7 YEAST GENETICS
21 Suppressor analysis is a proven method to identify interacting genes
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Missense mutations change a single codon so as to cause the replacement of one amino acid by another in a protein sequence.
- A high-copy-number suppressor
allows a mutation in one gene to be suppressed by overexpression of a
second gene that is present on a high-copy-number plasmid.
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A classic approach to identifying
interacting genes is the isolation and analysis of extragenic
suppressors. An extragenic suppressor is a mutation in a gene that is
distinct from the gene with the initial mutation. Extragenic
suppressors of an interesting mutation are identified in two general
steps.
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The first step is the isolation of
"revertants" of the original mutant — that is, strains that no longer
have the original mutant phenotype. We can isolate such revertants by
either a screen or a selection. A revertant strain could arise by
either true reversion of the mutation or by a second mutation in an
interacting gene, an extragenic suppressor, that compensates for the
defect of the original mutation. One example of this type of extragenic
suppressor was given earlier when we described the epistasis tests. In
that case, a mutation in one gene suppressed the defect caused by a
mutation in another gene.
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Figure 7.25
A true revertant will be completely linked to the
initial mutation, producing all PD tetrads. An extragenic suppressor
mutation will segregate from the initial mutation, resulting in PD,
NPD, and TT tetrads.
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True revertants and extragenic
suppressors are distinguished by the second step. This is a genetic
cross of the "revertant" by a wild-type strain, followed by tetrad
analysis, shown in Figure 7.25.
For true revertants, all tetrads will be PD tetrads, since all spores
will inherit a wild-type allele. In contrast, for extragenic
suppressors, a different pattern will be seen because there will almost
certainly be recombination between the suppressor mutation and the
original mutation. Therefore, some spores will inherit only the
original mutation. If the original and suppressor mutations are
unlinked, the ratio will be the expected 1:1:4 for PD:NPD:TT. The
analysis of suppressor mutations has been an extremely powerful tool in
genetic analysis of yeast, as well as of other organisms, particularly
prokaryotes. (For two good reviews of suppressor analysis in yeast see 2909 and 2920.)
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Figure 7.26
Suppression between interacting proteins. In the example shown, two proteins, A and B, normally interact. In the a
mutant, this interaction is impaired and there is a mutant phenotype.
However, a suppressor mutation that alters B restores the interaction
with a, resulting in a wild-type phenotype.
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The nature of the suppressor mutation will often depend
on the type of mutation that is being suppressed. As Figure 7.26 shows,
a missense mutation
that alters the conformation of a protein might be suppressed by a
compensating change in an interacting protein. In this case, then, the
suppressor mutation identifies an interacting gene product, often the
goal of suppressor analysis. In contrast, a deletion mutation that
removes the coding region of a gene cannot be suppressed by a change in
an interacting protein. Only a "bypass" suppressor that compensates for
the complete loss of the initial gene product can suppress a deletion
mutation. Informational suppressors will often suppress nonsense
mutations. We must take all of this into consideration when planning a
project to study a gene by suppressor analysis, and must carefully
consider the molecular nature of the initial mutation.
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In addition to extragenic suppressors, a second type of
suppressor that we can screen for is called a high-copy-number suppressor.
This screen identifies genes that, when overexpressed, suppress the
defect caused by the mutation in the initial gene. For example, imagine
that a mutation weakens an interaction between two proteins because it
reduces the affinity between the mutant protein and the second protein.
This results in a mutant phenotype. If the level of the second protein
is now increased, it might overcome the reduced affinity, resulting in
a wild-type phenotype. High-copy-number suppression can occur by other
mechanisms as well. Screens for high-copy-number suppressors are done
by using an S. cerevisiae genomic library in a
high-copy-number (2-micron) vector to transform the mutant of interest.
The transformants are then screened for those with a wild-type
phenotype. The plasmids in these candidates are isolated and tested to
see if they contain the gene corresponding to the mutant gene (not the
desired class) or to a different gene that might be a high-copy-number
suppressor. A gene in the latter category would likely encode a protein
that would interact with the mutant protein.
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Genes identified by suppressor analysis
can be cloned and studied using all the types of genetic, molecular,
and biochemical approaches described in this chapter. Often, the study
of a gene identified as a suppressor will shed light not only on the
suppressor but on the gene containing the initial mutation. Suppressor
analysis is a powerful tool for yeast geneticists.
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Last Revised on January 27, 2004
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2909 Forsburg, S. L.
(2001).
The art and design of genetic screens: Yeast.
Nat. Rev. Genet. 2, 659-668.
PubMed Journal
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2920 Prelich, G.
(1999).
Suppression mechanisms: themes from variations.
Trends Genet. 15, 261-266.
PubMed Journal
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©Jones and Bartlett Publishers (2007)
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