FIG. 7.
Specificity controls for binding of MopA and MopB to anfA promoter DNA. (A) Use of an internal region of anfA (anfAintern) as negative control for DNA mobility shift assays. The control DNA fragment (anfAintern) (Table 2) was PCR amplified, 32P labeled, and incubated with either 7.5 μM MopA or MopB in the absence (−Mo) or presence (+Mo) of molybdenum. (B) Use of unlabeled anfA promoter fragments as specific competitor DNA. MopA or MopB (2.5 μM) and 32P-labeled anfA promoter fragments were mixed with a 400-, 800-, or 1,600-fold excess of unlabeled competitor DNA (compared to the labeled probe). (C) Use of anfAintern in competition assays. MopA or MopB (2.5 μM) and 32P-labeled anfA promoter fragments were mixed with a 550-, 1,100-, or 2,200-fold excess of unlabeled anfA internal fragments (compared to the labeled probe). All reactions were performed with 5 fmol 32P-labeled PanfA probes.