Gene replacement was confirmed by sequencing. One Spcs clone possessing the desired mutation was designated KD1113. Total
RNA was prepared from S. mutans strains as described previously (Shibata et al., 1999) and cDNA was generated via reverse transcription using Multi-Scribe reverse transcriptase and a random primer (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. RNA samples lacking reverse transcriptase were included as controls to ensure that the results were not due to DNA contamination. Quantitative real-time PCR was performed using the StepOne real-time PCR system (Applied Biosystems) in a final volume of 20 μL containing 10 ng cDNA, 10 μL 2 × Quantitect SYBR Green PCR master mix (Qiagen), and 10 pmol each primer (Table S1; Korithoski et al., 2007). PCR conditions were 95 °C for 15 min, followed by 40 cycles of 94 °C for Veliparib datasheet 15 s, 60 °C for 30 s, and 72 °C for 30 s. All data were normalized against to 16S rRNA gene as an internal standard. The fold-change in expression was determined using the 2−ΔΔCt method (Livak
& Schmittgen, 2001). Total RNA was isolated from UA159 as described in real-time RT-PCR analysis and then purified using the RNeasy Mini Kit (Qiagen). Subsequent procedures, including sample labeling and hybridization for DNA microarray, were performed by NimbleGen Systems Inc. (Madison, WI) and GSK2118436 GeneFrontier Inc. (Tokyo, Japan). Twenty perfectly matching 24-mer probes for individual genes were used
for hybridization. DNA probes Calpain were amplified from S. mutans UA159 genomic DNA using IGR793F and IGR793R primers (Table S1). PCR products were separated on 2% agarose gels and isolated. DNA probes were 3′-labeled with digoxigenin (DIG) using the DIG Gel Shift Kit 2nd Generation (Roche, Mannheim, Germany), with minor modifications according to the manufacturer’s instructions. Briefly, DNA probes (3.85 pmol) were mixed with 1 μL of 1 mM digoxigenin-11-ddUTP (DIG-ddUTP), 400 U of terminal transferase, 4 μL of 25 mM CoCl2, 4 μL of 5 × labeling buffer (1 M potassium cacodylate, 125 mM Tris-HCl, 0.125% bovine serum albumin; pH 6.6), and 10 μL sterile water (total volume 20 μL), and incubated at 37 °C for 15 min. Purified protein (500 ng) and 31 fmol digoxigenin-labeled DNA probe were incubated at room temperature for 15 min in a reaction mixture containing 20 mM Hepes (pH 7.6), 1 mM EDTA, 10 mM (NH4)2 SO4, 1 mM dithiothreitol, 0.2% (w/v) Tween 20, 30 mM KCl, 1 μg poly [d(I-C)], and 100 ng Poly l-lysine. Nucleoprotein complexes were resolved on 6% nondenaturing polyacrylamide gels at 150 V and then transferred to a nylon membrane (ATTO, Tokyo, Japan) for 30 min at 400 mA. The membrane was rinsed briefly in washing buffer [0.1 M maleic acid, 0.15 M NaCl, 0.