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.
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