Fig 1.

pH-responsive expression profile of an antisense sRNA, 5′ureB-sRNA, in H. pylori by Northern blotting. (A and C) Total RNAs from wild-type (WT) H. pylori 43504 and a transformant with pTM-P_cagA-5′ureB-sRNA(+) that expressed a high level of 5′ureB-sRNA (sRNA+) were harvested after treatment at pH 7.4 (lanes 1) and pH 4.5 (lanes 2) for 30 min. RNA samples (5 μg) were separated in 6% polyacrylamide-urea gels and then transferred to Zeta-Probe GT membranes. The sRNA was detected with an oligonucleotide sense probe (5′-ureB 2-1S) corresponding to the 5′ ureB with overnight exposure (A), and the ureAB transcripts were detected with a strand-specific oligonucleotide antisense probe (5′-ureB 2-1AS) with 30 min of exposure (C). The gels (stained with ethidium bromide) are shown as loading controls (right panels). (B and D) Relative transcript levels normalized to the corresponding intensity of 23S and 16S rRNAs for ∼290-nt 5′ureB-sRNA (B) and intact 2.7-kb and truncated 1.4-kb ureAB transcripts (D) from wild-type H. pylori 43504 and sRNA overexpression strains under different pH conditions. Arbitrary transcript units were determined by separately quantifying 16S and 23S rRNAs in ethidium bromide-stained gels and ureAB sRNAs from Northern blots at distinct settings for contrast and background subtraction of each sample. Since the bands representing each transcript in the hybridized blots and the bands representing 23S and 16S rRNAs in ethidium bromide-stained gels were quantified in two separate measurements with different settings in contrast and background subtraction, the arbitrary transcript units shown in the bar graphs do not reflect the actual relative levels between the transcripts tested and rRNAs.