The goals of the proposed research are to understand the function of components of the centrosome, including the centrioles/basal bodies, in the organization of the flagellar apparatus, the regulation of certain aspects of flagellar function, and the positioning of the cleavage furrow for cytokinesis. The experiments described in this proposal use Chlamydomonas genetics as well as biochemical and molecular biology approaches to probe the function of centrosomal components. Mutations in the UNI3 gene of Chlamydomonas affect basal body structure and result in cells with only one flagellum. The UNI3 gene has been isolated and characterized by Dr. Dutcher and appears to encode a potentially new member of the tubulin superfamily. Antibodies will be generated against the UNI3 gene product and will be used for protein localization studies in Chlamydomonas as well as in other organisms. Site directed mutational studies of the UNI3 gene are proposed to understand the function of the UNI3 gene product. Genetic studies to probe interactions between the UNI3 gene product and other components of the centrosome are also described. Dr. Dutcher also proposes to delete the Chlamydomonas gamma tubulin gene which she and other labs have cloned. The two flagella of Chlamydomonas are functionally distinct in several ways which is in some way related to their being templated from different basal bodies. Cellular phototaxis arises, in part, from the differential activation of specific dynein motors in the two flagella in response to a light induced signal transduction pathway. Phototactic mutants have been isolated and characterized. Several of these mutants are effected in components of the I1 inner dynein arm. Dr. Dutcher proposes experiments to test the hypothesis that these dynein motors are the targets for the asymmetric regulation of flagellar waveform during phototaxis. The phototactic defect in two new mutants isolated by Dr. Dutcher (ida7 and ida8) may be associated with an increased level of phosphorylation of a 138 kDa protein of the I1 inner dynein arm. One hypothesis is that these mutants are defective in a flagellar phosphatase enzyme which is important for regulating the phosphorylation of the 138 kDa protein. Dr. Dutcher proposes a number of experimental approaches to test this hypothesis. Specifically, a phosphatase activity is being isolated and characterized from wild type and mutant flagella. In addition, a number of new screens are proposed to characterize phototactic mutants already in hand, and new ones to be generated. These screens include one to look at flagellar waveform and another to characterize flagellar phosphatase activity in vitro. Finally, an in vitro assay of flagellar microtubule sliding is proposed to characterize dynein arm activity in mutant strains. The BLD2 gene of Chlamydomonas encodes a product essential for basal body assembly. When this gene is mutated, cells have gross defects in basal body assembly and cleavage furrow placement. Dr. Dutcher has performed a chromosome walk toward BLD2, starting with molecular markers closely linked to the gene. This walk will be continued, until recombination break points on both sides of the BLD2 gene are identified. Clones that potentially carry BLD2 sequences will be tested for their ability to rescue the bld2 mutant phenotype. Subcloning, screening of cDNA libraries, and sequencing of BLD2 carrying clones will be done to characterize the BLD2 gene. Antibodies will be made against the BLD2 gene product for immunofluorescence studies to localize the protein. New bld2 mutations, particularly nulls, will be sought, and pseudoreversion analysis will be performed to identify intra- and extragenic suppressors of the original bld2 mutant. As well as characterizing the flagellar phenotypes of these new strains, the placement of the cleavage furrow in these cells will be studied. Finally, the function of centrin will be tested. Centrin is a calcium binding protein which plays a role in positioning and segregating basal bodies, in microtubule severing, and in flagellar motility. To understand better the role of centrin in the cell, new conditional mutant alleles will be generated by site directed mutagenesis of specific regions of the centrin gene (VFL2). Pseudoreversion analysis will be done to identify suppressors of conditional centrin alleles which may identify components of the centrosome which physically interact with centrin. Also, the yeast two-hybrid screen will be performed, using the full length centrin cDNA and two deletion derivatives fused to the yeast Gal4p DNA binding domain as """"""""baits"""""""" and Chlamydomonas cDNAs fused to the transcription activation domain of Gal4p as """"""""prey."""""""" Gal4p activation of downstream reporter sequences will be used to identify cDNA clones which encode gene products which physically interact with centrin.
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