We achieved significant progress in understanding the molecular mechanisms regulating the transcription of yolk protein precursor genes (YPP) in the mosquito fat body. Several key target and regulatory genes were cloned and characterized. Using the Vg promoter, we engineered the first transgenic mosquito with the blood meal-activated systemic immunity. The regulatory regions of Vgand VCP will be dissected to identify minimal sites responsible for the timing and level of their expression. We made an important step towards understanding the molecular mechanism underlying the anautogenic repression of YPPgenes by discovering a novel GATA (GATAr) factor which is a unique represser inhibiting activation of YPPgenes during previtellogenesis. In this proposal, we will test the role of GATAr in the anautogenic repression of YPP genes by utilizing the Sindbis virus vector to produce RNAi knockouts of this factor. We plan to clone a GATA activator (GATAa) responsible for turning on the YPPgenes, likely by replacing the GATAr at the GATA cis elements of YYP gene regulatory regions. At the state of arrest, a putative GATAa accumulates in the fat body cytoplasm. Its nuclear translocation coincides with a blood meal, suggesting involvement of a unique .nutrition-activating signal transduction cascade in this process, which will be investigated. We showed that in the mosquito fat body, the expression of the Lp gene encoding the major lipid carrier protein, lipophorin (Lp) is constitutive, but elevated during vitellogenesis. We will clone the Lp gene and study how this pattern offal;body-specific expression is regulated. We will isolate regulatory regions of the lipophorin receptor (LpR) gene, encoding the fat body- specific isoform (LpRfb), which is highly expressed in the postvitellogenic period, characterize its structure and determine what transcription factors are required for its expression. Completion of these goals will permit us to engineer transgenic mosquitoes producing effector molecules before, during and after the vitellogenicperiod at the predictable levels. Knowledge of the molecular mechanisms underlying the blood meal-mediated cycles of repression-activation of the YPPgenes will serve for exploration of adaptation to hematophagy, leading to the establishment of genetic means for disruption of the anautogenic development of eggs.
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