Presentation on theme: "Grow on 5-FOA Gene 1 knockout Gene 2 knockout Gene 1 knockout"— Presentation transcript:
1Grow 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 shuffleTransform double knockout strain with LEU2 and HIS3plasmids with gene mutations (Protocol 1)ARSCENLEU2Mutant Gene 1HIS3MutantGene 2URA3Gene 1Gene 1 knockoutGene 2 knockoutGene 1 knockoutGene 2 knockoutURA3ARSCENGene 1Gene 2Grow on5-FOAARSCENLEU2Mutant Gene 1ARSCENHIS3MutantGene 2Outcome: System to analyze genetic interactions between essential splicing factorsQuestions answered: Do two splicing factors interact genetically?Can one mutation suppress the phenotype of another mutation?Can reveal mechanisms of core splicing factors
2Plasmid shuffle analysis in yeast Figure 1Plasmid shuffle analysis in yeastGene knockoutURA3ARSCENWT GeneARSCENHIS3Mutant GeneURA3ARSCENWT GeneGrow on5-FOAGene knockoutARSCENHIS3Mutant GeneWhat is growth phenotype of the gene mutation?
3Synthetic enhancement/lethal analysis in yeast Figure 2Synthetic enhancement/lethal analysis in yeastGene 1 knockoutGene 2 knockoutURA3ARSCENGene 1Gene 2ARSCENHIS3MutantGene 2ARSCENLEU2Mutant Gene 1URA3ARSCENGene 1Gene 2Grow on5-FOAGene 1 knockoutGene 2 knockoutARSCENLEU2Mutant Gene 1ARSCENHIS3MutantGene 2What is growth phenotype of two viable mutants combined?
4Figure 3 Negative Control Positive Synthetic lethal No synthetic pRS415-mutant 1gene 2pRS413pRS415pRS415-WTpRS413-mutant 1gene 1pRS415-mutant 2pRS413-WTpRS415-WT gene 2pRS413-WT gene 1NegativeControlPositiveSyntheticlethalNo syntheticEnhancement