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Gene Expression and Signaling Pathways in Yeast . Classical Genetics Genotype: The genetic makeup of an organism Phenotype: The observed “behavior” Basic Assumption: Genotype phenotype Environmental Responses (Yeast) Condition Response Pheromone “smooing” (mating) Filamentous growth (foraging) Nutrient depletion Osmotic stress (high salt concentration) Glycerol production, cell wall repair Cell wall damage Cell wall repair Genetic Screens Search for genes whose removal (KO) disrupt desired response Classical genetic screens identified many genes that are involved in yeast response to environmental cues. Can we understand these processes better? Central Dogma Regulation of expression of genes is crucial Understanding regulatory processes is a central problem of biological research Transcription Translation RNA Protein Measuring RNA production Sample 1 >> Sample2 Sample 1 << Sample2 Sample 1 Sample2 Yeast Signaling Pathways Goals Comprehensive map of genes involved in different responses Understanding the function of different components in the pathway Meta-goal: Utility of gene expression for studying signaling pathways Yeast Mating MATa haploid Pheromone a-factor Secreted by MATa Sensed by MATα α-factor Secreted by MATα Sensed by MATa MATα haploid Diploid Genes Involved in Mating Response MATa +α-factor (50nM for 30min) vs MATa Each dot – average of 4 repeats Statistical test finds • > 200 genes induced (red) • > 200 genes repressed (green) Does Dosage Matter? Strong correlation Results are reproducible Ste2 Ste2 is a necessary receptor for pheromone response Ste12 The whole response appears to be derived from pathwaydependent activation of Ste12 Far1 What is the relation between Ste12 and Far1 ?? Essentially all gene repression requires Far1 Most of these genes required for G1 cell cycle phase Rst1/2 (Dig1/2) Rst1/2 KO induces genes involved in both pheromone response and filamentous growth Pathway interference Global View Goals Closer look at HOG1 pathway Activity of HOG1 Roles of components upstream of HOG1 Hyperosmotic Stress High salt solution (or other osmolyte) water drawn out of the cell; salt enters the cell Cells shrink: damage to cell wall and membrane; increased protein concentration; problems with pressure for budding etc. Increased salt concentration: changes in protein/protein and protein/DNA interactions Hyperosmotic Stress Response Production of an osmolyte: Glycerol is synthesized to balance osmolarity inside/outside Change in metabolism to accommodate this Removal of salt: Upregulation/downregulation of appropriate transporters for NaCl, KCl and other ions Dealing with stress: Expression of chaperones and other general stress response genes Shutting down non-essential stuff HOG1 pathway cytoplasm General Stress Factors Pbs2 Hog1 Smp1 Msn1 Hog1pp Sko1 Hot1 Msn2/4 ? Msn2/4 Nucleus Step1: Calibrating the System Maximal change Hog1 dependent genes 579 Hog1 dependent genes 2-fold response to KCl 3-fold response to Hog1Δ Both in at least two time points (Note differences from previous paper) Hog1 dependent genes Induced in Hog1Δ Repressed by Hog1 Genes of mating and filamentous growth pathways FUS1, STE2, TEK1, PGU1,… Hog1 dependent genes Repressed in Hog1Δ Induced by Hog1 Either with or without KCl Hog1 dependent genes Expression in Hog1Δ changes in later stages Require Hog1 for late stage recovery Mostly secondary effects Teasing Out Components Hog1Δ = Pbs2 Δ Teasing Out Components Hog1Δ = Pbs2 Δ Ssk1Δ + Ste11Δ • Additional inputs to Pbs2? • Changes in pheromone pathway (Ste11) Teasing Out Components Ssk1Δ + Ste11Δ Ssk1Δ + Sho1Δ • Sho1 “leaker” than Ste11 • Additional inputs to Ste11? Teasing Out Components WT Ssk1Δ Ste11Δ Sho1Δ • Single KO have little effect on response • Are these receptor redundant? Redundancy and dosage