REGULATION OF FLAGELLAR ASSEMBLY IN CHLAMYDOMONAS

该提案描述了一个项目，以了解细胞如何调节一个复杂的，含有微管的细胞器，鞭毛的组装。虽然调控组装的单线态微管，如在有丝分裂纺锤体，已被深入研究，几乎没有什么是已知的细胞如何调节组装更稳定的双微管纤毛和鞭毛，或三重微管的基体和中心粒。我们将使用不能正确控制鞭毛长度的突变体作为切入点来剖析鞭毛组装的调节。四个不相关基因的突变已被证明会导致细胞失去对鞭毛长度的控制。 四个不相关基因的突变已被证明会导致细胞失去鞭毛长度控制。 突变体的鞭毛长到正常长度的四倍。矛盾的是，这些突变体中的一些在截肢后鞭毛的再生长中表现出严重的缺陷。在前一个项目期间，我们克隆了其中三个基因，LF 2，LF 3和LF 4。该提案描述了使用广泛的技术来表征LF基因、它们的蛋白质产物以及它们与之相互作用以控制鞭毛长度的蛋白质的实验。将克隆LF 1基因，并对所有四个LF基因的基因和全长cDNA进行测序。蛋白质产物将定位于野生型和突变型细胞中。强大的抑制筛选将用于分离每个基因的新的突变等位基因，其特定目标是识别用于交互式抑制筛选和结构/功能研究的错义突变。分子和遗传技术将用于识别和表征在鞭毛长度调节中与LF基因产物相互作用的蛋白质。鉴于纤毛和鞭毛的结构和蛋白质组成的高度保守性，该项目应该为人类这些细胞器的组装提供有用的见解。

This proposal describes a project to learn how cells regulate the assembly of a complex, microtubule-containing organelle, the flagellum. Although the regulation of assembly of singlet microtubules, such as those in the mitotic spindle, has been intensively studied, almost nothing is known about how cells regulate the assembly of the more stable doublet microtubules of cilia and flagella, or the triplet microtubules of the basal bodies and centrioles. We will use mutants which do not properly control their flagellar length as an entry point to dissect the regulation of flagellar assembly. Mutation in four unlinked genes have been shown to cause cells to lose flagellar length control. The mutations in four unlinked genes have been shown to cause cells to lose flagellar length control. The mutants grow flagella of up to four times normal length. A number of these mutants, paradoxically, show severe defects in the regrowth of flagella after amputation. During the preceding project period we cloned three of these genes, LF2, LF3 and LF4. This proposal describes experiments to use a broad range of techniques to characterize the LF genes, their protein products and the proteins with which they interact to control flagellar length. The LF1 gene will be cloned, and the gens and full length cDNAs for all four LF genes will be sequenced. The protein products will be localized in wild-type and mutant cells. Powerful suppressor screens will be used to isolate new mutant alleles of each of the genes, with a particular goal of identifying missense mutations for interactive suppressor screens and for structure/function studies Both molecular and genetic techniques will be used to identify and characterize proteins which interact with the LF gene products in the regulation of flagellar length. Given the high degree of conservation of both the structure and protein composition of cilia and flagella, this project should provide useful insights into the assembly of these organelles in humans.