Journal: Journal of Bacteriology

Article Title: Analyzing the Regulatory Role of the HigA Antitoxin within Mycobacterium tuberculosis

doi: 10.1128/jb.00454-10

Figure Lengend Snippet: FIG. 5. Recombinant HigA protein directly and specifically binds to the higB P2 promoter. Protein-DNA binding assays were performed between pure refolded HigA-His protein and the 68-bp 32P-labeled higB P2 promoter probe (23 fmol) using increasing amounts of protein (A), unlabeled higB P2 promoter fragment (identical to the probe) as specific competitor (B), unlabeled higB P1 promoter fragment as nonspecific competitor (C), and anti-HigA antibody (D). The concentrations of recombinant protein used were 3.5, 7, 14, and 28 M for lanes 2 to 5, respectively, 0 M for lanes 1 and 17, and 56 M for all other lanes. Competitor DNA fragments were added in 5-, 10-, 100-, and 200-fold molar excess of the labeled probe (lanes 8 to 11 and 13 to 16). Anti-HigA antibody was used at final dilutions of 1 in 5,000, 500, 50, and 5 for lanes 19 to 22, respectively.

Article Snippet: For competition or supershift assays, the appropriate amount of unlabeled competitor DNA or rabbit anti-HigA antibody (raised and purified by Cambridge Research Biochemicals Ltd. against a synthesized peptide equivalent to residues 112 to 129 of the HigA protein) was added to the reaction mixture.

Techniques: Recombinant, Binding Assay, Labeling

Journal: Journal of Bacteriology

Article Title: Analyzing the Regulatory Role of the HigA Antitoxin within Mycobacterium tuberculosis

doi: 10.1128/jb.00454-10

Figure Lengend Snippet: FIG. 6. Identification of the HigA DNA-binding motif and the effect of mutating this motif on HigA binding and higB P2 promoter activity. (A) The wild-type and mutated sequences of the 68-bp higB P2 promoter fragment. The experimentally determined P2 promoter transcriptional start site (45) and putative 10 and 35 sequences are in boldface and boxed, respectively. The identified perfect palindromic DNA motif is underlined, with mutated bases highlighted in gray. (B) DNA-binding assays were performed between pure refolded HigA-His protein (0 and 56 M for lanes marked and , respectively) and the 32P-labeled mutated higB P2 promoter probe (23 fmol). (C) Protein-DNA-binding assays were performed between pure refolded HigA-His protein (56 M) and the 32P-labeled wild-type higB P2 promoter probe (23 fmol) using increasing amounts of unlabeled mutated higB P2 promoter fragment as competitor (5-, 10-, 100-, and 200-fold molar excess of the labeled probe for lanes 2 to 5, respectively). (D) -Gal assays were performed on cell-free protein extracts of wild-type M. tuberculosis H37Rv and the higB-Rv1957 deletion strain containing the promoter-lacZ fusion plasmids pASF32 (wild-type higB P2 promoter) and pASF58 (mutated higB P2 promoter) alongside the empty vector control (pEJ414). The data presented are averages of the results for three biological replicates, each assayed in duplicate, and error bars represent standard deviations. The -Gal activity level for the higB-Rv1957 deletion strain containing pASF32 was significantly higher than the equivalent wild-type-strain level (, P 0.05; Student’s t test). The -Gal activity level for the higB-Rv1957 deletion strain containing pASF58 was not significantly different from the equivalent wild-type-strain level (P 0.05; Student’s t test).

Article Snippet: For competition or supershift assays, the appropriate amount of unlabeled competitor DNA or rabbit anti-HigA antibody (raised and purified by Cambridge Research Biochemicals Ltd. against a synthesized peptide equivalent to residues 112 to 129 of the HigA protein) was added to the reaction mixture.

Techniques: Binding Assay, Activity Assay, Labeling, Plasmid Preparation, Control

Journal: Journal of Bacteriology

Article Title: Analyzing the Regulatory Role of the HigA Antitoxin within Mycobacterium tuberculosis

doi: 10.1128/jb.00454-10

Figure Lengend Snippet: FIG. 7. HigA does not bind to the next-most-closely related motif within the genome. (A) Sequence of the 68-bp fragment containing the DNA motif (underlined) between whiB5 and Rv0023, with the two bases mismatched to the HigA DNA-binding motif highlighted in gray. (B) Protein- DNA binding assays were performed between pure refolded HigA-His protein (0 and 56 M for lanes marked and , respectively) and the 32P-labeled whiB5-Rv0023 probe (23 fmol). (C) Protein-DNA-binding assays were performed between pure refolded HigA-His protein (56 M) and the 32P-labeled wild-type higB P2 promoter probe (23 fmol) using increasing amounts of unlabeled whiB5-Rv0023 fragment as competitor (5-, 10-, 100-, and 200-fold molar excess of the labeled probe for lanes 2 to 5, respectively).

Article Snippet: For competition or supershift assays, the appropriate amount of unlabeled competitor DNA or rabbit anti-HigA antibody (raised and purified by Cambridge Research Biochemicals Ltd. against a synthesized peptide equivalent to residues 112 to 129 of the HigA protein) was added to the reaction mixture.

Techniques: Sequencing, Binding Assay, Labeling