Transcript Genetika Molekuler (8)

Genetika Molekuler (8)
Sutarno
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Lecture #6 Notes (Yeast Genetics)
LECTURE 6: HOW TO DISTINGUISH SUPPRESSION MECHANISMS
Know as much as possible about the original allele
The most critical decision is the starting allele(its not always predictable which allele will be best, but you can at least stack the odds in your favor)
missense, nonsense, or frameshift allele? Promoter mutation? Null? (different major suppressor classes expected for each of these)
If you don’t / can’t sequence it, is full length protein being made? (by antibodies)
If it’s a Ts- allele, is the protein stable?
Is it in a known domain?
If the structure is known, is it on the surface?
Is the suppressor tightly linked to the original mutation?
If so, then intragenic. Ballgame.
Does it suppress a null allele?
If so, then it is by definition a bypass allele.
Rules out some models (interaction, increasing the amount or activity of the original protein)
Specificity
Is it allele-specific suppression? (does it suppress a null, mutations only in one domain, weak vs strong alleles, or only the original allele?)
Frequetly used as an argument for direct interaction, but not necessarily true
Is it gene-specific?
Does it suppress other mutations with similar phenotype?
If it is part of a complex, does it suppress other mutations in that complex?
The more tested, and the closer the phenotype, the better
example: can test whether a suppressor of a cdc mutant suppresses other cdc mutants, but better if those mutants are defective in the same part of the cell cycle
Dominant or recessive?
Allows some interpretation of the mechanism (likely due to gain or loss of function), just as with any other mutation
What is the phenotype of the suppressor by itself?
Critical test to determine the relationship between the two genes.
Expect the suppressor by itself (in a wild type background for the original locus) to have a phenotype related to that of the original gene. That phenotype could be the
same or opposite to the original mutant.
The possible outcomes:
Related phenotype……………………best case scenario
No phenotype………………………..not necessarily a problem
Completely unrelated phenotype……scary…start considering another project
Does a null mutation in the suppressor suppress?
Then suppression is due to a loss of function. Ballgame.
These tests are extremely important, because when you clone SUP+ (especially if it doesn’t look like anything), your major clue to what it is doing is going to come from
these tests!
Another type of suppressor hunt strategy: Suppression by overexpression
Transform with a high copy number library, and select plasmids that suppress when present at high copy number
Hadwiger…..suppressors of cdc28 (kinase), got CDC28, CLN1, CLN2 (cyclins)
Transform with a library that contains genes expressed from an inducible promoter
GAL1p  OFF on glucose, ON in galactose