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. 2014 May 21;42(12):8008–8023. doi: 10.1093/nar/gku431

Figure 2.

Figure 2.

Cwc2 F183D is lethal with U2-C22 substitutions and U6-A79G at 37°C. (A) The secondary structure of U2-U6 helix I and the U6 ISL are displayed with the location of U2-U22 and U6-A79 noted by a dashed circle. (B) The plasmid shuffle assay was performed in the CWC2KO/U2KO strain containing the wild-type CWC2 and U2 genes on a URA3 marked plasmid. The strain was co-transformed with mutant Cwc2 and U2 alleles on LEU2 and HIS3 marked plasmids, respectively. One in five serial dilutions of yeast, with an initial OD600 of 1, were grown on plates containing 5-FOA at 16, 25, 30 and 37°C for 3–5 days (5 days for plates at 16 and 25°C), selecting against the wild-type URA3 plasmid to determine the growth phenotype of combining two viable mutations. (C) The plasmid shuffle assay was performed in the CWC2KO/U6KO strain containing the wild-type CWC2 and U6 genes on a URA3 marked plasmid. The strain was co-transformed with mutant Cwc2 and U6 alleles on LEU2 and HIS3 marked plasmids, respectively. One in five serial dilutions of yeast, with an initial OD600 of 1, were grown on plates containing 5-FOA at 16, 25, 30 and 37°C for 3–5 days (5 days for plates at 16 and 25°C), selecting against the wild-type URA3 plasmid to determine the growth phenotype of combining two viable mutations.