This proposal describes experiments to continue a genetic analysis of the regulation of the synthesis and assembly of the flagellar apparatus of the unicellular green alga Chlamydomonas reinhardtii. Three interrelated areas of research are described. (1) Long flagella mutants. Ten different mutants, mapping to three unlinked genes (lfl, lf2, and lf3) cause the flagella of Chlamydomonas to grow to excess length. Many new long flagella mutants will be isolated, to saturate genetically the loci regulating flagellar length. Revertants and temperature conditional pseudorevertants will be isolated for mutants at each locus, to identify suppressor mutations acting on long flagella mutants. Experiments are described to identify the gene products of the three known genes, and to characterize any ultrastructural defects in the flagella or basal bodies caused by the lf mutations. (2) Characterizing the Uni linkage group. The Uni linkage group (ULG) of Chlamydomonas is defined by a large number of genes involved in flagellar and basal body assembly and function. This concentration of genes affecting the flagellar apparatus, plus the unusual genetic properties of the linkage group, such as genetic circularity, make it an attractive subject for molecular analysis. Experiments to isolate and characterize genomic DNA clones from the Uni linkage group are described, taking advantage of newly described herbicide- resistance mutations which map to a single locus near unil on the ULG. (3) Synthesis defective mutants. Experiments are described to continue a genetic analysis of a number of mutants which are defective in the regulation of synthesis of flagellar proteins. The number of genetic loci which can cause this unusual defect, and their interactions, will be determined by linkage and complementation analysis. The health relatedness of this research arises from the conservation of flagellar and basal body structure and biochemistry among eukaryotes. This research will lead to a greater understanding of the assembly of these essential organelles, and the regulation of gene expression which produces flagellar proteins. Basal bodies are integral parts of the centrosome, and so may have an important role in the processes of mitosis and cytoskeleton assembly. Molecular clones obtained from the Uni linkage group may facilitate the molecular analysis of basal body function in higher organisms.
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