FIG. 4.

Analysis of readthrough products in a Cbf5p-depleted strain. (A) Schematic representation of the Tet::Cbf5 allele (top of the panel) and growth of the Tet::Cbf5 (gray squares) and isogenic (black squares) strains following transfer to doxycycline (Dox)-containing medium (bottom of the panel). Cell density (optical density at 600 nm [OD₆₀₀]) was measured at the indicated time, and the cultures were periodically diluted to be continuously kept in exponential growth. (B) Northern hybridization for the detection of CBF5 mRNA and 35S and 18S rRNAs from the Tet::Cbf5 strain (lanes 0 to 24 h); U6 was used as a normalization control. (C) Northern analysis of snoRNAs (box H/ACA, snR30, snR10, snR5, and snR189; box C/D, snR13) and of the control U6 snRNA. (D) RT-PCR analysis was carried out with set of primers described below the panel on total RNA extracted from following strain: Tet::Cbf5, grown in a complete medium (lanes 0) and then shifted in doxycycline-containing medium for 24 h (lanes 24); ssu72-2, grown at the permissive (lanes 25°C) and restrictive temperatures for 2 h (lanes 37°C); and CTK1 and ctk1Δ (lanes CTK1 and ctk1Δ). No products were observed when reverse transcriptase was omitted during cDNA synthesis (data not shown).