SACCHAROMYCES CEREVISIAE MICROTUBULE CYTOSKELETON

使用生物化学方法来鉴定 酿酒酵母微管细胞骨架，我们现在要确定 这些成分在有丝分裂、减数分裂和核中的作用 核聚变。此外，我们最近完成了基因的遗传选择 增强了β-微管蛋白突变体的功能，承诺提供 对微管稳定性调节机制的独特见解 在活体内。我们从学习中学到的原则将适用于所有人 真核细胞，因为微管的性质高度 保守的。这些具体问题将得到解决： MAP27、MAP38和MAP50在体内有哪些功能？活体内 三个生化鉴定的酵母图谱MAP27、MAP38和MAP38的功能 MAP50，将从遗传学上阐明。已经展示了MAP38和MAP50 通过免疫荧光与核内和核内 体内的细胞质微管。因此，确定功能 在这两种蛋白质中，将被给予最高的优先级。MAP27有GTP- 具有约束力的活动，表明它可能调节或确保适当的 微管的组装。编码MAP27、MAP38和MAP50的基因 已经被隔离了。利用体外诱变和基因替换，我们 将构建这些基因的突变等位基因。每种基因在体内的作用 MAP将使用四个公认的和高度敏感的工具来阐明 酵母菌体内微管功能的测定。一旦遗传基因 MAP功能分析完成后，生化活性 它们在体内的作用将被确定。 通过抑制A基因确定的基因在体内有什么功能 依赖苯菌灵的β-微管蛋白突变？我们已经确定了 可以突变或过度表达以抑制tub2的基因- 150β-微管蛋白突变体。这些基因很可能编码新的蛋白质。 在体内调节微管组装的物质。同样敏感的基因 MAP27、MAP38、MAP27、MAP38、 和MAP50基因(上面)将被用来阐明 抑制基因。双重突变分析将确定 每种蛋白质都在有丝分裂纺锤体等过程中起作用 组装，以及哪些蛋白质亚群一起发挥调节作用 微管在细胞周期中组装。 总而言之，这些研究将为这些机制提供新的见解 辅助蛋白通过与活细胞中的微管蛋白相互作用来实现 调节染色体分离、核迁移和 核聚变。

Having used biochemical approaches to identify components of the Saccharomyces cerevisiae microtubule cytoskeleton, we will now determine how these components function during mitosis, meiosis, and nuclear fusion. In addition, our recently completed genetic selections for genes that enhance the function of a beta-tubulin mutant promise to provide unique insights into mechanisms used to modulate microtubule stability in vivo. The principles learned from our studies will apply to all eukaryotic cells because the properties of microtubules are highly conserved. These specific questions will be addressed: What are the in vivo functions of MAP27, MAP38 and MAP50? The in vivo functions of three biochemically identified yeast MAPs, MAP27, MAP38 and MAP50, will be genetically elucidated. MAP38 and MAP50 have been shown by immunofluorescence to associate with both intra-nuclear and cytoplasmic microtubules in vivo. Therefore, determining the functions of these two proteins will be given the highest priority. MAP27 has GTP- binding activity suggesting that it might regulate or insure the proper assembly of microtubules. The genes that encode MAP27, MAP38 and MAP50 have been isolated. Using in vitro mutagenesis and gene replacement, we will construct mutant alleles of these genes. The in vivo roles of each MAP will be elucidated using four well-established and highly sensitive in vivo assays of microtubule function in yeast. Once the genetic analysis of the MAP functions is completed, the biochemical activities that underlie their in vivo roles will be determined. What are the in vivo functions of genes identified by suppression of a benomyl-dependent beta-tubulin mutation? We have already identified genes that can either be mutated or overexpressed to suppress the tub2- 150 beta-tublin mutant. These genes are likely to encode novel proteins that regulate microtubule assembly in vivo. The same sensitive genetic tests for in vivo microtubule function referred to for the MAP27, MAP38, and MAP50 genes (above) will be used to elucidate the roles of the suppressor genes. Double mutant analysis will determine the order in which each of the proteins acts in processes such as mitotic spindle assembly, and which subsets of proteins function together to regulate microtubule assembly during the cell cycle. In total, these studies will provide novel insights into the mechanisms by which accessory proteins interact with tubulin in living cells to mediate such functions as chromosome segregation, nuclear migration and nuclear fusion.