The ability to adapt to osmotic stress is an essential process in all living systems. Phylogenetically diverse organisms adapt to osmotic stress by using a conserved mechanisms: the accumulation of intracellular """"""""compatible solutes"""""""" to prevent the loss of cellular water. It is also reasonable to assume that the expression of many genes is modulated in response to hyperosmotic stress. Cellular responses to high osmolarity are also of key importance in renal physiology in higher organisms, including man. For example, mammalian renal medullary cells must cope with interstitial NaCl and urea concentrations that increase greatly and vary widely during anti-diuresis. The long-term objective of this research is to examine the physiology of osmotic adaptation in the eukaryotic yeast Saccharomyces cerevisiae with the goal developing a biologically and relevant, microbial model of renal cell function. Research described in this proposal seeks to test the hypothesis that cellular responses observed in yeast are analogous to the responses known to occur in renal cells. We chose to concentrate our efforts on the metabolism of choline during osmotic stress and the osmo- protective role played by this compound and its derivatives betaine and glycerophosphorylcholine (GPC). To that effect, three specific aims will be pursued. 1) Determination of the osmo-balancing roles played by betaine, choline and/or GPC during hyperosmotic stress. A combination of HPLC, osmometer and glycerol assays will be used to determine the relative concentration of choline and its derivatives (GPC and betaine) in the osmotically stressed cell. 2) Determination of betaine biosynthetic activity in yeast. Yeast contain detectable amounts of betaine that increase in osmotically stressed cells supplemented with choline. We will conduct in vivo and in vitro assays to determine the presence of betaine biosynthetic activity. 3) Analysis of ALD gene expression and function. We will use Northern analysis and phenotypic characterization of deletion in ALD genes by examining growth under osmotic stress, betaine synthesis and HPLC analysis. Students will be trained in microbial physiology and molecular biology using a technique such as HPLC, microscopy, enzyme assays, radioactive assays, PCR, Northern blotting. Students will be encouraged to participate in the formulation of hypotheses, design of experiments and data analysis to enable them to successfully pursue careers in biomedical sciences.
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