第一个补助金是目前即将到期的3年研究，针对原始具体目标1和2。 我们在第一部分的研究进展顺利，意外发现JH酯酶基因显然是一个更复杂的复合核心启动子，在鳞翅目和蚊子C7-10细胞中发挥作用。 我们在这里描述了第二部分剩余资金的研究，涉及原始提案的原始特定目标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始表征，这些分子赋予核心启动子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：具体目标1 -使用蛋白质分级分离和配体结合方法分离与重要的转录活性启动子基序结合的蛋白质（最初5年提案的具体目标3）具体目标2 -从T. ni和使用这些克隆从蚊子cDNA文库中分离相应的克隆（最初5年提案的具体目标4）具体目标3 -克隆自T. NI和蚊子在各自的细胞系，并为发育，组织和激素的特异性在热带医学和寄生虫学的NIH使命支持旨在研究和控制昆虫疾病媒介的实验项目。 拟议中的关于基因的复合启动子的研究，其精确控制是特别重要的，将首次确定这种启动子的诊断成分和特性，用于在蚊子和其他医学上重要的昆虫中识别和破坏。

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.