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. 2003 Oct;71(10):5640–5649. doi: 10.1128/IAI.71.10.5640-5649.2003

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Copyright © 2003, American Society for Microbiology

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FIG. 4. — (A) Schematic representation of the glyA-orfA intergenic region showing the probes used for mobility shift experiments. Probe B101IR contains the entire AT-rich IR sequence and extends from nucleotides −39 to −166, where +1 is the transcriptional initiation site of P_orf. Probe B101 lacks the IR region and extends from −77 to −166. Probe B180 extends from −87 to −218, and probe B154 extends from −111 to −218. (B and C) For mobility shift experiments, 2 ng of biotinylated ltx promoter fragments B101 (B) and B101IR (C) was incubated with 7.5 μg of a protein extract derived from A. actinomycetemcomitans 652 and electrophoresed in 5% polyacrylamide gels. Competition experiments were carried out as described above in the presence of 2 to 10 ng of unlabeled B101 probe. Lanes 1, labeled probe; lanes 2, labeled probe and protein extract; lanes 3, labeled probe, extract, and 2 ng of unlabeled B101; lanes 4, labeled probe, extract, and 5 ng of unlabeled B101; lanes 5, labeled probe, extract, and 10 ng of unlabeled B101. (D) DNA probes B101IRL and B101IR20 correspond to B101IR but contain altered IR sequences. These probes were amplified from the recombinant plasmid constructs IRL and IR20 (Fig. 3). Biotinylated probes B101IR (lanes 1 to 4), B101IRL (lanes 5 to 8), and B101IR20 (lanes 9 to 12) were incubated with 7.5 μg of A. actinomycetemcomitans protein extract in the absence and presence of unlabeled B101IR probe (2 and 5 ng) and electrophoresed in 5% polyacrylamide gels. Lane 1, labeled B101IR; lane 2, B101IR and extract; lane 3, B101IR, extract, and 2 ng of unlabeled B101IR; lane 4, B101, extract, and 5 ng of unlabeled B101IR; lane 5, labeled B101IRL alone; lane 6, B101IRL and extract; lane 7, B101IRL, extract, and 2 ng of unlabeled B101IR; lane 8, B101IRL, extract, and 5 ng of unlabeled B101IR; lane 9, labeled B101IR20 alone; lane 10, B101IR20 and extract; lane 11, B101IR20, extract, and 2 ng of unlabeled B101IR; lane 12, B101IR20, extract, and 5 ng of unlabeled B101IR. (E) Probes B180 and B154 lack 10 and 36 bp, respectively, from the 3′ end of probe B101. Biotinylated B180 and B154 were incubated with 30 μg of A. actinomycetemcomitans protein extract and electrophoresed in 5% polyacrylamide gels. Lane 1, labeled B154; lane 2, B154 and extract; lane 3, labeled B180; lane 4, B180 and extract.

FIG. 4. — (A) Schematic representation of the glyA-orfA intergenic region showing the probes used for mobility shift experiments. Probe B101IR contains the entire AT-rich IR sequence and extends from nucleotides −39 to −166, where +1 is the transcriptional initiation site of P_orf. Probe B101 lacks the IR region and extends from −77 to −166. Probe B180 extends from −87 to −218, and probe B154 extends from −111 to −218. (B and C) For mobility shift experiments, 2 ng of biotinylated ltx promoter fragments B101 (B) and B101IR (C) was incubated with 7.5 μg of a protein extract derived from A. actinomycetemcomitans 652 and electrophoresed in 5% polyacrylamide gels. Competition experiments were carried out as described above in the presence of 2 to 10 ng of unlabeled B101 probe. Lanes 1, labeled probe; lanes 2, labeled probe and protein extract; lanes 3, labeled probe, extract, and 2 ng of unlabeled B101; lanes 4, labeled probe, extract, and 5 ng of unlabeled B101; lanes 5, labeled probe, extract, and 10 ng of unlabeled B101. (D) DNA probes B101IRL and B101IR20 correspond to B101IR but contain altered IR sequences. These probes were amplified from the recombinant plasmid constructs IRL and IR20 (Fig. 3). Biotinylated probes B101IR (lanes 1 to 4), B101IRL (lanes 5 to 8), and B101IR20 (lanes 9 to 12) were incubated with 7.5 μg of A. actinomycetemcomitans protein extract in the absence and presence of unlabeled B101IR probe (2 and 5 ng) and electrophoresed in 5% polyacrylamide gels. Lane 1, labeled B101IR; lane 2, B101IR and extract; lane 3, B101IR, extract, and 2 ng of unlabeled B101IR; lane 4, B101, extract, and 5 ng of unlabeled B101IR; lane 5, labeled B101IRL alone; lane 6, B101IRL and extract; lane 7, B101IRL, extract, and 2 ng of unlabeled B101IR; lane 8, B101IRL, extract, and 5 ng of unlabeled B101IR; lane 9, labeled B101IR20 alone; lane 10, B101IR20 and extract; lane 11, B101IR20, extract, and 2 ng of unlabeled B101IR; lane 12, B101IR20, extract, and 5 ng of unlabeled B101IR. (E) Probes B180 and B154 lack 10 and 36 bp, respectively, from the 3′ end of probe B101. Biotinylated B180 and B154 were incubated with 30 μg of A. actinomycetemcomitans protein extract and electrophoresed in 5% polyacrylamide gels. Lane 1, labeled B154; lane 2, B154 and extract; lane 3, labeled B180; lane 4, B180 and extract.