FIGURE 1.

Many tRNA fragments in S. Typhimurium are base paired in cells. (A,B) RNA was isolated from the indicated rnaΔ strains using cold (4°C) phenol/chloroform/isoamyl alcohol, subjected to ethanol precipitation, and resuspended in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl. After mixing with equal volumes of 95% formamide, 5 mM EDTA, and 0.02% bromophenol blue, the RNA was heated to 95°C for 5 min, fractionated in denaturing gels, and subjected to northern blotting to detect 5′ halves of tRNA^Phe(GAA) (A) and tRNA^Leu(UAG) (B). In lane 7, the heating step was omitted. (C,D) The resuspended RNA described in A–B was mixed with 1/3 vol of 25% glycerol and 0.02% bromophenol blue, fractionated in native gels, and analyzed by northern blotting to detect 5′ halves of tRNA^Phe(GAA) (C) and tRNA^Leu(UAG) (D). In lane 7, the RNA was heated to 95°C for 5 min before loading. (E,F) After heating and fractionation in denaturing gels as in A–B, the indicated RNAs were subjected to northern blotting to detect 5′ halves of tRNA^Trp(CCA) (E) and tRNA^His(GUG) (F). (G,H) The same RNAs as in E and F were fractionated in native gels and subjected to northern blotting to detect 5′ halves of tRNA^Trp(CCA) (G) and tRNA^His(GUG) (H). (I–L) The northern blots in A–B and C–D were reprobed to detect 3′ halves of tRNA^Leu(UAG) and tRNA^Trp(CCA) after fractionation in denaturing (I,J) or native gels (K,L).