The goal of this proposal is to characterize factors in the regulatory cascade that mediate 20-hydroxyecdysone (20E) action in the mosquito. We cloned a cDNA encoding ecdysteroid receptor (AaEcR) and characterized its expression during vitellogenesis in the mosquito Adedes aegypti. Recently, we also cloned a cDNA encoding for the mosquito Ultraspiracle homolog (AaUSP) which is a heterodimeric partner of ecdysteroid receptor. We further demonstrated that AaEcR and AaUSP, expressed from cloned cDNAs by in vitro transcription/translation, are capable of hormone and DNA binding as a functional heterodimer. The 20E-responsive gene coding for mosquito vitellogenic carboxypeptidase (VCP) was also cloned. However, potential hormone response elements present in two allelic VCP genes are different from that of the consensus sequence for the ecdysone response element. Furthermore, experiments utilizing cycloheximide, an inhibitor of protein synthesis, showed that 20E action on the mosquito yolk protein genes for vitellogenin and VCP is indirect and likely mediated by a regulatory cascade similar to that in Drosophila. These studies have set the stage for elucidating the molecular mechanism of regulation of gene expression by 20E in the mosquito. In this proposal we plan to analyze AaEcR by cloning isoform-specific cDNAs and gene(s) and studying their expression. Possible isoforms of AaUSP and its gene(s) will also be analyzed. Both AaEcR and AaUSP will be expressed in a baculovirus system and their purified proteins studied with respect to biochemical properties and ligand (20E) binding. We will investigate binding properties of AaEcR/USP complexes to hormone-response elements of 20E-regulated genes from mosquito and Drosophila. Finally, we will identify transcription factors mediating 20E response in the mosquito. The search for alternative strategies of mosquito control is based on the identification of processes and genes that govern development, reproduction, or vectorial capacity. Elucidation of how key regulatory molecules such as ecdysteroid receptor govern the expression of genes essential to mosquito development and reproduction is of fundamental importance to these efforts.
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