Grow on5-FOA

ARS

CEN

LEU2

Mutant Gene 1

ARS

CEN

HIS3

MutantGene 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

MutantGene 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 HIS3plasmids with gene mutations (Protocol 1)

Grow on5-FOA

ARS

CEN

HIS3

Mutant Gene

URA3

ARS

CEN

WT Gene

Gene knockout

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

Grow on5-FOA

ARS

CEN

LEU2

Mutant Gene 1

ARS

CEN

HIS3

MutantGene 2

URA3

ARS

CEN

Gene 1 Gene 2

Gene 1 knockout

Gene 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

MutantGene 2

ARS

CEN

LEU2

Mutant Gene 1

Figure 2

Figure 3

pRS415-mutant 1gene 2

pRS413

pRS415

pRS415-WTgene 2

pRS413-mutant 1gene 1

pRS415-mutant 1gene 2

pRS415-mutant 2gene 2

pRS413-WTgene 1

pRS413-mutant 1gene 1

pRS415-WTgene 2

pRS413-mutant 1gene 1

pRS413-mutant 1 gene 1

pRS415-mutant 2gene 2

pRS413-WTgene 1

pRS415-WT gene 2

pRS413-WT gene 1

NegativeControl

PositiveControl

Control

Syntheticlethal

Control

Control

No syntheticEnhancement

Control