Download Suppressors

17 Oct, 2011
Epistasis
In learning how genes interact we need to look at double-mutants
The double mutants may have one of 4 phenotypes:
1) Both mutant phenotypes of single mutants—genes act independently of one
another
2) The double mutant may exhibit phenotype WT or close to WT—this type of
interaction will be suppression
3) The double mutant may exhibit a more severe phenotype –enhancement (we
did not cover in lectures)
4) The double mutant may exhibit only one of the two mutant phenotypes-this
type of interactions is epistasis
Epistasis-the ability of one gene to mask the activity of another gene.
Since gene activity is inferred looking at a mutant that lacks the WT activity for that
gene, epistasis refers to the ability of one mutant gene to mask the activity of
another mutant gene.
Epistasis is often used to place genes affecting the same biological process into a
logical pathway.
Technique:
1) Make a double mutant and compares it to phenotypes of single mutants.
In budding yeast, mutations that affect general amino-acid control fall into two
classes: tra3 and aas.
tra3 single mutant has constitutive activation of aa synthesis. This means that TRA3
function is to negatively regulate aa synthesis.
aas single mutants cannot activate aa synthesis. This means that AAS (AAS1, AAS2,
AAS3) function is to positively regulate aa synthesis.
To place these genes into a logical pathway double mutants were made:
tra3; aas3 phenotype of aas3-no aa synthesis. So aas3 is epistatic to tra3
tra3; aas1 phenotype of tra3-constant aa synthesis. So tra3 is epistatic to aas1
tra3; aas2 phenotype of tra3-constant aa synthesis. So tra3 is epistatic to ass2
As a result we cannot conclude that either TRA phenotype or AAS phenotype is
inherently epistatic.
AAS1
AAS2
TRA3
AAS3
AA biosynthesis
We cannot infer relationship between AAS1 and AAS2 to infer their relationship,
because double mutant is not informative. Double mutant has the same phenotype
as single mutant.
As a result in order to perform epistasis to infer relationship between two genes,
single mutants have to have at least some difference in phenotypes.
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Suppressors
The motivation for most suppressors screens is to find more genes that affect the
same biological process as the original gene.
Two types of suppression:
I. Suppression – a mutation in one gene alleviates the defect of a mutation in another
gene—“low-copy” suppressor.
II. Suppression – overexpression of a wild-type copy of a gene alleviates the defect of
a mutation in another gene—“high-copy suppressor”. In this case the wild-type
allele of a gene is acting as an extragenic suppressor.
Low-copy supressors involved getting a second mutation in the genome.
Once the suppressor is found, the first question:
If this suppressor defines a different gene?
If this mutation in the same gene it is an intragenic suppressor, which can be true
revertant or pseudo-revertant, which you are not interested in.
To figure this out, you need to perform mapping. You should remember how to
perform mapping from the last review.
High-copy suppressors screen involves the use of a wild-type cloned gene.
Over-expression of WT copy one gene ameliorates the effect of a mutation in
another gene.
Technique: high-copy suppression, when a high-copy plasmid or constitutive or
inducible promoters are used to overexpress a suppressor gene.
High-copy suppression has many advantages over conventional suppressor studies.
The genes screened for the high copy-suppression have already been cloned.
Suppose you have a mutant and you want to identify more genes that a affect the
same biological process:
You make a genomic library from WT strain
Use 2μ plasmid with a selection marker
Transform your mutants
Select ones that have WT phenotype
What kind of suppressor genes you expect to see:
1) WT allele of the original mutation (CDC28)
2) Genes, product of which directly interact with your mutant gene and restore
its WT biochemical activity.
3) Genes that are functionally-related to your gene, but not interact with it.
Over-expression of this gene provides enough functional product to
overcome the loss of the first gene. These are bypass suppressors.
How one could distinguish a suppressor 2) from a bypass suppressor 3)?
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A bypass suppressor allow suppression of null allele—it does not need a residual
activity of the first mutant gene to restore WT phenotype.
Example:
TUB1and TUB3 –tubulin genes, they are paralogs
TUB1 is essential—yeast cannot grow and divide
TUB3 is not essential
You can build 2 different models and test them:
1) TUB3 isoform is not involved in growth and cell division, although homologous,
TUB3 might be functionally distinct from TUB1
OR
2) TUB1 and TUB3 have similar functions, but their level of expression is different.
Mutant tub3Δ is fine, because TUB1 provides enough product to substitute for its
loss.
To test theses hypotheses:
Varying the dose of the genes can test these two hypotheses. Under the first
hypothesis, changing the dose of the gene will have no effect. Under the second
hypothesis increasing the dose of TUB3 should suppress a tub1 mutant.
Perform high copy suppression experiment:
Have a strain tub1 (ura3 Δ, trp1 Δ) carrying TUB1 on URA3 plasmid.
You want to get rid of this plasmid later (since TUB1 is essential).
What promoter and plasmid would you use—native if and low-copy CEN plasmid?
Transform it with TUB3 on a 2μ plasmid with TRP1 as a selection marker.
Plate on +5FOA, -trp
TUB1 CEN plasmid should be lost.
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