Fig. 3.

Genetic screen for mutations in Utp6 that confer CS. (A) Schematic for the design of the genetic screen for mutations in Utp6. Gap repair using a mutagenized library of utp6 with the p415GPD expression vector was performed in yeast with the endogenous UTP6 under a galactose-inducible promoter. Double-transformants were selected by growth on glucose plates lacking leucine. Colonies were re-struck onto new plates, then replica plated to test for growth at 17°C. Colonies that grew normally at 30°C, but not at 17°C, were further validated as described in Champion et al., 2008. (B) Results of the genetic screen for mutations in Utp6. Indicated are strains derived from YPH499 GAL::3HA-UTP6, each containing a plasmid expressing either wild type (WT) or mutated (cs-1 to cs-22) Utp6. Cells were plated in serial dilutions on glucose plates lacking leucine and grown at either 30°C or 17°C to test for CS. Plasmid inserts were sequenced, and mutations found in Utp6 are listed in Table II.