This application describes studies on regulation of ribosome biosynthesis during mosquito reproduction, in the context of an overall effort to develop molecular tools to genetically interfere with disease transmission. Our research integrates in vitro studies with mosquito cell lines and in vivo studies with the intact organism to 1) understand at the molecular level, key physiological processes that control mosquito reproduction, 2) clone and characterize the genes involved, and 3) use the cloned genes to develop effective gene transfer procedures and genetic control strategies. A particular focus of this phase of research is identification of promoter elements that mediate coordinate control of gene expression, with an immediate goal of establishing basic information for construction of synthetic modular promoters to control gene expression in transfected cells and transformed mosquitoes. Mosquito reproductive physiology is central to acquisition and transmission of human pathogens by mosquito vectors. Ribosome biosynthesis provides the protein synthetic machinery essential to egg production, and the coordinate control of RNA and ribosomal protein genes with respect to one another and in response to the metabolic needs of the organism provides a model for manipulation of genes transcribed by RNA polymerases I and II. Because ribosomal genes are expressed in all cells, elucidation of molecular aspects of their regulation will facilitate extension of gene transfer technologies to the mosquito embryo. Finally, in transformed Drosophila, it has already been shown that genetic disruption of ribosome biosynthesis interferes with normal egg production.
The specific aims i nclude physiological studies on ribosomal protein and rRNA gene expression in the adult female mosquito during the acquisition of """"""""competence"""""""" following emergency from the pupa, and during the vitellogenic cycle initiated by the blood meal. This study will include psoralen crosslinking to distinguish between active and inactive genes, and analysis of the recruitment of mRNA from ribonucleoprotein particles into actively translating polysomes. Cis- and trans-acting regulatory elements from the genes we have cloned will be identified using transfected cells, gel retardation, and nuclear run-on techniques. Cloning efforts will focus on rp S6 and its regulatory elements. Phosphorylation of this highly conserved ribosomal protein is associated with control of protein synthesis, and reduced expression of rp S6 in Drosophila leads to disruption of immunity functions. These studies will contribute new information and analytical approaches to investigate gene expression and provide molecular infrastructure essential to disruption of disease transmission in genetically transformed mosquitoes.
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