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

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Presentation on theme: "Grow on 5-FOA ARS CEN LEU2 Mutant Gene 1 ARS CEN HIS3 Mutant Gene 2 URA3 ARS CEN Gene 1 Gene 2 Gene 1 knockout Gene 2 knockout Gene 1 knockout Gene 2 knockout."— Presentation transcript:

1 Grow on 5-FOA ARS CEN LEU2 Mutant Gene 1 ARS CEN HIS3 Mutant Gene 2 URA3 ARS CEN Gene 1 Gene 2 Gene 1 knockout Gene 2 knockout 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 Overview: Use of double knockout yeast strains to analyze genetic interactions by plasmid shuffle 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 Transform double knockout strain with LEU2 and HIS3 plasmids with gene mutations (Protocol 1)

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

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

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


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