FIG. 3.

Conditional alleles of CET1 mutated in the catalytic region. (A) cet1 point mutants. The upper table lists the coding changes and the phenotypes of three mutant alleles. ts, temperature sensitive, unable to grow at 37°C; cs, cold sensitive, unable to grow at 16°C. The lower schematic shows the location of these coding changes relative to three motifs (motifs A, B, and C [15]) conserved among Cet1, C. albicans Cet1 (44), and Ctl1, a second RNA triphosphatase from S. cerevisiae (29, 30). (B) Conditional phenotypes. Mutants were introduced into cells by plasmid shuffling and spotted on new plates for 2 days at 30 and 37°C and for 6 days at 16°C. (C) Temperature-sensitive mutants in the catalytic region are unstable at the nonpermissive temperature. Extracts were prepared from cells grown at the temperatures indicated as described in the legend to Fig. 2 and tested for Cet1 protein by immunoblotting. Lanes: 2 and 3, wild-type CET1; 4 and 5, cet1-401; 6 and 7, cet1-438. Lane 1 is extract from YSB710 containing only Cet1(205–549).