My long term objectives are to identify new types of cytoskeletal-associated proteins in the cortex of the Drosophila embryo and to understand their role in guiding the rapid nuclear divisions that occur in this region. My general strategy is to identify mutations that specifically disrupt the late blastoderm divisions occurring in the cortex of the Drosophila embryo. I believe that many of these mutations will be in genes coding for cytoskeletal-associated proteins. By employing newly developed cellular and molecular techniques, I will determine the function of these proteins in the nuclear division process as a complement to more conventional biochemical studies of the Drosophila cortex. I propose seven specific aims to identify and characterize these cytoskeletal components of the Drosophila embryo: 1.Mutant screen-I will screen the existing collections of P-element induced maternal-effect mutations for those that specifically disrupt the cortical nuclear divisions.2.Revertant analysis and mapping of the mutations- To confirm that the mutation is induced by a P-element I will mobilize the Inserted P-element to revert the mutant phenotype. In addition, I will map the mutation through deficiency analysis and also through in situ localization on the polytene chromosomes.3.Immunofluorescent characterization- Through immunofluorescence on fixed embryos at different stages, I will characterize the nuclear, centrosome, actin and tubulin distributions in the mutant embryos.4.Real-time analysis- Using micro-injected fluorescently-tagged proteins, I will follow the dynamics of the of nuclei, centrosomes, actin and tubulin through multiple nuclear cycles in living mutant embryos.5.Isolating and characterizing the gene- The P-element induced mutations allow rapid cloning of the flanking DNA sequences. After full length cDNAs are obtained, northern and DNA sequence analysis will be performed.6.Generating antibodies- The cDNAs will be subcloned into an expression vector and the fusion protein generated used to generate antibodies to facilitate various cellular studies.7.Biochemical studies- I will determine if any of the mutations I am studying are disrupted in the actin and tubulin-associated proteins they have identified.
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