ASSEMBLY OF BASAL AND FLAGELLA OF CHLAMYDOMONAS

衣藻基部和鞭毛的组合

• 批准号: 2177418
• 负责人: PAUL A. LEFEBVRE
• 金额: $ 13.81万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177418
• 关键词: Chlamydomonas; chickens; complementary DNA; eukaryote; flagellum; gene mutation; genetic mapping; genetic regulation; image processing; kinetosome; laboratory rabbit; molecular biology; molecular cloning; mutant; nucleic acid sequence; plant genetics; protein biosynthesis; sectioning; transposon /insertion element

This proposal seeks to take advantage of newly developed procedures for transformation and gene tagging in Chlamydomonas to study how cells regulate the assembly of basal bodies and flagella. Compared to our understanding of the assembly of singlet cytoplasmic microtubules, little is known about the assembly of the doublet and triplet microtubules which comprise basal bodies, centrioles, and flagella. A series of mutants with defective flagellar assembly will be isolated by insertional mutagenesis; transforming DNA integrates randomly in the Chlamydomonas genome, frequently generating mutants from which the affected gene can be cloned using the plasmid DNA sequence as a hybridization probe. The specific aim of this proposal is to isolate and characterize mutants of three different types. 1) Flagella-less mutants. Mutants which are viable but unable to assemble flagella have been previously described, and are easily identified by their immotile phenotype. A large number of such mutants will be collected and placed in complementation groups. The wild-type gene corresponding to each group will be cloned and the gene product will be characterized. Full length cDNAs will be isolated and sequenced, and antibodies specific to each gene product will be prepared and used for subcellular localization. This same approach will be used for two other classes of mutants. 2) Uniflagellar mutants. Four mutants, mapping to a single locus, uni1, have been shown to have a structural defect in the basal body, such that only one flagella can be assembled on most cells. The single flagellum is found almost exclusively on the older basal body of the pair, indicating that the mutant lesion may delay maturation of the flagella beyond one cell cycle. 3) Length control mutants. A number of mutants, mapping to four genes, have cells with extra-long flagella, up to four times normal length. These mutants identify an active mechanism used by cells to limit the growth of their flagella. The mutants were characterized during the current project period, and candidate insertional mutants have been identified. The molecular characterization of the gene products should permit the dissection of a mechanism whereby cells can monitor the extent of assembly of an organelle, and carefully control the assembly process. Basal bodies, centrioles and flagella are very widely distributed among eukaryotic cells, yet they are arguably the least understood organelles in terms of their mechanism of assembly. The health relevance of this research lies in the opportunity it provides to describe the assembly control mechanisms in molecular detail. These studies of assembly control in Chlamydomonas may serve to illuminate cellular homeostatic mechanisms in the same way that studies of ras homologues in a simple model system like yeast have illuminated ras function in humans.

该建议旨在利用新开发的程序 衣原体中的转化和基因标记以研究细胞如何 调节基底体和鞭毛的组装。与我们相比 了解单线细胞质微管的组装， 关于双线和三重脚的组装知之甚少 包括基底体，中心元和鞭毛的微管。一个 将隔离具有缺陷鞭毛组件的一系列突变体 通过插入诱变；转换DNA在 衣原体基因组，经常产生突变体 可以使用质粒DNA序列克隆受影响的基因作为A 杂交探针。该提议的具体目的是隔离 并表征三种不同类型的突变体。 1）无鞭毛 突变体。可行但无法组装的突变体 先前已经描述过，很容易被其Immotile识别 表型。将收集大量此类突变体 在互补组中。对应于每个的野生型基因 将克隆组，并将基因产物进行表征。满的 长度cDNA将被分离和测序，抗体特异性 每个基因产物将被准备并用于亚细胞 本土化。 同样的方法将用于其他两个类别 突变体。 2）Uniflagellar突变体。四个突变体，映射到一个 基因座uni1已显示在基础上具有结构性缺陷 身体，使大多数细胞只能组装一个鞭毛。这 单个鞭毛几乎完全在较旧的基体上发现 这对，表明突变病变可能会延迟成熟 鞭毛超过一个细胞周期。 3）长度控制突变体。一个数字 突变体的映射到四个基因，具有超长鞭毛的细胞， 最多四倍正常长度。这些突变体确定了一个活动 细胞用来限制其鞭毛生长的机制。 这 突变体在当前项目期间的特征，以及 已经确定了候选插入突变体。 分子 基因产品的表征应允许解剖 细胞可以监视组装程度的机制 细胞器，并仔细控制组装过程。基础， Centrioles和Flagella在真核生物中分布非常广泛 细胞，但可以说，它们是最不了解的细胞器 它们的组装机制。这项研究的健康相关性 这是它提供了描述装配控制的机会 分子细节的机制。这些关于装配控制的研究 衣原体可以阐明细胞稳态机制 就像在简单模型系统中研究RAS同源物的方式一样 就像酵母一样，在人类中具有照明的RAS功能。