Figure 2.

Effects of Dbr1-Ala mutations on intron levels in vivo. RNA was prepared from Δdbr1 cells containing wild-type DBR1 (WT), the indicated DBR1-Ala mutant alleles, the plasmid vector with no insert (Δdbr1), or plasmids that express Dbr1, mDbr1 and SpDbr1 under the transcriptional control of the constitutive TPI1 promoter. Total RNA (15 µg) was separated on formaldehyde/agarose gels and transferred to membranes. Sequential hybridizations were carried out using ³²P-labeled DNA probes specific for PGK1 mRNA and the intron sequences of RPS13 and ACT1. Hybridized ³²P-labeled probe was visualized by autoradiography and quantified with a phosphorimager. In each sample, the relative signal measured with the intron probe was divided by the PGK1 signal to control for loading. This number was set to 100% for Δdbr1. The intron level for each of the mutants was normalized to that of Δdbr1. The average of 2–4 measurements (using 4 intron RNAs and 2 control mRNAs; see Materials and Methods) is given as % intron accumulation.