Review Questions yeast lecture 30.03.2015
1. What processes are required for successful mating to occur? (1)
Signaling to the partner
Sensing of the partner
Orientation towards the partner
Synchronization of Cell cycle
Fusion (cell/nuclear)
2. For what processes in higher eukaryotic organisms can the yeast mating response be used as a model? (1)
Intercellular signaling. The pathway is analogous/Homologous to cell-cell signalling in higher eukaryotes.
Also environmental signaling.
3. What feature of α-factor was exploited to identify yeast mutants that were unable to mate? What “trick” was used
to allow for complementation analysis of the mutants obtained ?(2)
Yeast cells arrest growth in G1 phase when exposed to mating pheromone. Mutants were collected that
were insensitive to mating pheromone at 34oC and sterile at that temperature, but not 22oC.
4. What are the main steps/factors involved in the mating signaling cascade (disregard polarization)? (3)
Mating pheromone (a/α-factor) binds the 7-transmembrane receptor (Ste2p or Ste3p); the heterotrimeric
G-protein (Gpa1p, Ste4p, Ste18p) dissociates and recruits the Ste5 scaffold protein with associated
kinases, initiating the phosphorylation cascade involving Ste20p MAPKKKK, Ste11p MAPKKK, Ste7p
MAPKK, Fus3p MAPK. Fus3p phosphorylates the Ste12p transcription factor, which activates
expression of genes required for mating.
5. What problem do you notice when you compare different yeast signaling pathways? Which mechanisms are used
in yeast to solve this problem? (2)
Shared components should interfere with specificity of each pathway. In yeast, Fus3p kinase probably
inactivates the target of Kss1p target to prevent invasive growth. Specific scaffold proteins may play a
role
6. Describe the main steps required for cell polarization! (2)
Cell surface site determination using intrinsic (e.g.for cell budding) or extrinsic (e.g. for mating) cues.
Marking of cell surface by ”Landmark protein”. Establishment of polarity by activation of small GTPase
proteins in vicinity of the landmark. Cytoskeletal re-orientation/polarisation (actin and other polarized
components.
7. What are the “landmark proteins”/polarity cues in the mating versus budding? (1)
Mating: Activated mating factor receptor(s) at the point of highest pheromone concentration
Budding: Bud 10, Bud8, Bud9, placed in relations to the previous budding event
8. What is the transcriptional state in α-cells (1)? In a cells (1)? In a/α diploids (1)? (With regard to a specific, αspecific, haploid specific genes)?
α-cell: α-1 activator activates α-specific genes, α2 repressor represses a-specific genes; haploid-specific
genes and RME1 gene are transcribed . a-cell: a-specific genes, haploid-specific genes and RME1 gene are
transcribed. In a/α diploids, α2 represses a –specific genes, α2 and a1 repress α1(  no α-specific gene
expression) and haploid specific genes, RME1 is not transcribed can enter meiosis
9. Which cell switches mating type after budding, mother or daughter cell, and which factor plays a pivotal role in
the process of determining which cell switches?(2)
The mother cells switches. The Ash1p is the determining factor. ASH1 mRNA is actively localized in the
daughter bud and encodes a DNA binding protein that negatively regulates HO nuclease expression
10. What are the mating type loci in yeast and what information do they contain? (1)
HML: α-cassette, HMR: a-cassette; MAT: α-cassette or a-cassette, depending on mating
type
11. How is mating type cassette donor preference achieved in a or α cells?(2)
a-cells: RE is active( nucleosomes not firmly positioned on DNA); high homology of HML flanking
regions with MAT flanking regions causes switch to α; α cells: RE is inactive by Mcm1p/ α2 via
nucleosome positioning, only HMR is available as a recombination donor
12. What is heterochromatin?(1)
Heterochromatin is “a specialized chromatin structure that blocks expression of most genes within the
silenced domain, irrespective of which activator or RNA polymerase is used”
13. Outline the steps involved in establishment of silencing in mating type loci! (3)
Rap1p binds to its target site sites and recruits Sir3p and Sir4p (Nucleation)
After establishment of Rap1p/Sir2p/Sir3/Sir4p interaction:
Sir2p deacetylates histones (marking for silencing)  more Sir3p, Sir4p recruited via interaction with
deacetylated histones. More Sir2p recruitment via Sir4p. Further histones deacetylated. More Sir3p,
Sir4p recruited via interaction with deacetylated histones…….Spreading via interaction with modified
(hypoacetylated) Histones/Nucleosomes
14. Explain “Epigenetic Inheritance”!(1)
Epigenetic Inheritance: “Heritability of two different states in otherwise identical cells”
Ability of a specialized chromatin structure (or protein structure) to template its own reformation
15. How were long-lived yeast mutants isolated? (2)
Longevity in yeast is associated with increased stress resistance. A short-lived, starvation
sensitive yeast strain was mutagenized, and selection for starvation resistant clones led to
the isolation of mutants with an increased life span
16. How was the uth2-42 mutation cloned? (2)
A mating deficiency in this mutant was associated with the longevity phenotype of the
uth2-42 mutation. The mutant was transformed with a genomic yeast library, and the
transformants replica plated onto a lawn of a yeast strain of the opposite mating type.
After allowing for mating, the cells were replica plated onto media selecting for diploids.
The diploid colonies (able to mate) harbored the complementing plasmid.
17. How is SIR2 involved in the aging of a yeast cell? (2)
SIR poteins are required for silencing of extrachromosomal rDNA circles that accumulate in older cells.
The cell cycle slows down. Silencing of other regions is disturbed, and less SIR complex is available for
DNA repair.