Synthesis and Degradation of Ribosomes in the Mosquito This renewal application builds upon a longstanding interest in regulation of ribosome biosynthesis during mosquito reproduction. Our goals are defined in the broader context of overall efforts by many laboratories to develop molecular tools to interfere with transmission of disease by vector mosquitoes. The research has been facilitated by effective integration of in vitro studies using cultured mosquito cell lines and in vivo studies with vitellogenic mosquitoes, and includes some of the earliest applications of gene transfer technology to mosquito cells in culture. In continuing this work, we plan to focus particularly on ribosomal protein S6, the major phosphorylated protein on the eukaryotic ribosome. Based on an existing cDNA, we will use recombinant DNA techniques including the polymerase chain reaction to complete the rpS6 genomic DNA structure, including 1 to 2 kb upstream and downstream of the coding region. In this analysis we will learn whether mosquito rpS6 genes resemble the Drosophila homolog in having alternative third exons. Using primers based on the cDNA, we will undertake a limited phylogenetic analysis to explore whether a previously undescribed C-terminal similarity to histone H1 protein is shared among two closely related groups of insect vectors. The 5'-flanking sequences of the mosquito rpS6 gene will be compared to those of rpL8 and rpL34, and sites common to all three genes will be defined by functional assays in transfected cells. Proteins that interact with shared regulatory elements will be identified using gel retardation and related techniques. We will identify the sites of rpS6 phosphorylation, and investigate conditions for manipulating the state of rpS6 protein phosphorylation in cultured cells and in mosquito fat body. In addition, we will examine whether the histone-like C-terminal extension encoded by the rpS6 cDNA is phosphorylated at serine residues, and whether it is processed to generate a small, phosphorylated ribosomal protein that we have previously described in mosquito fat body. Physiological studies in the female mosquito will integrate our understanding of ribosomal protein and rRNA gene expression with the well-studied hormonal events that orchestrate the reproductive cycle. These studies provide cloned genes, molecular approaches, and characterization of regulatory processes that can potentially be manipulated to interfere with disease transmission by blood-feeding arthropods.
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