PCR, Southern transfer and hybridization, and Ψ sequence analysis of wild-type and ΔrluD::kan mutant E. coli strains. (A) PCR reaction products were generated using genomic DNA as template. Primers used contain sequences homologous to the 5′ and 3′ ends of the rluD gene retained in the ΔrluD::kan mutant strain (forward primer 5′-GAAGCAGTATATATGGCACAACGAGTACAG CTC and reverse primer 5′-GGGAAGCTTTCATAACCAGTCCAC TTCATC). (Lanes 1,3) wild-type MG1655; (lanes 2,4), ΔrluD::kan. (Lanes 1,2) undigested; (lanes 3,4) reaction products digested with 20 units of HindIII (NEB) at 37°C for ≥16 h. Left side of the graph, sizes (kb) of the molecular weight markers. Right side of the graph, sizes (kb) of the DNA bands for each strain. (B) Southern transfer and hybridization were performed using genomic DNA incubated with 20 units of BamHI (NEB) at 37°C for 3 h to achieve partial digestion. Probes were generated from the wild-type MG1655 PCR product by digestion with 10 units NruI (NEB) and 5 units SphI (NEB) to generate the 806-nt probe specific for the center of rluD and a mixed probe specific for the 5′ and 3′ ends of rluD, respectively. Wild-type MG1655 (lanes 1); ΔrluD::kan (lanes 2); (panel I) hybridization with the 806-nt probe; (panel II) the same filter stripped; (panel III) the filter subsequently hybridized with the mixed 5′ and 3′ probes. (C) Pseudouridine sequencing analysis of 23S rRNA from MG1655 and ΔrluD::kan. Total RNA was isolated from the indicated strains and examined for Ψ (Ofengand et al. 2001).
