The long-term goal of our research remains to exploit budding yeast, Saccharomyces cerevisiae, as a model for studying eukaryotic cell biology. Since the sequencing of its genome in 1996, it has become the leading model system for functional genomics. As progress is made in defining the core biological processes (and genes and proteins that carry them out), attention has begun to shift to understanding how they are regulated and coordinated so as to allow cells to maintain homeostasis over a staggering variety of alternative and often rapidly changing environmental circumstances, often referred to as "system-level biology". Our goals are to develop and apply combinations of genomic and classical genetic as well as microbial physiology approaches in order to gain understanding of the systematic organizational features that allow yeast to maintain homeostasis when challenged by massive or subtle changes in their circumstances. The specific aims proposed have in common experimental designs and methods, some newly developed in our laboratory, that combine genome-scale (e.g. DNA micro arrays) technology with more classical physiological (e.g. chemostats) and genetic (e.g. mutant and suppressor screens, experimental evolution) approaches. They are: (1) to characterize genome-wide gene expression changes associated with the maintenance of nutritional homeostasis in the face of differing and/or changing environmental conditions. (2) To study the response of yeast to perturbations in the efficiency or activity of specific essential subcellular structures or systems, such as the actin and tubulin cytoskeletons, caused by well-characterized temperature-sensitive mutations. (3) To continue to study pathway structure and 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. (4) To continue development of technology that will allow better detection, quantitation, mapping and recovery of mutations that contribute to fitness, be they the result of experimental evolution or more classical genetic methods (e.g. suppressor or synthetic lethal screens). [unreadable] [unreadable]