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. 2010 Nov 13;39(6):2221–2233. doi: 10.1093/nar/gkq898

Figure 3.

Figure 3.

Identification of Slh1 as a genetic partner of the RBG complexes. (A) Description of the screen for gene having a negative synthetic effect in a Δrbg1Δrbg2 strain. A Δrbg1 Δrbg2 ura3-52 yeast strain containing an URA3-plasmid carrying a RBG1 allele was grown on a CSM-URA plate to stationary phase. Colonies from the plate were resuspended in water, plated on YPDA solid medium (7 × 107 cells /plate) and UV-irradiated. After incubation at 30°C for 48 h, colonies were scraped from the irradiated YPDA plates, resuspended in water and plated on CMS plates (2 × 103 cells/plate; 40 plates). After incubation at 30°C for 48 h, CSM plates were replica-plated on 5FOA plates. The original strain grows well upon loss of the URA3-plasmid [WT like] and is thus resistant to 5-FOA [5-FOAR]. Mutants unable to grow (or growing poorly) on 5-FOA plates [5-FOAS] were retained as putative candidates containing a mutation with a negative synthetic interaction (*si) with the double deletion Δrbg1Δrbg2. To eliminate false positive, 5-FOAS candidates were transformed with a LEU2-plasmid carrying a RBG1 allele. Those were plated on 5-FOA plates and only 5-FOAR transformants were kept for further analysis. (B) The three synthetic slow growth strains isolated in the screen carry a recessive mutation. The original isolates and diploids resulting from the cross of these isolates with an isogenic Δrbg1Δrbg2 strain were streaked in parallel with a Δrbg1/Δrbg1; Δrbg2/Δrbg2 isogenic diploid on YPDA plates and incubated 3 days at 30°C to monitor growth. (C) Meiotic segregation analysis indicates that a single gene is responsible for the synthetic slow growth. Diploid obtained by crossing the three synthetic slow growth strains isolated in the screen with a Δrbg1Δrbg2 strain were sporulated and tetrads were dissected on YPDA plates. Pictures were taken after 4 days at 30°C. Tetrads are numbered 1, 2 and 3 and spores are labeled a, b, c and d. (D) Growth phenotype of yeast strains carrying combinations of deletions of RBG1, TMA46, RBG2 and GIR2 and SLH1 genes. Serial dilutions of exponential liquid cultures were spotted on YPDA plates and incubated 3 days at 30°C.