Figure 6.

Mutations in the Cwc2 zinc finger and RRM are lethal in vivo due to reduced expression of the mutant protein. A haploid yeast strain with the chromosomal CWC2 gene knocked-out and CWC2 on a URA3 marked plasmid (pRS416-Cwc2) was created. This strain was transformed with either a HIS3 marked plasmid alone (pRS413) or pRS413 with wild-type Cwc2 with three C-terminal FLAG tags (Cwc2-3FLAG) or Cwc2-3FLAG with the indicated Cwc2 mutations in the RRM domain (A) or mutations of conserved N-terminal residues including the zinc finger domain (B). Single colonies from these transformations were streaked onto 5-FOA to select against the wild-type pRS416-Cwc2 plasmid. The plates were photographed after incubation at 30°C for 3 days. (C) Immunoblot with the anti-FLAG antibody and the G6PDH antibody (against Zwf1 protein, as a loading control) of extracts from strains containing the pRS416-Cwc2 wild-type plasmid and the pRS413-Cwc2-3FLAG wild-type or mutant plasmid.