The present proposal aims to explore the utilization of the regulatory regions of genes, which are highly expressed in a tissue-specific manner and which are stimulated by a blood meal (BM), for creating a blood- triggered systemic immune response in the malaria mosquito. Systemic immune factors, circulating in the mosquito hemolymph, could midgut wall through the migration of sporozoites to the salivary glands. Thus, in the fat body it will be most desirable to activate a high level of immune factor secretion by the time of oocyst formation, 24-30 hour post blood meal (PBM). Ideally suited for this purpose are yolk protein precursor (YP) genes, which are highly expressed in the mosquito female fat body in response to a BM and which reach maximum expression at 24-30 hour PBM. Powerful YP gene promoters will be cloned from Anopheles gambiae and utilized for expression of transgenic immune factors. Continuous immune factor production during the post-vitellogenic period will be important for acting on maturing oocysts and migrating sporozoites. To achieve this goal, regulatory regions of genes expressed later in vitellogenesis will be utilized. This proposal rests on recent achievements in studies of Aedes aegypti, a model for vector research. Elucidating of the molecular basis of BM-activated gene expression in the Aedes fatty body will aid in the cloning of homologous genes from the Anopheles mosquito. During the initial five years of this proposal, we intend to clone the regulatory regions of the YP genes, vitellogenin (Vg) and vitellogenic carboxypeptidase (VCP). To obtain regulatory regions of genes activated late in vitellogenesis in the Anopheles fat body, we propose to clone genes involved in lipid metabolism. The pro-lipophorin gene (LP) and a homologue to the tissue-specific Aedes aegypti HNF-4 gene are expressed late in vitellogenesis. In addition, we will utilized differential display for identification and cloning of other late genes. Anopheles gene promoters will be tested for BM activation and specific expression via genetic transformation in the Aedes system and later via Anopheles gene transformation. Finally, once Anopheles promoter- reporter constructs have been characterized, we will test BM-activation of defensin, driven by Anopheles Vg, VCP, LP, and HNF-4 promoters.
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