Figure 3. A Cryptic Transcript Is Produced from the 3′ End of GAL10 .

(A) Northern analysis of RNA from wild-type and Reb1 BSΔ mutant cells grown in rich media containing 2% glucose or 2% galactose. Probes are GAL10 sense, GAL10 antisense, GAL1, ACT1. Bands marked # in galactose samples are GAL10 mRNA, detected since small amounts of antisense RNA probe are produced by template strand switching (“turnaround transcription”) at the end of the DNA substrate during in vitro transcription.

(B) Total and oligo(dT) affinity selected RNA from wild-type and Reb1 BSΔ mutant cells hybridized with GAL10 sense and ACT1 probes.

(C) Cap-dependent 5′ RACE analysis of total RNA performed using a GeneRacer kit with primer GAL10F2. Products were cloned and sequenced to determine transcriptional start site.

(D) Schematic of GAL10 ncRNA 5′ ends as determined by 5′ RACE.

(E) Quantification of transcript half-life after addition of 2% galactose to cells growing exponentially in raffinose medium. RNA was probed for GAL10 antisense, GAL10, GAL1, and ACT1. Quantification represents data from three cultures; y axis is in arbitrary units; error bars indicate ±1 standard error.

(F) GAL10 antisense signal from 20 mg FT4 wild-type RNA compared to signal from in vitro transcribed truncated GAL10 antisense RNA in 20 mg of wild-type RNA. Panels showing GAL10 antisense and the in vitro transcribed RNA derive from the same exposure of the same blot, and no differential processing has been applied.

(G) Northern analysis of GAL10 antisense in TRAMP mutants. Cells were grown to mid-log phase at 25° in rich media containing 2% glucose, except the trf4Δ GAL-trf5 strain,which was grown on 2% galactose prior to a 24 hr shift to 2% glucose. RNA analysis as in (E). *p < 0.05 for Student’s t test of wild-type versus mutant.