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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) Gene 1 knockout Gene 2 knockout ARS ARS CEN CEN ARS URA3 HIS3 CEN LEU2 Mutant Gene 2 ARS CEN Mutant Gene 1 URA3 Grow on 5-FOA Gene 1 knockout Gene 2 knockout ARS ARS CEN CEN HIS3 LEU2 Mutant Gene 1 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 Figure 1 Plasmid shuffle analysis in yeast Gene knockout ARS ARS CEN CEN HIS3 URA3 Mutant Gene WT Gene ARS CEN Grow on 5-FOA URA3 WT Gene Gene knockout ARS CEN HIS3 Mutant Gene What is growth phenotype of the gene mutation? Figure 2 Synthetic enhancement/lethal analysis in yeast Gene 1 knockout Gene 2 knockout ARS ARS CEN CEN ARS URA3 HIS3 CEN LEU2 Mutant Gene 2 Mutant Gene 1 ARS CEN Grow on 5-FOA URA3 Gene 1 knockout Gene 2 knockout ARS ARS CEN CEN HIS3 LEU2 Mutant Gene 1 Mutant Gene 2 What is growth phenotype of two viable mutants combined? Figure 3 Negative Control Positive Control Control pRS413 pRS413-WT gene 1 pRS415-WT gene 2 Control pRS413-mutant 1 gene 1 pRS415 pRS415-WT gene 2 pRS413-WT gene 1 pRS413-mutant 1 gene 1 pRS415-mutant 2 gene 2 pRS415-mutant 1 gene 2 pRS413-mutant 1 gene 1 pRS415-mutant 2 gene 2 No synthetic Enhancement pRS413-WT gene 1 pRS413-mutant 1 gene 1 pRS415-WT gene 2 Control pRS415-mutant 1 gene 2 Control Synthetic lethal
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