MOLECULAR BIOLOGY OF MICROTUBULE INTERACTING PROTEINS

描述：拟议研究的长期目标是 鉴定和鉴定在细胞周期中具有重要功能的蛋白质 微管的组装和活动。大多数这些蛋白质和 它们的功能没有被描述，但它们可能在 测定微管的许多基本细胞活动。这个 在这笔赠款的支持下，研究人员的早期工作导致了这一发现 一种被称为伽马微管蛋白的蛋白质。伽马微管蛋白与 但不同于相互作用形成的α和β微管蛋白 被称为微管蛋白的异源二聚体。自从伽马微管蛋白被发现以来， 来自几个实验室的数据表明，伽马微管蛋白在 在真核生物中，它主要存在于微管组织中 真菌和动物细胞的中心(MTOC)。这导致了 假设γ-微管蛋白是微管组装所必需的 但是关于γ-微管蛋白的功能，它在 调节微管组装及其与微管的相互作用，以及 它与MTOCs其他组成部分的互动仍有待回答。 拟议工作的第一个目标是分离和表征 丝状真菌中条件致死的伽马微管蛋白突变 尼杜拉曲霉。这样的突变体很难分离出来 常规方法因此提出了“丙氨酸扫描”的方法。这些 条件性突变应该是澄清伽马的有用工具 微管蛋白功能及假逆转分析用于鉴定 伽马微管蛋白相互作用蛋白。变种人将接受检查以 确定突变对核分裂、有丝分裂、有丝分裂的影响 纺锤体的形成、细胞质微管网络和定位 和γ微管蛋白的稳定性。 第二个目标是识别和表征相互作用的蛋白质。 用伽马微管蛋白。我们将采取两种方法。第一种方法 将涉及酵母双杂交筛选，而第二个将使用 类似于伽马发现中所用的伪反转分析 微管蛋白。由这两种互补方法中的任何一种确定的基因 将进一步表征，以确定其潜在角色 产物在微管中组装和发挥功能。 该提案的最终目的是研究一种新的微管蛋白 在线虫基因组计划中确定的。这一推测的基因产物 最初被鉴定为一种伽马微管蛋白，但进一步的分析表明 它与伽马微管蛋白的同源性只有40%-44%，与 其他微管蛋白。这一点有足够的不同，以至于提出了这一点 代表了一个新的微管蛋白家族。这将由第一个人调查 确定线虫是否有常规的伽马微管蛋白。如果 ，然后调查员打算搜索这一事件的同源物 在其他生物中发现了新的微管蛋白，并开始剖析其功能。

DESCRIPTION: The long-term objective of the research proposed is to identify and characterize proteins that have essential functions in the assembly and activities of microtubules. Most of these proteins and their functions have not been described but they likely have roles in determining the many essential cellular activities of microtubules. The investigator's earlier work supported by this grant led to the discovery of one such protein known as gamma tubulin. Gamma tubulin is related to but distinct from the alpha and beta tubulins which interact to form the heterodimer known as tubulin. Since the discovery of gamma tubulin, data from several labs have shown that gamma tubulin is ubiquitous among eukaryotes and it is found primarily in the microtubule organizing centers (MTOCs) of fungal and animal cells. This has led to the hypothesis that gamma tubulin is essential for microtubule assembly from MTOCs but questions about the function of gamma tubulin, its role in regulating microtubule assembly, its interactions with microtubules, and its interactions with other components of MTOCs remain to be answered. The first aim of the proposed work is to isolate and characterize conditionally-lethal gamma tubulin mutations in the filamentous fungus Aspergillus nidulans. Such mutants have been difficult to isolate by conventional means so the "alanine scanning" approach is proposed. These conditional mutants should be useful tools for clarification of gamma tubulin function and for pseudoreversion analysis for identification of gamma tubulin interacting proteins. Mutants will be examined to determine the effects of mutations on nuclear division, mitosis, mitotic spindle formation, the cytoplasmic microtubule network, and localization and stability of gamma tubulin. The second aim is to identify and characterize proteins that interact with gamma tubulin. Two approaches will be taken. The first approach will involve a yeast two-hybrid screen while the second will use pseudoreversion analysis similar to that used in discovery of gamma tubulin. Genes identified by either of these complementary approaches will be characterized further to determine the potential roles for their products in microtubule assembly and function. The final aim of the proposal is to investigate a novel tubulin identified in the C. elegans genome project. This putative gene product was originally identified as a gamma tubulin but closer analysis shows that it shares only 40-44% identity with gamma tubulins and less with other tubulins. This is sufficiently different to propose that this represents a new tubulin family. This will be investigated by first determining whether C. elegans has a conventional gamma tubulin. If identified, the investigator then intents to search for homologs of this novel tubulin in other organisms and begin to dissect its function.