FIG. 5.

The sad1-1 mutant is defective in splicing. (A) Total cell RNAs extracted from the sad1-1 strain (lane 3), three unrelated temperature-sensitive strains (lanes 1, 2, and 4) and the prp2-1 strain (lane 5) grown for 2.5 h at the nonpermissive temperature were resolved on an agarose-formaldehyde gel, transferred to nitrocellulose, and hybridized with a radioactively labelled fragment of the RP29 gene. As a control, RNAs isolated from the prp2-1 strain grown at the permissive temperature (23°C) are presented (lane 6). The positions of migration of the RP29 pre-mRNA and mRNA are indicated. (B) The sad1-1 strain or an isogenic wild-type strain was transformed with either vector alone, a plasmid containing the coding region of the β-galactosidase gene under the control of the GAL10 promoter, the same plasmid in which an intron deriving from the RP51A gene was introduced in the lacZ coding region (intron 1) (62), or the same plasmid in which a synthetic intron was introduced in the lacZ coding region (intron 2) (28). The strains were either grown continually at 23°C or grown for 30 min at 37°C. In either case, galactose was added subsequently and growth was continued for 2 h at the same temperature. Levels of β-galactosidase (bgal) activity produced in these cells are shown (in duplicate).