: Genes coding for the cuticular proteins (CPs) of Anopheles gambiae will be identified and temporal and spatial patterns of expression of each gene, or group of related genes, will be determined. CPs are among the most abundant multigene families in insects. Over half of the sequences identified to date have a conserved motif, the R&R consensus, which confers the ability to bind chitin. This motif is found in two forms, one associated with proteins from soft, the other with proteins from hard cuticles. CP motifs known from other insects will be used in computer analyses of the Anopheles genome. Once putative CPs are identified, their temporal patterns of expression will be determined with quantitative RT-PCR. Then the spatial location of representative mRNAs will be determined with in situ hybridization on histological sections of animals of different developmental stages. Finding mRNAs in the epidermis underlying a forming cuticle will be taken to indicate that the candidate proteins are destined for secretion into the cuticle. Localization will also provide a correlation of primary protein structure with cuticle type. To learn if putative CPs that lack the R&R consensus also bind chitin, cDNAs for representative proteins will be expressed, their protein products purified and tested for binding on chitin columns. Further information on proteins in cuticle, including those that are not solubilized by strong denaturing agents, will be obtained by using shotgun proteomic methods to identify peptides that can be released by proteases from intact and extracted cuticles. This first comprehensive analysis of the CPs of a single species will clarify the roles that diverse types of CPs play in the structure and function of cuticle, especially as the sequences identified will also be subject to analysis of secondary structure and further structural modeling. Such information that extends from the genome of Anopheles gambiae to the constituents and construction of its cuticle may point the way to new strategies to control this formidable vector.
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