GENETIC CONTROL OF MICROTUBULE FUNCTION IN DEVELOPMENT

拟议工作的总体目标是研究分子机制 通过该方法， 胚胎形态发生，并检查差异基因的控制 指导这种发展过程的行动。 模型 系统是一个研究微管蛋白基因表达和微管功能 在黑腹果蝇的胚胎发育过程中。 特别是要 研究胚胎特异性β 3-微管蛋白的调节和功能 我们已经确定了一个基因，它只被合子基因组转录， 胚胎发育中期的一段短暂时期。 拟议实验的具体目标包括遗传和 微管组装中表达和功能的分子分析 胚胎特异性β 3-微管蛋白亚基：（1）突变的分离 β 3微管蛋白的结构基因 突变将通过以下方法分离： 筛选胚胎致命突变，这些突变不能补充a 缺陷染色体，我们已经表明删除β 3-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 在胚胎发生过程中的已知基因型。 该方法提供 这种胚胎遗传标记以前在果蝇中缺失 这将使我们能够研究β 3-微管蛋白纯合子胚胎 在基因作用期间发生突变。 (2)已知突变的分析 其影响表达β 3-微管蛋白的组织。 定位 β 3-微管蛋白的原位杂交将通过原位杂交进行 我们已经构建了一个用于切片胚胎的信息特异性探针。 （三） 分离编码与以下物质相互作用的产物的基因中的突变： β 3微管蛋白。 (4)启动子融合实验将（a）使我们能够 检查不同β-微管蛋白基因产物的功能等效性， 从而间接地评估了导致 形成和多个微管蛋白基因，和（B）允许一种分离 特异性影响时间调节的突变，或 β 3-微管蛋白表达的组织特异性。 (5)审查 β 3-微管蛋白基因周围的染色质结构， 和基因表达的“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.