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(A) The Δ tdh1 Δ tdh2 Δ tdh3 strain MR173 containing the plasmid p( URA3 )- Eco GAP was transformed with the indicated HIS3 plasmids. Transformants were selected, grown overnight, and spotted in a five-fold serial dilution on SC media containing 5′FOA for counter-selection of the URA3 plasmid or on SC media as control. Plates were then incubated for 3 days at 30°C. (B) Similar experiment as described in (A), but using Δ rki1 yeast expressing the human RKI1 orthologue (Rpi1) from the URA3 plasmid. (C) Δ tdh1 Δ tdh2 Δ tdh3 yeast overexpressing Sir2 or its mammalian homologues, human <t>SirT1,</t> human SirT2, and mouse SirT2, were processed as in (A). (D) Similar to (C), but overexpressing the mutant proteins Sir2 H364Y and Sir2 P394L . (E) The yeast strain MR110, in which chromosomal TPI1 is deleted and yeast TPI1 is expressed from an URA3 episome, was transformed with the indicated HIS3 plasmids and processed as described above. (F) Similar experiment using the quadruple deletion strain Δ tdh1 Δ tdh2 Δ tdh3 Δ zwf1 . (G) Similar to (E), but using yeast strain MR101, which is isogenic to MR110, but expresses human instead of yeast Tpi1 from the URA3 episome.
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(A) The Δ tdh1 Δ tdh2 Δ tdh3 strain MR173 containing the plasmid p( URA3 )- Eco GAP was transformed with the indicated HIS3 plasmids. Transformants were selected, grown overnight, and spotted in a five-fold serial dilution on SC media containing 5′FOA for counter-selection of the URA3 plasmid or on SC media as control. Plates were then incubated for 3 days at 30°C. (B) Similar experiment as described in (A), but using Δ rki1 yeast expressing the human RKI1 orthologue (Rpi1) from the URA3 plasmid. (C) Δ tdh1 Δ tdh2 Δ tdh3 yeast overexpressing Sir2 or its mammalian homologues, human SirT1, human SirT2, and mouse SirT2, were processed as in (A). (D) Similar to (C), but overexpressing the mutant proteins Sir2 H364Y and Sir2 P394L . (E) The yeast strain MR110, in which chromosomal TPI1 is deleted and yeast TPI1 is expressed from an URA3 episome, was transformed with the indicated HIS3 plasmids and processed as described above. (F) Similar experiment using the quadruple deletion strain Δ tdh1 Δ tdh2 Δ tdh3 Δ zwf1 . (G) Similar to (E), but using yeast strain MR101, which is isogenic to MR110, but expresses human instead of yeast Tpi1 from the URA3 episome.

Journal: PLoS ONE

Article Title: Interfering with Glycolysis Causes Sir2-Dependent Hyper-Recombination of Saccharomyces cerevisiae Plasmids

doi: 10.1371/journal.pone.0005376

Figure Lengend Snippet: (A) The Δ tdh1 Δ tdh2 Δ tdh3 strain MR173 containing the plasmid p( URA3 )- Eco GAP was transformed with the indicated HIS3 plasmids. Transformants were selected, grown overnight, and spotted in a five-fold serial dilution on SC media containing 5′FOA for counter-selection of the URA3 plasmid or on SC media as control. Plates were then incubated for 3 days at 30°C. (B) Similar experiment as described in (A), but using Δ rki1 yeast expressing the human RKI1 orthologue (Rpi1) from the URA3 plasmid. (C) Δ tdh1 Δ tdh2 Δ tdh3 yeast overexpressing Sir2 or its mammalian homologues, human SirT1, human SirT2, and mouse SirT2, were processed as in (A). (D) Similar to (C), but overexpressing the mutant proteins Sir2 H364Y and Sir2 P394L . (E) The yeast strain MR110, in which chromosomal TPI1 is deleted and yeast TPI1 is expressed from an URA3 episome, was transformed with the indicated HIS3 plasmids and processed as described above. (F) Similar experiment using the quadruple deletion strain Δ tdh1 Δ tdh2 Δ tdh3 Δ zwf1 . (G) Similar to (E), but using yeast strain MR101, which is isogenic to MR110, but expresses human instead of yeast Tpi1 from the URA3 episome.

Article Snippet: Human Rpi1 and SirT1 coding sequences were amplified from a human fetal cDNA library (Clontech), mouse SirT1 from a mouse testis cDNA library (Clontech), K. lactis GDP1 from p1696 , SIR2 from pAR14 , Hst2 from BY4741-, and Eco GAP and lac Z from E. coli genomic DNA (strains Xl1blue and GM2929, respectively).

Techniques: Plasmid Preparation, Transformation Assay, Serial Dilution, Selection, Incubation, Expressing, Mutagenesis

Plasmids used in this study.

Journal: PLoS ONE

Article Title: Interfering with Glycolysis Causes Sir2-Dependent Hyper-Recombination of Saccharomyces cerevisiae Plasmids

doi: 10.1371/journal.pone.0005376

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: Human Rpi1 and SirT1 coding sequences were amplified from a human fetal cDNA library (Clontech), mouse SirT1 from a mouse testis cDNA library (Clontech), K. lactis GDP1 from p1696 , SIR2 from pAR14 , Hst2 from BY4741-, and Eco GAP and lac Z from E. coli genomic DNA (strains Xl1blue and GM2929, respectively).

Techniques: