Figure 4. Northern blot analysis of the C. violaceum ohr genes.

(A) Total RNA was extracted from exponential-phase cultures of wild type ATCC 12472 strain under uninduced conditions (UN) and after exposure to 200 µM tBOOH, 200 µM CuOOH, 200 µM H₂O₂, 3% NaCl and 4% ethanol (EtOH) for 15 min. RNA was separated on 1.5% agarose gel, transferred to a nylon membrane and hybridized with radiolabeled probes specific to ohrA, ohrR or ohrB. (B) ohrA induction by lipid hydroperoxides. Wild type cells were exposed to 50 µM and 200 µM linoleic acid hydroperoxide (LaOOH) or to 50 µM oleic acid hydroperoxide (OaOOH) for 15 min prior to Northern blot. (C) Time course of ohrA and ohrR induction. RNA samples from wild type cells untreated (0) or treated with 100 µM tBOOH for the indicated times (0 to 90 min) were hybridized with probes specific to ohrA or ohrR. Ethidium bromide staining of rRNA was used as a loading control and is shown below each Northern blot experiment. The sizes of the bands were estimated by ethidium bromide staining of the molecular weight marker Low Range RNA Ladder Riboruler (Fermentas).