The microtubule organizing center of eukaryotic cells is responsible for the nucleation and organization of microtubules. The microtubule organizing center has two distinct morphological components; they are the centrioles and the pericentriolar material. The role of centrioles and their structural equivalent basal bodies are not well understood. Centrioles and basal bodies are morphologically and biochemically complex. Basal bodies are needed for the assembly of cilia and flagella. Cilia line the nasopharynx, trachea, and bronchi to move mucus to remove contaminants from the lungs and play an important role in reproduction in the oviduct and in the movement of the sperm. During embryogenesis, motile cilia are needed for determination of the left-right axis during development. Centrioles are also needed for the current positioning of the cleavage furrow during cytokinesis in Chlamydomonas. To understand the mechanisms of assembly as well as the function of these organelles, a genetic dissection will be continued to characterize mutations that disrupt the assembly of basal bodies substructures in Chlamydomonas. Delta-tubulin, the newest member of the tubulin superfamily, is required for the assembly of triplet microtubules and is localized to basal bodies and centrioles. The isolation of suppressors and enhancers of tud1 mutations will identify genes whose products interact with delta-tubulin. The BLD2 gene is also required for the assembly of basal bodies. Several complementary approaches will be taken to clone the BLD2 gene. The Bldg gene product will be localized in the cell and its role in the cell cycle examined. Mutant strains with structurally abnormal basal bodies suggest a role for basal bodies in some aspect of microtubule dynamics and stability. A collection of mutants will be characterized with cell and molecular techniques to probe the role of basal bodies in regulating cellular and spindle microtubules. Serial section electron microscopy and two-dimensional gel electrophoresis will be used to begin a correlation of substructures in the basal body and their biochemical composition that will be used to identify genes in the future.
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