Transcript let-60(gf)

Genetic interaction and interpretation of genetic interactions
- Biosynthetic pathway/ genes acting in different steps.
-Order genes in a genetic pathway - studies on yeast mating
-pheromone response
- Epistasis analysis using null mutations- The GAP story
- Epistasis analysis using gf mutations - The Ras suppressors
-Enhancer and synergistic effect between two alleles -The Ras pathway.
-Understanding at molecular level/biochemical level.
-Limitation of genetics
Pheromone response in yeast
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Adopted from Hartwell et al. Genetics
Genetics study of mating response
1. How do you want to start it?
2. What is the assay (phenotype) for the screen?
Makay and Manning 1978, Hartwell 1980
Isolated mutations in 12 genes that cause the
sterile phenotype. They named them ste1-12. All
with the non-response phenotype.
Let us go back to 1970s to re-live the genetics of yeast mating response
spread
Normal plates with yeast lawn
Ste1
Ste2
Ste3
Ste4
ste5
+ EMS
Makay and Manning 1978
Hartwell, 1980, get to ste 12 by isolating Ts alleles.
+ a factor, no growth
Now what would you do?
If we assume all these genes act in the same pathway.
Assuming ste1-12 mutations are all loss-of-function
mutations, can we use them to determine the order of
gene actions?
A: yes. B: No.
- If you made a double mutant containing ste1
and ste2, what would you see? What would you
learn?
A test
Signal
Signal
Gene A
signal
response
Gene A
signal
response
GeneA
A has
has aa gain-of-function
loss-of-function (hyperactive)
mutation
Gene
mutation
GeneA
A is
is aa negative
positive factor, the mutation should
IfIf Gene
enhance the
the signal
signal response
response
enhance
A: Yes.
Yes.
A:
B: No
No
B:
when two genes act in the same pathway, mutants with
opposite phenotypes mean one gene act as a positive
regulator, the other is the negative regulator.
Genetic epistasis
Epistatic: one effect masks the other
Epistasis is used to learn about the order of gene action
- indirect
- need to learn biochemistry to understand the
molecular action
- important to verify and biochemical assumption
Epistasis can only be done with two different
mutant phenotypes
Genes in the same pathway:
Mutations with opposite mutant phenotypes
Unable to mate
?
always want to mate
Blinder et al. 1989 Cell:
Mating constitutive = haploid lethal (please go read the paper)
Use cleaver sector synthetic lethal screen isolated many
haploid lethal mutations that are unable to mate.
Several mutations define the Ga gene.
About trimeric G protein
GDP
GDP
GTP
receptor
a


GTP
a

RGS
Pi
?
?

a or , who interacts with the downstream target?
Who activates the target, a or 
Ga (-): hyper response, always want to mate
Ga
mating response
G (-): No mating response
G
mating response
Ga(-) & G(-): No mating response
A:
G
Ga
mating response
C: not sure
B:
Ga
G
mating response
Getting tricky
Ga (-): constitutive mating
G (-): non-mating (ste mutants)
Ga (-) + G (-): no mating.
A:
B:

Ga

Target
Ga
C:
Target
Could be either
Epistasis with lf mutations
Receptor

Ga
Target
lf
lf
lf
lf

Receptor
Ga
lf
lf
lf
lf
 is required for the target activation
Target
About trimeric G protein
GDP
GDP
GTP
receptor
a


