MICR 302 Lecture Notes - Lecture 14: Pheromone, Mating Of Yeast, Fus3
1 May 2018
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Lecture 14
S. cerevisiae lifecylcle:
1. a mating event happens between two mating types: Mat-a and Mat-alpha (almost genetically
identical, physiologically are identical)
2. they mate through conjugation
-cell wall and PM fuse, contents and the nuclei fuse and you go from 1n to 2n chromosomal
genes)
-NOTE: this is the preferred form, THIS IS DIPLOID (ensures there are two copies of the
genes incase one gets destroyed)
3. if it finds itself in an environment without fermentable carbon or no nitrogen, it will undergo
sporulation
4. sporulation results in 2 Mat-a and 2 Mat-alpha HAPLOID cells packaged into an ASCUS
-very robust cell wall that ensures they last indefinitely, also keeps spores close for when they
need to mate.
Mating is co-ordinated by Pheromones
1. Shoo Formation
-since yeast are non-motile they grow in a polarized way up the concentration gradient
towards production of peptides
-Mat-a produce Ste2 receptors that receive alpha-factors form mat-alpha cells
-Mat-alpha produce Ste3 receptors that receive a-factors from mat-a cells
2. Membrane Fusion
3. Nuclear migration and fusion
NOTE: there is a species of wasps that eat these spores and in the stomach of the wasp exist
gucanases that break down the ascus and liberate the haploids.
—> gut of wasp = nightclub for yeast to mingle that would normally never see each other which
introduces genetic diversity
Pheromone Response Pathway
1. GPCRs
-7 transmembrane regions
-activated PAK, MEKK, MEK and then MAPK (these are diffusible and phsohprylate targets to
change their activity)
-a scaffold keeps things together to help amplify the signal (kinases in close proximity)
2. MAPK pathway
-massive amplification of signals
-a series of phosphryl-events create a pool of activated MAPK to hit targets depending on
events (HITS FUS3)
3. Singling
a) specificity
b) amplification
c) attenuation
GPCR (7 transmembrane receptors)
-diverse processes
-all eukaryotic
-popular drug targets (40% of pharmaceuticals
find more resources at oneclass.com
find more resources at oneclass.com
The Adrenaline Receptor
-GPCRs are discriminated form others by their extracellular loops which follow a lock-and-key
mechanism inducing great specificity
-Adrenaline receptor is activated by epinephrine hitting the GPCRs
-binding of the ligand causes a conformational change which transmits the signal to the inner
side of the plasma membrane.
MAPKs in yeas - a paradigm for human MAPKs
-STE11—> Ste7 —> Fus3/Kss1 due to pheromone response
Specificity —>
-theere are different MAPK pathways depending on the stimulus
-this makes them a good proxy for studying a fundamental process
RECALL: yeast cells easy to use due to their easy transformation and efficient homologous
recombination
Pheromone detection: Changin Gene Expression
Certain pathways induce factors for mating:
1. cell fusion (Fus3)
2. Nuclear Fusion (KAR1)
3. a CDK inhibitor (FAR1)
-cell cycle is ARRESTED after mating pheromone detected so that the cell does not undergo
mitosis
-only want ONE copy of the genome before mating
-inhibitor stops the cells in G1 so that only cells in G1 (one genome) will proceed to mating
Ste12 = key transcription factor (sterile 12)
-sequestered by dig 1 and 2 bound proteins
-dig proteins are phosphorylated by MAPK to activate Ste12 which induces mating
pheromones.
Signal Attenuation is built-in
-since the adrenaline rush stops wuickly, we must be able to turn off the signal
-the cell ensures that with activation attenuation follows using Msg5, a phosphatase that
dephopshrylytes the MAPK to turn OFF the signal (turns of Fus3, thats the MAPK)
-the limited pool is decreased which conserves energy
Sexual Reproduction: yeast vs. humans
Mitosis:
-parallel to situations that happen in humans
-mating creates the first diploid zygote cell
-germ cells undergo mitosis to make a 2n —> 4n and then the haploid sperm meets egg.
RECALL WASPS AND HOW THEY BREAK DOWN ASCUS AND LET SPORES MINGLE N
MATE.
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find more resources at oneclass.com
Medellian Genetics —> sex lifecycle v useful for genetics
Mendel’s Laws
1. Law of Dominance
-functional alleles will ALWAYS prevail over recessive alleles
-at a given locus A will always present phenotypically over a
2. Law of Segregation
- when you separate the 2 alleles through a cross, they will segregate randomly
3. Law of Independent Assortment
-independently assort themselves because they are on separate genes
ex: sporulation, alleles segregate independently and randomly to the 4 progeny following
germination
Tetrad analysis separates products of meiosis
-physically separating the products of meiosis using a light microscope
-first must digest ascus using enzymes, tetrahedron collapses and flattens into a cruciform —?
Tetrads
1. take the 4 spore-ascus and separate them in an ordered state:
a) using a glass needle (piece of fiberoptic cable) separate tetrads (each spore is visible)
b) pic them up and bash the ascus against an agar plate
Tetrad Dissection
-allows you to determine the relationship between 2 alleles (dormant vs. recessive)
1. take two haploids: one WT MAT-a with hypothetical gene (YFG1) and one MAT-alpha with a
k/o of YFG1 and anti fungal resistance
2. mate and create diploid that is heterozygous to the WT and k/o allele
3. 4 haploid spores form an ascus, meiosis shows independent segregation: 2 progeny will
have the WT and 2 have the k/o
2 = sensitive to G418, 2 = resistant to G418
—> tells you a single gene is responsible for the phenotype you are interested in.
Practical Yeast Molecular Microbiology and Genetic Engineering
-yeast genome = app 600 genes, 25% not studies and do not know their function, therefore
left with “dry” nomeclature
Genes or “open reading frames (ORFs)” are named according to location
Y (yeast)
D (a-p, 16 chromosomes)
R (right arm, relative to centromere)
356 (356th ORF from centromere, 001 = ON centromere)
W (watson strand, either W or C… arbitrary assignment)
Gain and loss of fiction experiments:
-major driver in genetic analysis
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Lecture 14: cerevisiae lifecylcle, a mating event happens between two mating types: mat-a and mat-alpha (almost genetically identical, physiologically are identical) they mate through conjugation. Cell wall and pm fuse, contents and the nuclei fuse and you go from 1n to 2n chromosomal genes) Note: this is the preferred form, this is diploid (ensures there are two copies of the genes incase one gets destroyed) 3. if it nds itself in an environment without fermentable carbon or no nitrogen, it will undergo sporulation: sporulation results in 2 mat-a and 2 mat-alpha haploid cells packaged into an ascus. Very robust cell wall that ensures they last inde nitely, also keeps spores close for when they need to mate. Mating is co-ordinated by pheromones: shoo formation. Since yeast are non-motile they grow in a polarized way up the concentration gradient towards production of peptides. Mat-a produce ste2 receptors that receive alpha-factors form mat-alpha cells.
