BASAL BODY DUPLICATION IN CHLAMYDOMONAS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人们对基本体和中心粒的组装知之甚少。为了更多地了解复制过程，细胞的生长和分裂周期可以通过光和暗的周期来同步，以检查基底复制的早期步骤。此外，我们还有两个可能特别感兴趣的突变体。第一种是野生型基体底部有无定形环的菌株，但突变型基体不组装微管。因此，研究早期步骤的特征是很重要的。第二种是RNA干扰菌株，其富含叶氨酸的重复蛋白的mRNA显著减少；它制造了太多的基底。该蛋白似乎是基底体/中心粒复制的负调节因子，在基底体分离中没有缺陷。 同步应变的特征将使用高压冷冻和冷冻替代材料，并嵌入塑料中，在连续的断层重建中重建整个基底复合体。到目前为止，已经有4张有丝分裂细胞的系列断层图像显示了发育中的前基底小体。它们的特征是单线微管的环，类似于草履虫中所描述的。此外，还将切割冷冻水合材料的冰冻切片，并对冰冻断层图像进行成像，以接近自然状态的方式获取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.