Despite the great importance of hematophagous insects as disease vectors, they have received relatively little attention from investigators utilizing modern molecular biological techniques. This proposal focuses on the insect gut, the first site of interaction between the insect and the parasites it transmits. The project has two broad objectives: (i) to develop means for interfering with the capacity of insects to transmit disease, and (ii) to gain a better understanding of the mechanisms that regulate gene expression in the insect gut. Specifically, we propose the following research plan. I) Functional characterization of a gut-specific promoter. Recently, we found that putative promoter sequences from the black fly carboxypeptidase gene can drive gut-specific expression of a reporter when transformed into Drosophila. In a separate set of experiments, we also found that a recombinant retrovirus can transfect Anopheles gut epithelial cells in vivo. While the transgenic Drosophila system is useful to assay tissue-specific gene expression, transfection of retroviruses into Anopheles is useful to measure induction of gene expression by a blood meal. To identify cis-acting promoter sequences required for gut-specific expression and expression induced by blood feeding, we will construct a series of promoter deletions, and assay for function by the two aforementioned in vivo assays. Interaction between promoter sequences and gut nuclear proteins will be investigated by the use of DNA mobility-shift assays. DNA-protein interactions will be further characterized by DNase I footprinting experiments. Cloning of genes encoding DNA-binding proteins will be accomplished by one of three approaches: direct screening of an expression library with a DNA probe, the yeast """"""""one-hybrid"""""""" genetic screen, or biochemical purification of the binding protein by DNA affinity chromatography. These experiments may inform on how genes with potential for blocking parasite transmission, can be expressed in the mosquito gut. II) Identification of genes expressed early after a blood meal. Following a blood meal, transcription of the major digestive enzymes is activated after a lag of 6-8 hours. There is reason to believe that induction of the late enzymes depends on the activation of intermediate gene products, including transcription factors, during the lag time. To identify genes whose transcription is activated early after the blood meal, we will compare gut RNAs isolated from mosquitoes prior to, and 2-4 hours after a blood meal, by use of the PCR-based """"""""differential display"""""""" technique. PCR fragments originating from the blood-induced mRNAs will be cloned, and the corresponding genes will be characterized by conventional approaches. The peritrophic matrix (PM), which forms late after a blood meal, constitutes a physical barrier for the diffusion of secreted products into the blood meal. The promoters of the """"""""early genes"""""""" identified by these experiments, may be used to drive the expression of gene products with anti-parasite activity, prior to PM formation.
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