Post on 21-Dec-2015
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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)
Nutrient depletion Filamentous growth (foraging)
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
Transcription
RNA
Translation
Protein
Regulation of expression of genes is crucial Understanding regulatory processes is a central
problem of biological research
Measuring RNA production
Sample 1 >> Sample2
Sample 1 << Sample2Sample 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
Pheromone a-factor
Secreted by MATa Sensed by MATα
α-factor Secreted by MATα Sensed by MATa
MATa haploid 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 pathway-dependent activation of Ste12
Far1
Essentially all gene repression requires Far1
Most of these genes required for G1 cell cycle phase
What is the relation between Ste12 and Far1 ??
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
Pbs2
Hog1
Hog1pp
cytoplasm
Nucleus
Smp1Msn1
Sko1 Hot1 Msn2/4
?
Msn2/4
GeneralStressFactors
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)
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
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