Yeast cells respond to an increase in external osmolarity by large scale changes in gene expression (1, 5, 7, 10, 11). The signal transduction pathways, including the HOG MAP kinase cascade (6), that mediate this response are fairly well understood. In contrast, almost nothing is known about how yeast cells, adapted to a hypertonic environment, respond to the hypotonic shock of a decrease in external osmolarity. Imagine the reaction of yeast growing on a drying grape when it suddenly starts raining!
Of possible responses to hypotonic shock are changes in expression of certain cell wall proteins, making the cell wall tougher, i.e., hard to burst. The goal of this proposed analysis is to determine the effect of hypotonic shock on yeast gene expression at the level of the whole genome, focusing on the role of the osmosensor Sln1 in mediating gene expression responses. Although most prior investigations of Sln1 show its role in response to hypertonic stress (4, 8), there is clear data suggesting its role in hypotonic responses as well (9, 12). Intriguingly, a limited study of gene expression (2) shows that Sln1 regulates expression of a few cell wall genes.
The analysis proposed here involves two pairs of different conditions, i.e., two arrays. Depending on the level of interest and the number of available arrays, these two pairs (arrays) can be independently pursued or done together by one group.
Array 1 procedure: a culture containing a single wild type strain (w303) is grown to log phase in 0.4x YEPD plus 1.2 M sorbitol and the culture split into equal volumes. One culture (the experimental, hypotonic shock culture) is diluted 1:8 into a flask containing 0.4x YEPD medium, the control culture diluted 1:8 into 0.4x YEPD plus 1.2 M sorbitol. The experimental culture will thus be exposed to a ~5x decrease in medium osmolarity with little or no change in basic nutrient levels. After 10 min, the cultures are centrifuged, RNA extracted from each cell pellet, each RNA separately labeled and mixed together for analysis of hybridization to a single genomic DNA array.
Array 2 procedure: two strains are each exposed to hypotonic shock (described above) and the relative gene expression compared between the two strains. The two strains are ssk1D and ssk1D sln1D, respectively. Separate cultures for each strain will be grown to a similar density in 0.2x YEPD plus 1.5 M sorbitol and then diluted 10x into medium containing 0.2x YEPD. After 5 min, the cultures are centrifuged, RNA extracted from each cell pellet, each RNA separately labeled and mixed together for analysis of hybridization to a single genomic DNA array. This array-based analysis will test whether there is Sln1-dependent expression of specific genes following hypotonic shock. Use of the ssk1D genetic background is required here because Sln1 has two separable functions: negatively regulating the HOG MAP kinase pathway (3, 4) and positively regulating cell wall gene expression (2). Without Sln1, cells constitutively activate the HOG MAP kinase pathway and die. Ssk1 is required for HOG MAP kinase activation in the absence of Sln1 (3) and therefore the ssk1D sln1D strain is fully viable. Ssk1 is not required for Sln1-dependent expression of cell wall genes (2).
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2. Li, S., S. Dean, Z. Li, J. Horecka, R. J. Deschenes, and J. S. Fassler. 2002. The eukaryotic two-component histidine kinase Sln1p regulates OCH1 via the transcription factor, Skn7p. Mol Biol Cell 13:412-24.
3. Maeda, T., M. Takekawa, and H. Saito. 1995. Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3- containing osmosensor. Science 269:554-8.
4. Maeda, T., S. M. Wurgler-Murphy, and H. Saito. 1994. A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369:242-5.
5. O'Rourke, S. M., and I. Herskowitz. 2003. Unique and redundant roles for HOG MAPK pathway components as revealed by whole-genome expression analysis. Mol Biol Cell.
6. O'Rourke, S. M., I. Herskowitz, and E. K. O'Shea. 2002. Yeast go the whole HOG for the hyperosmotic response. Trends Genet 18:405-12.
7. Posas, F., J. R. Chambers, J. A. Heyman, J. P. Hoeffler, E. de Nadal, and J. Arino. 2000. The transcriptional response of yeast to saline stress. J Biol Chem 275:17249-55.
8. Posas, F., S. M. Wurgler-Murphy, T. Maeda, E. A. Witten, T. C. Thai, and H. Saito. 1996. Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 "two-component" osmosensor. Cell 86:865-75.
9. Reiser, V., D. C. Raitt, and H. Saito. 2003. Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure. J Cell Biol 161:1035-40.
10. Rep, M., M. Krantz, J. M. Thevelein, and S. Hohmann. 2000. The transcriptional response of Saccharomyces cerevisiae to osmotic shock. Hot1p and Msn2p/Msn4p are required for the induction of subsets of high osmolarity glycerol pathway-dependent genes. J Biol Chem 275:8290-300.
11. Rep, M., M. Proft, F. Remize, M. Tamas, R. Serrano, J. M. Thevelein, and S. Hohmann. 2001. The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage. Mol Microbiol 40:1067-83.
12. Tao, W., R. J. Deschenes, and J. S. Fassler. 1999. Intracellular glycerol levels modulate the activity of Sln1p, a Saccharomyces cerevisiae two-component regulator. J Biol Chem 274:360-7.
and Intended Use
Created by B. Beason (email@example.com), Rice University, 29 February 2004
Updated 20 July 2006