Figure 1.

Transcription of the crs1 gene does not determine accumulation of the RNA. a, Transcriptional run-on (TRO) analysis of crs1 and intronless control genes over a meiotic time course using the F90 strain containing the pat1-114 mutation (see Supplementary Table 3 online, for the complete genotype), which undergoes ectopic meiosis at high temperature (34°C)²,¹². Cells were harvested at the times indicated after induction of meiosis and the rate of RNA synthesis measured as described previously¹³, with the modifications noted in Supplementary Information. Newly synthesized ³²P-UTP-labelled RNA was detected by hybridization to probes immobilized on nitrocellulose filters followed by PhosphorImager analysis. The decreased background in the 5-8 hr TRO assays reflects the lower overall incorporation during late meiosis (D. S. M. & J. A. W., in preparation), as equal amounts of RNA (by A₂₆₀) were applied to each filter. b, Quantitative real-time PCR (qPCR) analysis of crs1 RNA splicing over a meiotic time course. Total RNA was extracted from the F90 strain carrying a deletion of the crs1 gene² (see Supplementary Table 3 online for the complete genotype), and the levels of unspliced and spliced crs1 RNA at the times indicated after the temperature shift assayed as described in Supplementary Methods (online). The absolute amount of each species in aMol per μg RNA was determined by comparison to signals generated using known amounts of T7 transcripts. All assays were performed in triplicate and error bars indicating standard deviation are shown.