a

RGS
Pi
A:
GTP
Ga


Target
epistasis analysis using loss-of-function mutations: genetic control of programmed cell death
Results
Gene A
ced-9(+)
Gene B
ced-4(+)
Phenotype
Normal programmed cell death
ced-9(-)
ced-4(+)
Extra cell deaths
ced-9(+)
ced-4(-)
Cells that normally die survive
ced-9(-)
ced-4(-)
Cells that normally die survive
Conclusion: ced-4(-) phenotype is epistatic to that of ced-9(-)
Linear model
Parallel model
Death
signal
ced-9
On
Off
Off
Death
signal
No death
signal
Death
signal
Off
On
Off
cell death
No death
Extra death
No death
ced-9
On
Off
Off
Molecular Actions
ced-4
ced-4
Off
On
Off
CED-9
CED-4
CED-9
Factor X
cell death
Off
On
Off
No death
Extra death
No death
CED-4 is inactive
Activates CED-3
caspase for the
killing
A: linear
B: parallel
A second example of epistasis analysis using loss-of-function mutations: dauer larva formation
Results
Gene A
age-1(+)
Gene B
daf-18(+)
Phenotype
When starved, worms become dauer larvae
age-1(+)
daf-18(-)
Defective in dauer formation
age-1(-)
daf-18(+)
Constitutive dauer formation
age-1(-)
daf-18(-)
Defective in dauer formation
Conclusion: daf-18(-) phenotype is epistatic to that of age-1
Linear Model
signal
age-1
Dauer formation
daf-18
On
Off
Off
Off
On
Off
No dauer
Constitutive dauer
No dauer
age-1
signal
Factor X
Parallel Model
Dauer formation
daf-18
On
Off
Off
On
Off
On
Off
On
Off
AGE-1
PI3Kinase
signal
+P
Molecular actions
PIP3
PIP2
-P
DAF-18
PTEN
Binds and activates
AKT kinases to
prevent dauer
formation
A: linear
B: parallel
What if you only have positive factors?
- in case of mating response, you only have ste
genes, whose lf mutants are non-maters.
You can use gain-of-function mutations (hypermorph).
- gf mutations often generate opposite mutant
phenotypes as that of lf mutations in the same gene.
When a hyperactive (gf) mutant phenotype is the winner
signal
Results
pathway
function
Gene A
sem-5(+)
Gene B
let-60 (+)
Phenotype in vulval induction
3 of the 6 precursor cells are induced
sem-5(+)
let-60 (gf)
Extra cells induced (Multivulva)
sem-5(lf)
let-60 (+)
Less than 3 cells induced (Vulvaless)
sem-5(lf)
let-60 (gf)
Extra cells induced (Multivulva)
Model and
explanation
Conclusion: the let-60(gf)
phenotype is epistatic to that
of sem-5(lf)
In a given precursor cell
EGFR
signal
+
+
+/-
Sem-5
(GRB2)
SOS
On
Off
Off (lf)
Off (lf)
let-60
(Ras)
On
Off
Off
On (gf)
Vulval induction
Induction
No induction
No induction
induction
No parallel model
When a hyperactive (gf) mutant phenotype is the loser
Results
Gene A
lin-45 (+)
Gene B
let-60 (+)
Phenotype in vulval induction
3 of the 6 precursor cells are induced
lin-45(+)
let-60 (gf)
Extra cells induced (Multivulva)
lin-45 (lf)
let-60 (+)
Less than 3 cells induced (Vulvaless)
lin-45 (lf)
let-60 (gf)
Less than 3 cells induced (Vulvaless)
Conclusion: the lin-45 (lf) phenotype is epistatic to that of let-60 (gf)
In a given precursor cell
Model A
signal
+
+
+
Model B
let-60
(Ras)
lin-45
(Raf)
On
Off
Off (lf)
On
Off (lf)
On
Off
On
On (gf)
On (gf)
signal
Vulval induction
let-60/Ras
Gene X
Vulval
induction
On
Off
Off
On
Off
Induction
No induction
No induction
Induction
No induction
lin-45/Raf
+
+
+
On
Off
On
On (gf)
On (gf)
On
On
Off (lf)
On
Off (lf)
Induction
No induction
No induction
Induction
No induction
Biochemistry:
Ras directly binds
to raf for its
activation
Why do we do epistasis analysis?
- provide a critical guide for biochemical analysis
-Add significance to relationship based on biochemical
functions