FIG. 3.

I-PpoI mRNA is synthesized by pol I. (A) Experimental strategy used to determine which RNA polymerase is responsible for I-PpoI expression. Exon sequences are indicated by filled boxes; intron sequences are indicated by open boxes. Mutations at the I-PpoI target site on plasmid pNOY103R are indicated by an X in the 25S rDNA gene. (B) I-PpoI activity assay showing that I-PpoI protein is not expressed in the pol I ts strain at the restrictive temperature. Lanes: 1, p42 linearized with AvaII; 2 and 3, linearized p42 incubated with protein extract from wild-type (WT) strain INVSc2/I3/pNOY103R grown at 23°C; 4 and 5, linearized p42 incubated with protein extract from NOY401/I3/pNOY103R grown at 23°C; 6 and 7, linearized p42 incubated with protein extract from INVSc2/I3/pNOY103R grown at 37°C; 8 and 9, linearized p42 incubated with protein extract from NOY401/I3/pNOY103R grown at 37°C. For NOY401/I3/pNOY103R grown at 37°C, 1 and 2.5 μg of total protein were used. For the other protein extracts, 200 and 500 ng of total protein were used.