We are analyzing mutations that affect the function, the morphogenesis, and the inheritance of basal bodies with genetic, biochemical, immunological, and molecular techniques. Basal bodies or centrioles have been postulated to play a role in flagellar assembly and movement, chromosome segregation, cytokinesis, and the establishment of cell polarity. The isolation of mutations affecting basal bodies allows us to determine the processes that require intact basal bodies. Conversely, the isolation of mutations in these various cellular processes permit us to ask if basal bodies are affected. We identify loci by two formally different approaches. The first is the isolation of mutants that fail to complete these apparently basal body dependent processes correctly. The second is the isolation of mutants that interact with known basal body mutants are suppressors or dominant enhancers of the mutant phenotypes. The analysis of mutants will be facilitated by the isolation of a set of mutations in Chlamydomonas that permit mitotic diploid strains to re-enter the meiotic pathway. These mutations, which are defective in nuclear fusion, can be used to recover new mutant alleles and deletion mutations at identified loci. Genes that we identify by these approaches are assayed in several ways. We examine the mutant cells for changes in the polypeptide composition of isolated basal bodies and flagellar axonemes, for changes in the swimming patterns by light microscopy, for changes in the fidelity of chromosome segregation, and for changes in cellular organization by immunofluorescence and differential interference contrast light microscopy. We also study the inheritance of a new and unusual linkage group, linkage group XIX, that shows clustering of flagellar and basal body mutants and has a circular genetic map. To physically analyze this unusual chromosome, we are pursuing three molecular and immunological approaches. First, we are refining a DNA transformation system for Chlamydomonas to increase the frequency of transformation. We are attempting to isolate monoclonal antibodies against basal bodies polypeptides that we identify as the gene products of linkage group XIX loci. We will use these antibodies in conjugation with expression libraries in the bacteriophage Lambdagtll to isolate the Chlamydomonas coding sequences for these antigenic determinants. Thirdly, we are beginning to isolate circular species of DNA to look for the physical counterpart to the circular genetic map of linkage group XIX.
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