The long term goals are to understand the basic mechanism of dynein force production, to determine the roles of individual outer arm dynein subunits in flagellar movement, and to learn how dynein is assembled at the correct places in the flagellar axoneme. These studies will use Chlamydomonas flagellar dynein as a model system, because Chlamydomonas is the only organism available that allows a combination of classical genetic, molecular genetic, biochemical, and physiological approaches to be brought to bear on these important questions. Recent molecular genetic studies have identified several motifs and regions, including four P loops and a region proposed to be involved in ATP-dependent microtubule binding, that are highly conserved in all dynein heavy chains (DHCs). To learn more about the roles of these highly conserved regions, they will be modified by site-directed mutagenesis of a gamma DHC minigene. The mutated minigenes will then be transformed into a gamma-DHC null mutant and the expressed modified subunits isolated and characterized in vitro with regard to their ATP-hydrolytic, ATP-binding, microtubule-binding, and force-generating properties. The results will provide the first definitive information on the functions of those regions most likely to be part of the DHC motor domain. To determine the roles of each of the three different DHCs in the Chlamydomonas outer arm, a set of 3 mutants, each of which is defective for a different DHC, will be studied to determine how loss of a particular chain affects interdoublet sliding and axonemal bending in vivo. In addition, the individual subunits will be isolated and their force- generating properties examined in vitro under various conditions. The results will indicate if each subunit is capable of force generation, and if so, if it functions at all times or only during certain behavioral responses. A 70 kDa axonemal protein has been identified that may determine the site to which the outer arm binds. A cDNA encoding this protein will be cloned. Studies will be carried oat to determine how the protein interacts with the dynein, and how it is bound to the doublet microtubule. Other proteins essential for outer arm assembly will be identified by the cloning of tagged genes from new outer arm-less strains generated by insertional mutagenesis. There are multiple species of inner arm dynein, and they may be differentially distributed along the axoneme. Definitive characterization of inner arm organization has been precluded by the lack of specific probes for different inner arm species. Cloned DNAs encoding different inner arm DHCs will be used to generate fusion proteins, which in turn will be used to make antibodies to determine the distribution of the inner arm DHCs along the axoneme. Because the basic structure of DHCs has been conserved throughout evolution, the fundamental information obtained from these studies will be applicable to the dyneins of all organisms, including man. Consequently, knowledge derived from these studies will increase our understanding of human diseases such as immotile cilia syndrome, in which the dynein arms are frequently lacking. The studies also will provide a basis for understanding such important processes as sperm maturation and capacitation, which are necessary for fertilization and which ultimately must involve changes in the functioning of the dynein arms.
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