SOD suppressor strategy
Yeast suppressor screen protocol.
This assay can be performed with yeast with your haploid gene deletion of choice on plates of your choice. Other conditions could also be used, such as growth at 37C instead of drug for example if your mutant is sensitive to elevated temperature.
For the example, we are looking for suppressor mutations that allow the sod1 mutant to grow in the presence of paraquat (a superoxide producing drug). sod1 mutants are normally very sensitive to paraquat. supp=suppressor.
1. Grow cells Overnight in YPD liquid culture
2. Spread varying amounts of cells onto plates containing drug at various concentrations. Also plate onto YPD containing no drug, so that you can determine the normal viability and growth rate of the strain.
3. Let grow at 30C from 2-4 days
4. Select colonies that grow. You do not want the plate to be overgrown with cells (called a lawn). Verify the phenotype by comparing growth on YPD+paraquat plates to wildtype and the original mutant strain. The suppressor containing strain should grow more similar to the wildtype than the original mutant strain.
example of verification that the suppressor enhances the growth of sod1 mutant on YPD+paraquat
5. After the suppressor has been verfifed as described in step 4, characterize the suppressor by mating the sod1supp to the original mutant (sod1) but of the opposite mating type. Also mate the parent strain (e.g, sod1 without the suppressor) to the opposite mating type.
- To determine if the suppressor is recessive or dominant, test the paraquat sensitivity of the sod1supp/sod1 diploid and compare to sod1/sod1 diploid strain (without suppressor).
- If the sod1/sod1supp diploid is sensitive, then the suppressor is recessive. If the diploid is resistant, then the suppressor is dominant.
6. Determine if the suppressor is in a single gene, sporulate the sod1supp/sod1 diploid and for each tetrad that was sporulated, look for the pattern of 2 sensitive and 2 resistant to paraquat. This pattern indicates that the suppressor mutation is in a single gene.
6. Example of 2 to 2 segregation
7. Determine complementaion groups by crossing different suppressors with each other to determine if they are in the same gene (e.g., cross sod1suppA with sod1suppB, sod1suppA with sod1suppC and all other possibilities). Test the resistance of these newly created diploid strains to paraquat.
- If two different suppressors are both recessive:
- the diploid of the two crossed suppressor strains will be resistant if the suppressors are in the same gene
- or they will be sensitive if the suppressors are in different genes
8. Identify suppressors by collecting serveral spores which have been backcrossed and send them to be sequenced. Sequencing will require the isolation of genomic DNA perhaps using a Qiagen Blood and Tissue
DNA isolation kit (Qiagen catalog # 69504 69504) or something similar.
8. Suppressor identification