The proposal focuses on the molecular mechanisms which establish asymmetries in the oocyte of Drosophila melanogaster. During oogenesis the posterior follicle cells send a signal of unknown nature to the developing oocyte. This signal leads to a polarization of the oocyte cytoskeleton, which then directs the localization of determinants such as oskar and bicoid RNA to the anterior and posterior poles of the egg. The investigator has previously identified a gene, mago nashi (mago) which plays a role in the signal transduction mechanism by which the oocyte receives the polarizing information from the posterior follicle cells. A second protein, Tsunami (Tsu) has been found to interact with Mago in yeast 2-hybrid screens. The proposal is aimed at providing insight into the in vivo function of both Mago and Tsu in Drosophila. The first goal of the proposal is to map domains of the Mago protein which are necessary for function and subcellular localization. Point mutations and deletions will be introduced into the Mago protein and the cellular distribution of these mutant proteins will be monitored. The protein will also be targeted to specific cellular locations, and the phenotypes of such artificially localized proteins will be determined. Tsunagi is a previously uncharacterized gene, and goal 2 of the proposal is the characterization of the role of tsu in development. Mutations in tsu will be searched for among the pre-existing mutants in the region. Such mutations will then be further characterized. The expression pattern of tsu RNA and protein will also be determined. Given that Tsu contains RNA binding motifs SELEX procedures will be performed in an attempt at defining RNA sequences that are bound by Tsu. If potential target proteins can be identified they will be further studied. The third goal of the proposal addresses the question whether Mago and Tsu interact with each other in vivo, and if these experiments are positive, the domains required for the interaction will be determined. (In the supplemental material that was submitted in September, the PI reports that Tsu and Mago indeed can indeed be co-immunoprecipitated from fly extracts). The fourth goal of the proposal involves screens for additional genes that act in the mago pathway. These screens have already been initiated and some promising candidates have been isolated, and will be further studied.
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