sRNA hybridization and phenotypic analysis of 5-FOA-resistant epimutants.

(A) sRNA hybridization of epimutants and revertants from an rdrp3 mutant background, before (R–resistant) and after 5 (P5) or 10 (P10) passages without selection. P, rdrp3Δ parental strain (MU439). Blots were hybridized with antisense-specific probes against pyrF, pyrG, or 5S rRNA (loading control). (B) sRNA blot of epimutants and revertants from an rdrp1 mutant background, before (R–resistant) and after 5 passages without selection (P5). P, rdrp1Δ parental strain (MU419). Blots were hybridized with antisense-specific probes against pyrF, pyrG or 5S rRNA (loading control). (C) Phenotypic analysis of one representative epimutant, before and after reversion. A pyrF epimutant (E2) is shown before and after 5 (P5) and 10 (P10) passages without selection, grown on MMC media, MMC supplemented with uridine and uracil, and MMC supplemented with 5-FOA, uridine, and uracil. pyrG-, a known mutant of pyrG, served as a negative control; P, rdrp3Δ parental strain (MU439). (D) A pyrG epimutant (E4) is shown before and after 5 (P5) passages without selection, grown on MMC, MMC supplemented with uridine and uracil, and MMC supplemented with 5-FOA, uridine, and uracil. P, rdrp1Δ parental strain (MU419).