FIG. 6.

Extracts made from sad1-1 cells do not splice a pre-mRNA substrate in vitro. Extracts were prepared from two sad1-1 isolates, from a prp2-1 strain, and from a wild-type (wt) strain grown either at the permissive temperature (lanes 1 to 7) or for 2.5 h at the nonpermissive temperature. Four microliters of extract was incubated with a radioactive actin pre-mRNA for 30 min at 25°C. In lanes 5, 6, 7, 12, 13, and 14, 2 μl each of the indicated extracts was used. Subsequently, RNA was deproteinized and analyzed on a denaturing polyacrylamide gel. The positions of migration of the pre-mRNA substrate, the exon 1 and lariat intermediates, and the mRNA and intron lariat products of the reaction are indicated.