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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。中心体促进染色体传递的保真度，其数量和结构的畸变在大多数肿瘤中是明显的。中心粒是中心体复制和组织的重要组成部分，中心体是中心体组织的核心结构。形成纤毛和鞭毛的基体类似于中心粒，具有保守的分子组成和结构组织。新生中心粒和基体组装的初始事件突出表现为形成非晶态圆盘和侧轮，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组分数量有限的限制。为了确定产生这些组装早期步骤的成分，我们将采用两种方法从四膜虫富含基底体的口腔器官中分离这些结构蛋白。首先，我们将采用增加盐浓度的方法逐步提取和分离基础体蛋白。从口腔器械框架中丢失的溶解结构将通过电子显微镜确认。含有侧翻蛋白的馏分将会受到MuDPIT的影响。在第二步中，将从四膜虫中分离出基础体和生化制剂，并置于侧轮重组条件下。这些方法利用了一种能够在体外进行侧翻重组的制剂（Gavin, 1984）。类似于基础体和中心粒组装的早期步骤的结构被重新组装，并进行质谱分析（MudPIT），以确定车轮组装所需的基础体成分。除了预测无定形圆盘蛋白和侧轮蛋白外，这些研究还将确定一些已知的和潜在的新型基底体和中心粒成分。

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.