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Recall from the last lecture
From Boone, Bussey and Andrews, 2007
Epistasis….infer the order of function
A
ADE3
B
ADE1
C
ADE13
ADENINE
Adenine biosynthesis pathway in yeast cells
ade1 mutants are Ade- but are also red because compound B is red
What is the phenotype of the double mutants:
ade3 ade1?
ade13 ade1?
We would say ade3 is epistatic (hides) ade1
Often you don’t have distinguishing phenotypes
Example – radiation sensitive mutants
YFG1
YFG2
YFG1
YFG2
or
Compare single mutants to double mutant
Quantitative analysis of genetic interactions
•Mutations can have subtle effects on cells
•Suppose mut1 grows a little bit slowly 90% as well as WT
•Suppose mut2 also grows a little bit more slowly ()
•If the genes acted independently, then expect (.8 x .9=.72) 72% as well as WT
•Example- Collins et al. Nature 446:806
•“Functional dissection of protein complexes involved in yeast chromosome
biology using a genetic interaction map.”
•Choose the genes based on GO Process annotation
•Measure the growth rates of all single and double mutant combinations
•Can get “aggravating” (synthetic sick) or “alleviating” (suppression) combinations
Design
• Choose 754 mutants based on GO annotations
•Mostly non-essential but some essential genes mutated as “DAMP” alleles
•Essential genes where the 3’ UTR is removed making the mRNA unstable
•Hypomorphs
•754 x 754 matrix
•Each entry is a number corresponding to a score of how the double mutant
deviated from “expected” assuming the genes functioned independently
•Expectation is that if two things are doing the same thing,
they should have the same patterns (and vectors) of interactions
•Compare data from protein-protein interaction map (TAP purifications)
•Expectation is that proteins in same complex are doing the same thing
1 complex
Identical pattern
of interactions
for all subunits
2 different complexes
Identical pattern
of interactions
except for blue subunit
Additional interactions
for blue subunit
Chemical-Genetic Profiling
•Useful for determining mode of action of a drug
•Screen the collection of strains for mutants that are sensitive
•Do NOT identify the molecular target in the screen
Drug
A deletion mutant that is sensitive is missing a protein
But the target is still present (that’s why the cells are sensitive)
Every strain that is sensitive still has the target
Target
Drug
Target
Buffer
Chemical Genomics
Target
SGA
Buffer
Screen for benomyl sensitive strains
Represent genes that buffer the effect of the drug
YPD
Benomyl
Sublethal
[benomyl]
Benomyl-a benzimidazole drug that binds -tubulin (Tub2)
and depolymerizes microtubules
Get slide about barcoding etc
Deletion Mutants
SGA data
+ chemical-genetic
profiling benomyl
Deletion mutants
SGA
Queries
Microtubule
&
Spindle Function