IDENTIFICATION OF EARLY BASAL BODY ASSEMBLY PROTEINS FROM THE ORAL APPARATUS OF

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。中心体促进染色体传递的保真度，其数量和结构的异常在大多数肿瘤中都很明显。中心粒是中心体复制和组织的组成部分，是中心体组织的核心结构。纤毛和鞭毛的形成与中心粒相似，具有保守的分子组成和结构组织。新生的中心粒和基本体组装的最初事件突出表现为形成无定形的圆盘和侧翻，随后是富含微管的叶片形成桶状结构。中心粒的研究受到已知成分数量有限的限制。为了确定在组装过程中产生这些早期步骤的成分，我们将利用两种程序从四膜虫富含基体部的口腔器官中分离这些结构蛋白。最初，我们将使用增加的盐浓度来逐步提取和分离基础体蛋白。从口腔器械框架中丢失的增溶结构将通过电子显微镜进行确认。含有侧翻蛋白的组份随后将受到泥坑的影响。在第二个程序中，将从四膜虫中分离出富含生物体的生化制剂，并将其置于侧翻重组条件下。它们利用一种能够在体外进行侧翻重新组装的制剂(Gavin，1984)。重组类似于基体部和中心粒组装早期步骤的结构，并进行质谱分析(MudPIT)以确定侧翻组装所需的基体部组件。除了预测无定形的圆盘和车轮蛋白外，这些研究还将确定一些已知的和潜在的新的基体和中心粒成分。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Centrosomes promote the fidelity of chromosome transmission and aberrations in their number and structure is evident in the majority of tumors. Integral to centrosome duplication and organization, centrioles are a core structure around which centrosomes organize. Basal bodies, for the formation of cilia and flagella, are analogous to centrioles with a conserved molecular composition and structural organization. Initial events in the assembly of nascent centrioles and basal bodies are highlighted by the formation of an amorphous disk and cartwheel followed by the microtubule rich blades to form a barrel structure. Centriole study is constrained by the limited number of known components. To identify the constituents that generate these early steps in assembly, we will utilize two procedures for isolating these structural proteins from the basal body rich oral apparatus of Tetrahymena. Initially, we will use increasing salt concentrations to extract and isolate the basal body proteins in a stepwise fashion. Solubilized structures lost from the oral apparatus framework will be confirmed by electron microscopy. Fractions containing cartwheel proteins will then be subjected to MuDPIT. In the second procedure, basal body enriched, biochemical preparations will be isolated from Tetrahymena and placed into cartwheel reassembly conditions. These utilize a preparation that enables cartwheel reassembly, in vitro (Gavin, 1984). Structures resembling early steps in basal body and centriole assembly were reassembled and subjected to mass spectrometry (MudPIT) to identify basal body components necessary for cartwheel assembly. In addition to predicting amorphous disk and cartwheel proteins, these studies will identify a number of known and potentially novel basal body and centriole components.