GENETIC CONTROL OF MICROTUBULE FUNCTION IN DEVELOPMENT

拟议工作的总体目标是检查分子机制。 通过它来构建三维亚细胞结构 胚胎形态发生，并考察差异基因的控制 指导这种发展过程的行动。模型 系统研究微管蛋白基因表达和微管功能 在黑腹果蝇胚胎发育过程中。特别是，我们将 检测胚胎特异的β3-微管蛋白的调节和功能 我们已经鉴定的基因，它仅由受精卵基因组转录 胚胎发育中期的短暂时期。 拟议中的实验的具体目标包括遗传和 微管组装过程中表达和功能的分子分析 胚胎特异的β3-微管蛋白亚基：(1)突变的分离 在β3-微管蛋白的结构基因中。突变将通过以下方式分离 筛查不能补体A的胚胎致死突变 我们已经显示的缺失染色体会缺失β3-微管蛋白基因。 我们已经开发了一种普遍适用的识别胚胎的策略 在胚胎发育过程中的已知基因。此方法提供了 此前在果蝇中缺失的胚胎遗传标记 文献，并将使我们能够研究β3-微管蛋白纯合子胚胎 基因作用期间的突变。(2)已知突变的分析 影响表达β3-微管蛋白的组织。本土化 β3-微管蛋白的研究将通过原位杂交 我们已经构建了用于切割胚胎的消息特异性探针。(3) 分离编码与之相互作用的产物的基因突变 β3-微管蛋白。(4)启动子-融合实验将(A)使我们能够 检测不同β-微管蛋白基因产物的功能等价性 因此间接评估了导致 形成和多个微管蛋白基因，以及(B)允许一种方法分离 特定影响时间或时间调节的突变 β3-微管蛋白表达的组织特异性。(5)考试 β3-微管蛋白基因周围的染色质结构 以及基因表达的“关闭”。

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.