MICs were determined as described previously (Sim et al., 2010). Western blot analysis of the chloramphenicol acetyl transferase (CAT) protein was performed as described previously (Kim et al., 2009). To measure steady-state levels of mutant bdm′-′cat mRNA, cDNA was synthesized using a Prime Script first-strand www.selleckchem.com/products/lee011.html cDNA synthesis kit (Takara) using 1 μg of total RNA isolated from E. coli cells expressing mutant bdm′-′cat mRNA as a template. Then, real-time PCR was performed in a C1000 Thermal Cycler (BioRad) using SYBR Premix Ex Taq (Takara) with the synthesized
cDNA as a template. The primers used were: 5′-ATGTTTACTTATTATCAGGCAG and 5′-TTAAAGCGTAGGGTGCTGGCCAC for bdm, 5′-TGACGAAGTTGACGTTGCTC and 5′-CTTCCAGGTGCAGAGTGTCA for rpsA. Both a primer extension analysis and an in vitro RNA cleavage assay were performed as described previously (Sim et al., 2010). In this study,
RGFP966 clinical trial bdm loop RNA transcripts were synthesized using PCR DNA as a template. PCR DNA was synthesized using two primers, T7 bdm loop F (5′-TAATACGACTCACTATAGGGGCATGGTGTTGTCACTG) and bdm +175R (5′-TTGCTGGTAGATATCAC), and the template DNA was pBRS1 or pBRS1, which contained mutations at the RNase III cleavage sites. Synthesized bdm loop RNA transcripts were either 5′-end labeled with [γ-32P]ATP (3000 mCi mmol−1) and T4 polynucleotide kinase (Takara) or 3′-end labeled with [5′-32P]pCp (3000 mCi mmol−1) and T4 RNA ligase (New England Biolab), separated in 4% polyacrylamide
gels containing 8 M urea. The transcripts were eluted from the gel via mixing in a buffer containing 30 mM Tris-HCl, pH 7.9, 10 mM NaCl, 0.1% sodium dodecyl sulfate, and 0.1 mM EDTA, pH 8.0, for 16 h and were all purified using phenol–chloroform extraction and ethanol precipitation. His-tagged RNase III purification and cleavage assays were performed as described previously (Amarasinghe et al., 2001). Briefly, 1 pmol of labeled RNA was incubated with 0.5 μg of purified RNase III in the presence of 0.25 μg mL−1 of yeast tRNA (Ambion) and 20 U of RNaseOUT™ (Takara) in cleavage buffer (30 mM Tris-HCl, pH 7.9, 160 mM NaCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, pH 8.0). Cleavage reactions were initiated by adding 10 mM MgCl2 after 5 min of incubation at 37 °C. Samples were removed at the designated time intervals, mixed with an equal volume of Gel Loading Buffer II (Ambion), denatured at 65 °C for 10 min, and separated on an 8% polyacrylamide gel containing 8 M urea. EMSAs were performed as described previously (Pertzev & Nicholson, 2006). In these assays, Mg2+ was replaced by Ca2+, promoting substrate binding to RNase III while preventing substrate cleavage. Briefly, 5′-end-labeled RNA was incubated at 37 °C for 10 min with RNase III in a buffer containing 30 mM Tris-HCl, pH 8.0, 160 mM NaCl, 10 mM CaCl2, 0.1 mM EDTA, 0.1 mM dithiothreitol, 5% glycerol, and yeast tRNA (5 μg mL−1).