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ARS. ARS. Gene 1 knockout. Gene 2 knockout. CEN. CEN. HIS3. HIS3. ARS. ARS. CEN. CEN. ARS. ARS. CEN. CEN. URA3. URA3. Gene 2. Gene 2. Gene 1. Gene 1. LEU2. LEU2. Mutant Gene 1. Mutant Gene 1. Gene 1 knockout. Mutant Gene 2. Mutant Gene 2. Gene 2 knockout.

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PowerPoint Slideshow about 'Grow on 5-FOA' - sydnee


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Presentation Transcript
slide1

ARS

ARS

Gene 1 knockout

Gene 2 knockout

CEN

CEN

HIS3

HIS3

ARS

ARS

CEN

CEN

ARS

ARS

CEN

CEN

URA3

URA3

Gene 2

Gene 2

Gene 1

Gene 1

LEU2

LEU2

Mutant Gene 1

Mutant Gene 1

Gene 1 knockout

Mutant

Gene 2

Mutant

Gene 2

Gene 2 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)

Grow on

5-FOA

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

slide2

ARS

ARS

Gene knockout

CEN

CEN

HIS3

HIS3

ARS

ARS

CEN

CEN

URA3

URA3

Mutant Gene

Mutant Gene

WT Gene

WT Gene

Figure 1

Plasmid shuffle analysis in yeast

Grow on

5-FOA

Gene knockout

What is growth phenotype of the gene mutation?

slide3

ARS

ARS

Gene 1 knockout

Gene 2 knockout

CEN

CEN

HIS3

HIS3

ARS

ARS

CEN

CEN

ARS

ARS

CEN

CEN

URA3

URA3

Gene 2

Gene 2

Gene 1

Gene 1

LEU2

LEU2

Mutant Gene 1

Mutant Gene 1

Mutant

Gene 2

Mutant

Gene 2

Figure 2

Synthetic enhancement/lethal analysis in yeast

Grow on

5-FOA

Gene 1 knockout

Gene 2 knockout

What is growth phenotype of two viable mutants combined?

slide4

Negative

Control

Positive

Control

Control

pRS413

pRS413-WT gene 1

pRS413-mutant 1

gene 1

pRS415

pRS415-WT gene 2

pRS415-WT

gene 2

pRS413-WT

gene 1

pRS413-mutant 1

gene 1

Synthetic

lethal

Control

pRS415-mutant 2

gene 2

pRS415-mutant 1

gene 2

pRS413-mutant 1

gene 1

pRS413-WT

gene 1

pRS415-mutant 2

gene 2

pRS415-mutant 1

gene 2

pRS413-mutant 1

gene 1

No synthetic

Enhancement

Control

pRS415-WT

gene 2

Control

Figure 3