CONTROL OF GENE ACTIVITY AND ORGANELLE BIOGENESIS

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Microtubules are ubiquitous in eukaryotic cells where they form diverse organelles involved in cell shape, cell motility, intracellular movements mitosis and meiosis. Among the most elaborate microtublar organelles are cilia and flagella, which, in humans, are found in the respiratory tract and the female reproductive tract, tissues with high incidences of cancer. Ciliary dysfunction can cause respiratory and fertility problems. The long term goals of this proposal are to understand the molecular mechanisms underlying microtubule diversity, with particular emphasis on the biogenesis and function of cilia. Hypotheses concerning the role of tubulin primary sequences, posttranslational modifications, microtubule organizing centers and microtubule-associated proteins in generating microtubule diversity will be tested. The lower eukaryote, Tetrahymena thermophila, is used as a model. Advantages of this system include the relatively small tubulin multigene superfamily and the recent development of methods for mass transformation of somatic and germinal nuclei enabling gene replacement and gene disruption. These methods will be used for a detailed mutagenic analysis of the function of alpha-and-beta-and gamma-tubulin and of their secondary modifications. The function of a newly discovered, unusually diverse beta-tubulin will be analyzed. Genes encoding microtubule associated proteins that affect microtubule stability in vivo will be clones, sequenced and their protein products will be characterized. 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 division, events that are critical for normal development and that are frequently abnormal in neoplastic cells.