The long-term goal of our research is to exploit budding yeast, Saccharomyces cerevisiae, as a model system for studying eukaryotic cell biology. It has been clear for many years that a major opportunity for yeast research is to fill out the """"""""parts list"""""""" for the core biological functions common to eukaryotes. The central focus for all the experiments remains understanding of the roles of the tubulin and actin cytoskeletons in maintaining the internal architecture of the cell as it grows and goes through the various stages of its life cycle. It is now well understood that the major cytoskeletal proteins are highly conserved among all eukaryotes, including the human, and that among their number are many whose homologs have been implicated in human disease. In addition to the application of classical genetic and cell biology methods, several of which were developed previously as part of this program, it is now proposed to exploit the complete genome sequence of this yeast by using DNA microarrays to follow patterns of gene expression.
The specific aims are: (1) To extend our program of associating yeast genes with cellular processes by following patterns of gene expression under diverse, but rigorously controlled, growth conditions in chemostats. Specific drugs or mutations that affect a broad range of different intracellular processes will be used to cause growth limitation. The effects of these diverse growth limitations on gene expression will then be used to infer functional and regulatory roles of individual genes. (2) To study pathway structure, regulatory interactions and networks systematically by allowing yeast to undergo adaptive evolution over hundreds of generations of steady state growth in defined media in chemostats. Adaptive evolution will be studied not only with respect to nutrition, but also with respect to limitations in particular cellular processes through the use of specific, stable deletion mutations in genes that impair the function of the cytoskeleton. (3) To study the effect of stable deletion mutations affecting sequence and/or chromosome stability on adaptive evolution in chemostats. These mutations are homologs of human genes in which defects are known to cause cancer or premature aging. (4) To pursue further ongoing studies of structure/function relationships in the tubulin and actin cytoskeletons. (5) To continue systematic, immuno-electron microscopy and real-time imaging characterization of the morphology, stability, and movements of subcellular organelles and cytoskeletal structures of yeast.
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