Journal:
Article Title: Transcriptional Activation of sclA by Mga Requires a Distal Binding Site in Streptococcus pyogenes
doi: 10.1128/JB.186.23.7847-7857.2004
Figure Lengend Snippet: Mga regulation of sclA in M1 strains. (A) A search of the serotype M1 GAS SF370 genomic sequence upstream of the known Mga-regulated sclA gene with the published Mga consensus binding sequence (15) revealed two potential Mga-binding sites. An alignment of PsclA Mga-binding site I (PsclA-I) and site II (PsclA-II) with published consensus Mga-binding sequence is shown. PsclA-II overlaps the predicted −35 hexamer of this promoter as described previously (13, 27). PsclA-I represents a novel site and is located farther upstream. Bold, capital letters and lines represent nucleotide identity to the Mga consensus binding sequence, while lowercase letters represent nonidentity. Asterisks indicate nucleotides in PsclA-I that are identical to the Mga-binding consensus but are not conserved in PsclA-II. (B) Northern blot analysis of total RNA isolated from M1 GAS strains SF370 (Mga+, lane 1), KSM165-L (Mga−, lane 2), MGAS166 (Mga+, lane 3), and MGAS166.165-L (Mga−, lane 4). Blots were hybridized with probes to emm and sclA amplified from the PCR primers listed in Table Table11.
Article Snippet: Blots were hybridized overnight at 50°C with 5 × 10 6 cpm of [α 32 P]dATP-labeled emm or sclA probe (RadPrime labeling system; Invitrogen) followed by two low-stringency washes at room temperature (RT) for 5 min and two high-stringency washes at 50°C for 15 min. Blots were visualized by exposure to a phosphorimaging cassette for 2 h. Probes were PCR amplified from a serotype M1 strain by using the primers listed in Table for emm and sclA .
Techniques: Sequencing, Binding Assay, Northern Blot, Isolation, Amplification
