PREPUPAL ACTIVATION OF JH ESTERASE GENE

本研究的长期目标是确定毛癣幼体激素酯酶基因在变态前蛹期精确调控表达的分子机制。这个竞争性更新是最初提议的5年研究项目的第二部分。在资助最初部分时，研究组先前决定，原定5年建议的具体目标应分为两笔连续拨款，第一笔拨款是就原定具体目标1和2进行的目前即将到期的3年研究。我们对第一部分的研究进展顺利，意外发现JH酯酶基因显然是作为一个更复杂的复合核心启动子组织的，并且在鳞翅目和蚊子的C7-10细胞中都是如此。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），以及赋予核心启动子作为复合核心启动子的特定属性的这些分子的下一个初始表征。因此，本建议的具体目标是：特异性目标1 -分离与重要的转录活性启动子基序结合的蛋白质；使用蛋白质分离和配体结合方法（原5年计划的特异性目标3）特异性目标2 -从T. ni中克隆这些分离的转录因子，并利用这些克隆从蚊子cDNA文库中分离出相应的克隆（原5年计划的特异性目标4）特异性目标3 -从T. ni和蚊子中克隆的因子在各自细胞系中的功能表征，并用于发育-美国国立卫生研究院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。对精确控制尤为关键的基因的复合启动子的研究将首次确定这些启动子的诊断成分和特性，以便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.