BASAL BODY DUPLICATION IN CHLAMYDOMONAS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 对基体和中心粒的组装知之甚少。 为了了解更多关于复制过程的信息，细胞生长和分裂周期可以通过光和暗的周期来同步，以检查基体复制的早期步骤。 此外，我们还有两个可能特别感兴趣的突变体。第一种是具有存在于野生型基体基部的无定形环的菌株，但突变基体不组装微管。因此，它的特性是重要的，看看早期的步骤。 第二种是RNA干扰菌株，其具有显著减少的富含亮氨酸的重复蛋白的mRNA;其产生太多的基体。 这种蛋白质似乎作为一个负调节器的基体/中心粒复制，并没有缺陷的基体分离。 将使用高压冷冻和冷冻替代并嵌入塑料中的材料表征同步菌株，以在连续断层扫描重建中重建整个基体复合体。迄今为止，4个系列断层扫描的有丝分裂细胞已成像，揭示发展probasal机构。其特征是单线态微管环，类似于草履虫中所描述的。此外，冷冻水合材料的冷冻切片也将被切割，冷冻断层图像将被成像，以接近天然状态的方式获得复制中间体的详细信息。

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. Little is known about the assembly of basal bodies and centrioles. To understand more about the duplication process, the cell growth and division cycle can be synchronized by cycles of light and dark to examine the early steps in basal body duplication. In addition, we have two mutants that may be of particular interest. The first is a strain that has the amorphous ring that is present at the base of wild-type basal bodies, but the mutant basal bodies do not assemble microtubules. Thus, its characterization is important to look at early steps. The second is an RNA interference strain that has significant reduction in the mRNA for a luecine rich repeat protein; it makes too many basal bodies. This protein appears to act as a negative regulator of basal body/centriole duplication and is not defective in basal body segregation. Synchronized strains will be characterized using material that has been high pressure frozen and freeze-substituted and embedded in plastic to reconstruct the entire basal body complex in serial tomographic reconstructions. To date, 4 serial tomograms of mitotic cells have been imaged that reveal developing probasal bodies. These are characterized by rings of singlet microtubules, similar to what has been described in Paramecium. In addition, cryosections of frozen hydrated material will also be cut and cryotomograms will be imaged to get detailed information of duplication intermediates in a manner close to the native state.