The long term goals are to understand the basic structure, assembly, function and regulation of flagellar outer arm dynein and the unusual cytoplasmic dynein DHC1b, which is essential for flagellar assembly. Chlamydomonas will be used as the model system for these studies because it allows a combination of genetic, molecular genetic, biochemical, and physiological approaches to be brought to bear on the problems being investigated. The outer dynein arm docking complex (ODA-DC), a recently discovered structure necessary for the outer arm to bind to the doublet microtubules, will be studied by scanning transmission electron microscopy and conventional electron microscopy to determine its structure in solution and in situ. Protein cross- linking studies are proposed to determine how the ODA-DC polypeptides interact with one another, and how they bind to the doublet microtubule and the outer arm. The cloning of the ODA10 gene, which is essential for outer arm assembly, will be completed; its product will be studied to determine its relationship to the outer dynein arm and the ODA-DC. The genes ODA5 and ODA8, the products of which are believed to interact with that of ODA10 to form a complex, will be similarly cloned and analyzed. Immunofluorescence microscopy will be used to determine the distribution of DHC1b in the cell body. A temperature-sensitive allele of the DHC1b gene will be isolated and exploited to test the hypothesis that DHC1b is the retrograde motor for intraflagellar transport (IFT), a movement of particles up and down the flagellum that is essential for flagellar assembly. The DHC1b dynein will be purified, its polypeptide composition determined, and its functional states investigated. Mutants with defects in retrograde IFT will be isolated and studied to learn more about the proteins essential for DHC1b function. Site-directed mutagenesis will be carried out to test the hypotheses that the first P loop of the gamma dynein heavy chain of outer arm dynein hydrolyzes ATP to generate force, and that at least one of the other P loops is an ATP-binding regulatory site that controls the gamma subunit's ATPase and force-generating activities. The results will provide a basis for understanding a wide range of human diseases, including immotile cilia syndrome, in which the dynein arms often are missing from the ciliary and flagellar axonemes, and diseases such as retinitis pigmentosa and Usher syndrome, which involve degeneration of cells in the retina and inner ear that have modified cilia.
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