The overall aim of the proposed work is to examine the molecular mechanisms by which three-dimensional subcellular structures are constructed during embryonic morphogenesis, and to examine the control of differential gene action by which such developmental processes are directed. The model system is a study of tubulin gene expression and microtubule function during embryogenesis in Drosophila melanogaster. In particular, we will examine the regulation and function of the embryo-specific Beta 3-tubulin gene we have identified which is transcribed by the zygotic genome only for a brief period in mid-embryogenesis.
The specific aims for the proposed experiments include both genetic and molecular analysis of the expression and function in microtubule assembly of the embryo-specific Beta 3-tubulin subunit: (1) Isolation of mutations in the structural gene for Beta 3-tubulin. Mutations will be isolated by screening for embryonic lethal mutations which fail to complement a deficiency chromosome which we have shown deletes the Beta 3-tubulin gene. We have developed a generally applicable strategy for identifying embryos of known genotype during the course of embryogenesis. This method provides the embryonic genetic marker previously missing from the Drosophila literature and will allow us to study embryos homozygous for Beta 3-tubulin mutations during the time of gene action. (2) Analysis of known mutations which affect the tissue in which Beta 3-tubulin is expressed. Localization of Beta 3-tubulin will be done by in situ hybridization of a message-specific probe we have constructed to sectioned embryos. (3) Isolation of mutations in genes which encode products which interact with Beta 3-tubulin. (4) Promoter-fusion experiments which will (a) allow us to examine functional equivalence of different Beta-tubulin gene products and hence indirectly assess the evolutionary pressures which led to the formation and multiple tubulin genes, and (b) allow a method to isolate mutations which specifically affect regulation of the timing or tissue-specificity of Beta 3-tubulin expression. (5) Examination of chromatin structure around the Beta 3-tubulin gene during both """"""""turn-on"""""""" and """"""""turn-off"""""""" of gene expression.
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