Download Mating of haploid strains

Mating of haploid strains
Cell signal transduction
a-pheromone
α-pheromone
a-pheromone
receptor
α-pheromone
receptor
a-cell
Transcription
of genes
needed for
mating
α-cell
Pheromone action in yeast
Pheromone signal-transduction cascade
•  Pheromone binds Gcoupled receptor
•  MAP Kinase cascade
•  Ste12p phosphorylated
•  Activated Ste12p
starts mating program
MATa/MATα
The MAT locus information
•  The MAT locus can encode three regulatory peptides:
- a1 is encoded by the MATa allele
- α1 and α2 are encoded by the MATα allele
•  Three regulatory activities: α1, α2, and a1-α2.
The products of the mating type
locus control gene expression
The concept of mutants
•  You have a complex back box and you would like to
understand how it works
•  Take a hammer, hit the box until it doesn’t work
anymore
•  Now you ask, what did I brake
•  Why doesn’t it work anymore if this particular part is
broken ?
Sterile mutants can monitor
the MAT status
•  Mutations have been identified at several loci that
produce a non-mating phenotype, called sterile
(STE).
•  The sterile mutations fall into three classes:
1. sterility only in a cells
- STE2, the α pheromone receptor
2. sterility only in α cells
- STE3, the a pheromone receptor
3. sterility in both a and α cells
- STE12, the general pheromone-responsive
transcription factor
MAT regulation in α cells
•  When the α allele is present at MAT, two genes are
expressed: MATα1 and MATα2,
•  Mutations in α1 affect only α-specific genes, such as
STE3.
•  MATα1 mutants prevent normal expression of STE3.
They do not affect other haploid specific genes or aspecific genes.
α1 is a positive regulator of α-specific genes
•  Mutations in α2 allow the expression of a-specific genes,
even in a MATα cell.
α2 is a negative regulator of a-specific genes
•  Consequently, in a MATα cell the α genes are expressed
while the a genes are not.
Saccharomyces as a model system
•  How do cells generate a mitotically stable,
complex, specific cell type?
⇒ same DNA, but different gene expression
states.
•  How do cells respond to environmental change
or information from other cells?
⇒  decision-making algorithms.
• How do cells maintain an undifferentiated state
“stem cell”?
⇒ non-equivalence of daughter cells at mitosis.
Homothallic and heterothallic strains
The mating type locus has
active an inactive cassettes
Cassette organization of
the mating type locus
Mating type switch by gene
conversion
Mating type switch
The HO endonuclease
•  DNA double strand break (DSB) occurs in late G1 after
START, in mother cells only
•  Brief period of HO transcription in mother cells only
•  In daughters expression of HO is repressed by Ash1
(asymmetric synthesis of HO)
•  Ash1 mRNA is asymmetrically deposited in daughter cells
•  Ho protein is rapidly degraded
•  GAL-HO is used for mating type switch
•  in diploids HO is repressed by MATa1/MATα2 heterodimer
ASH1 localization
movie
ASH1
localization
Figure 10-18: Mating type switch.
Prototrophic vs Auxotrophic
Uracil
kanMX6 E.coli kanamycin resistance gene
G418
Uracil
Diffusion/transport
Yeast nomenclature using the
ARG2 locus as an example
Gene
(italics)
Gene symbol
Definition
ARG2
A locus or dominant allele
arg2
Locus or recessive allele confering arg requirement
arg2-9
A specific recessive allele or mutation
ARG2-1
A specific dominant allele or mutation
arg2::URA3 Gene disruption in which insertion/replacement by
URA3 creates a null allele of ARG2.
Protein
(not italics)
Arg2p
Arg2 protein
Protein product of ARG2 gene
Systematic nomenclature YCR263c
How do you determine the mating
type of your yeast strain ?
How would you screen for mating
deficient mutants (ste) ?
The power of genetics
Leland Hartwell, Nobel Prize 2001
the cdc-screen
How would you screen for mutants that
affect the cell division cycle (cdc) ?