GENETIC CONTROL OF MICROTUBULE FUNCTION IN DEVELOPMENT

发育中微管功能的遗传控制

• 批准号: 3313904
• 负责人: ELIZABETH C. RAFF
• 金额: $ 13.91万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-09-01 至 1989-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3313904
• 关键词: DNA; Drosophilidae; chromatin; cytogenetics; developmental genetics; gene complementation; gene expression; gene interaction; gene mutation; genetic manipulation; genetic mapping; genetic markers; genetic promoter element; genetic regulation; genome; histogenesis; immature animal; in situ hybridization; microtubules; molecular cloning; molecular genetics; mutagen testing; nucleic acid hybridization; nucleic acid sequence; structural genes; temperature sensitive mutant; tissue /cell culture; tubulin

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.

拟议工作的总体目的是检查分子机制 通过该在此期间构建三维亚细胞结构 胚胎形态发生，并检查差异基因的控制 这些发展过程的指导行动。 模型 系统是微管蛋白基因表达和微管功能的研究 在果蝇中的胚胎发生期间。 特别是，我们会 检查胚胎特异性β3-微管蛋白的调节和功能 我们已经鉴定出的基因，该基因仅由合子基因组转录 在中期发生的短时间内。 提出的实验的具体目的包括遗传和 微管组装中表达和功能的分子分析 胚胎特异性β3微管蛋白亚基的元素：（1）突变的分离 在β3-微管蛋白的结构基因中。 突变将被隔离 筛选未能补充A的胚胎致死突变 我们显示的缺乏染色体删除了β3-微管蛋白基因。 我们已经制定了一种通常适用的策略来识别胚胎 在胚胎发生过程中已知的基因型。 此方法提供 果蝇先前缺少的胚胎遗传标记物 文献，将使我们能够研究纯合子β3微管蛋白的胚胎 基因作用时期的突变。 （2）已知突变的分析 影响β3微管蛋白的组织。 本土化 Beta 3-微管蛋白的原位杂交将进行 我们已构建到截面胚胎的消息特定探针。 （3） 隔离基因中编码与之相互作用的基因的突变 β3微管蛋白。 （4）启动子融合实验（a）允许我们 检查不同β-微管蛋白基因产物的功能等效性和 因此间接评估导致的进化压力 形成和多个微管蛋白基因，（b）允许一种分离的方法 特别影响时间调节的突变或 β3微管蛋白表达的组织特异性。 （5）检查 在两个“转到”期间，β3-微管蛋白基因周围的染色质结构 和基因表达的“关闭”。