Genetic analysis of basal body function

描述（由申请人提供）：在动物细胞中，微管由中心体组织，中心体是一种细胞器，具有一对中心粒，包裹在中心周围物质的基质中。每个细胞周期一次，中心体和中心粒在其中复制。这一事件对细胞分裂和基因组稳定至关重要。复制包括现有中心粒对的分离和在旧中心粒附近合成一个新的中心粒。新的中心粒对迁移到组装纺锤体的两极。中心粒可以成熟成基体，形成鞭毛和纤毛的模板。单细胞藻类衣藻由于其简单的细胞生物学特性和强大的遗传特性，为研究中心粒/基体提供了一个杰出的模型。在过去的几年里，我的实验室的研究已经帮助证明了基础体的复杂性，并确定了两个新的微管蛋白家族成员在中心粒/基础体复制和功能中的重要作用。这些研究留下了几个重要的问题，我们将解决其中的三个问题：1)。中心粒复制的早期事件需要什么分子？2）. 在细胞周期中参与中心粒成熟的分子有哪些？3）. 有丝分裂时中心粒是如何分离和迁移的？我们将利用衣藻的突变、抗体和RNA干扰来回答这些问题。我们将测试epsilon-和-微管蛋白在中心粒复制中的作用，以及epsilon-微管蛋白突变的几种抑制因子和增强因子的作用。我们假设至少四个基因，delta-微管蛋白，DT11， DT12和UN13，需要在M/G1过渡阶段将中心粒成熟为基体。我们将开发成熟的抗体标记物，并开始描述成熟过程中的事件和参与者。在有丝分裂前期，基小体转化回中心粒，中心粒分离、迁移和组织事件。利用RNA干扰，我们将测试几种在其他生物体中重要的蛋白质的作用，并利用gfp - γ -微管蛋白报告基因来筛选条件突变体，以跟踪中心粒分离和迁移。我们将使用“比较基因组学”来识别中心粒/基体的复制、成熟和迁移所需的其他基因。

DESCRIPTION (provided by applicant): In animal cells microtubules are organized by the centrosome, an organelle with a pair of centrioles enclosed in a matrix ofpericentriolar material. Once per cell cycle, the centrosome and centrioles within it duplicate. This event is critical to cell division and genome stability. Duplication involves the separation of the existing centriole pair and the synthesis of a new centriole adjacent to the old centriole. The new pairs of centrioles migrate apart to the poles of the assembling spindle. Centrioles can mature into basal bodies that template flagella and cilia. The unicellular alga, Chlamydomonas provides an outstanding model for investigations into centrioles/basal bodies because of its straightforward cell biological traits and powerful genetics. In the last few years, studies from my laboratory have helped to demonstrate the complexity of basal bodies and to identify critically important roles for two new tubulin family members in centriole/basal body duplication and function. These studies have left unanswered several important questions and we will address three of these questions: 1). What are the molecules needed for early events in centriole duplication? 2). What are the molecules involved in maturation of centrioles through the cell cycle? 3). How do centrioles separate and migrate at mitosis? We will employ mutations, antibodies, and RNA interference in Chlamydomonas to answer these questions. We will test the roles of epsilon- and eta-tubulin in centriole duplication as well as the roles of several suppressors and enhancers of epsilon-tubulin mutations. We hypothesize that at least four genes, delta- tubulin, DT11, DT12, and UN13, are needed to mature centrioles into basal bodies at the M/G1 transition. We will develop antibody markers for maturation and begin to characterize the events and players in maturation. Basal bodies are converted back into centrioles that separate, migrate, and organize events in preprophase of mitosis. Using RNA interference, we will test the roles of several proteins that are important in other organisms and with this knowledge screen for conditional mutants using a GFP-gamma-tubulin reporter to follow centriole separation and migration. We will mine using "comparative genomics" to identify additional genes that are needed for duplication, maturation, and migration of the centrioles/basal bodies.