CONTROL OF GENE ACTIVITY AND ORGANELLE BIOGENESIS

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Cilia and flagella are complex microtubular organelles that are nearly ubiquitous among eukaryotes. In humans they are found in the respiratory tract and in the female reproductive tract tissues with high incidences of cancer. Also in these tissues ciliary dysfunction can cause respiratory and fertility problems. The long-term goals of this proposal are to understand the molecular mechanisms underlying the biogenesis and function of cilia. The lower eukaryote, Tetrahymena thermophila, is used as a model. Advantages of this system include our recent findings that there is only one type each of alpha- and beta- tubulin proteins encoded by a single alpha and two beta genes, coupled with our newly developed methods for high frequency transformation, gene replacement and gene disruption using electroporation. These methods will allow extensive analysis of the function of alpha- and beta- tubulin and of their secondary modifications in cilia. Using newly developed vectors, genes encoding proteins essential for cilia formation and/or ciliary motility will be cloned by complementation of mutant function and sequenced; their protein products will be characterized and localized by in situ immunochemical methods. Because two of our initial three ciliary mutants also have a cell cycle phenotype, these studies should not only shed light on normal cilia biogenesis and the function of tubulins, but also on interrelationships between cytoskeletal functions and cell cycle progression, events that are critical for normal development and that are frequently abnormal in neoplastic cells.