Grow on 5-FOA Gene 1 knockout Gene 2 knockout Gene 1 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) ARS CEN LEU2 Mutant Gene 1 HIS3 Mutant Gene 2 URA3 Gene 1 Gene 1 knockout Gene 2 knockout Gene 1 knockout Gene 2 knockout URA3 ARS CEN Gene 1 Gene 2 Grow on 5-FOA ARS CEN LEU2 Mutant Gene 1 ARS CEN HIS3 Mutant Gene 2 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

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

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

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