cbc2-Y24A suppresses the Tgs1-dependence of HIS3-[PCH2] expression and PCH2 meiotic splicing. (A) Serial dilutions of yeast cells harboring the chromosomal HIS3-[PCH2] reporter gene were spotted in parallel on synthetic drop-out (SD) medium containing histidine (complete) or lacking histidine (–His) and incubated for 2 d at 30°C. The genotypes of the cells are indicated on the left. The HIS3-[PCH2] reporter is depicted at the bottom. The HIS3 ORF is colored gray. The PCH2 intron is colored black, and the nonconsensus branchpoint sequence is shown. (B) RNAs isolated from wild-type, tgs1Δ, cbc2-Y24A, and cbc2-Y24A tgs1Δ diploid SKY cells sampled 8 h post-transfer to sporulation medium were reverse transcribed with an oligo(dT) primer, and the cDNAs were PCR-amplified with primers flanking the intron of the chromosomal PCH2 gene (depicted at the bottom). The PCR products were resolved by native agarose gel electrophoresis and visualized by staining with ethidium bromide (the negative image is shown). The left lane shows the product of PCR-amplification of genomic DNA with the PCH2 primers, which is the same size as the RT-PCR product derived from the intron-containing PCH2 pre-mRNA. The positions and sizes (bp) of linear duplex DNA markers are indicated on the left. The positions of the RT-PCR products of unspliced and spliced PCH2 transcripts are indicated on the right.
