Grow on 5-FOA

1. ARS ARS Gene 1 knockout Gene 2 knockout CEN CEN HIS3 HIS3 ARS ARS CEN CEN ARS ARS CEN CEN URA3 URA3 Gene 2 Gene 2 Gene 1 Gene 1 LEU2 LEU2 Mutant Gene 1 Mutant Gene 1 Gene 1 knockout Mutant Gene 2 Mutant Gene 2 Gene 2 knockout Overview: Use of double knockout yeast strains to analyze genetic interactions by plasmid shuffle Transform double knockout strain with LEU2 and HIS3 plasmids with gene mutations (Protocol 1) Grow on 5-FOA Outcome: System to analyze genetic interactions between essential splicing factors Questions answered: Do two splicing factors interact genetically? Can one mutation suppress the phenotype of another mutation? Can reveal mechanisms of core splicing factors

2. ARS ARS Gene knockout CEN CEN HIS3 HIS3 ARS ARS CEN CEN URA3 URA3 Mutant Gene Mutant Gene WT Gene WT Gene Figure 1 Plasmid shuffle analysis in yeast Grow on 5-FOA Gene knockout What is growth phenotype of the gene mutation?

3. ARS ARS Gene 1 knockout Gene 2 knockout CEN CEN HIS3 HIS3 ARS ARS CEN CEN ARS ARS CEN CEN URA3 URA3 Gene 2 Gene 2 Gene 1 Gene 1 LEU2 LEU2 Mutant Gene 1 Mutant Gene 1 Mutant Gene 2 Mutant Gene 2 Figure 2 Synthetic enhancement/lethal analysis in yeast Grow on 5-FOA Gene 1 knockout Gene 2 knockout What is growth phenotype of two viable mutants combined?

4. Negative Control Positive Control Control pRS413 pRS413-WT gene 1 pRS413-mutant 1 gene 1 pRS415 pRS415-WT gene 2 pRS415-WT gene 2 pRS413-WT gene 1 pRS413-mutant 1 gene 1 Synthetic lethal Control pRS415-mutant 2 gene 2 pRS415-mutant 1 gene 2 pRS413-mutant 1 gene 1 pRS413-WT gene 1 pRS415-mutant 2 gene 2 pRS415-mutant 1 gene 2 pRS413-mutant 1 gene 1 No synthetic Enhancement Control pRS415-WT gene 2 Control Figure 3