CONTROL OF GENE ACTIVITY AND ORGANELLE BIOGENESIS

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The long-term goals of this proposal are to understand the molecular mechanisms underlying the biogenesis of cilia and their relationship to cell-cycle progression. The lower eukaryote, Tetrahymena thermophila is used as a model. A screen has been developed that yields conditional (ts) mutations in cilia regeneration, a surprisingly high percentage (2/5) of which are cell cycle arrest mutants. The first two of these mutants to be studied can be rescued by cytoplasmic exchange. A microinjection assay will be used to purify mutant gene products. Mutant genes will be cloned and used to develop a DNA-mediated transformation system that will, in turn, allow characterization of a large number of genes after cloning by complementation. Mutants affecting the single alpha-tubulin gene and extragenic revertants of mutations affecting cilia biogenesis and the cell cycle also will be isolated and characterized. A detailed analysis of alpha- and beta-tubulin gene organization and expression will be performed. By isolating and characterizing a large number of normal and mutant genes important in both cilia biogenesis and cell cycle regulation, it should be possible to elucidate mechanisms underlying both normal cytoskeletal function and cell cycle progression, events that frequently are abnormal in neoplastic cells.