Fig 5.

Alp7R is a DNA-binding protein that binds specifically at alp7C. (A) Fluorescence microscopy of strains expressing alp7R-gfp (agarose pads; scale bar equals 1 μm, all panels are at the same scale). Left, JP3223 (PY79 carrying pP_xylalp7R-gfp) in the presence of 0.5% xylose. Right, JP3322 (PY79 with an integrated copy of alp7C, carrying pP_xylalp7R-gfp) in the presence of 0.1% xylose. Top, cell membranes (FM4-64) and DNA (DAPI). Bottom, cell membranes and GFP. (B and C) Electrophoretic mobility shift assays (EMSA) of alp7C DNA by the Alp7R protein. See Materials and Methods. The number above each lane indicates the molar ratio of Alp7R protein to the DNA amplicon. (B) Competitor poly(dI-dC) was omitted from the binding reaction mixture. The high-molecular-weight material consists entirely of complex. (C) Competitor poly(dI-dC) was present in the binding reaction mixture at 25 μg ml⁻¹. At the higher Alp7R-to-alp7C ratios, the high-molecular-weight mate- rial contains both competitor and complex. (D) The percentage of unshifted DNA in each lane of panels B and C is plotted against the molar ratio of Alp7R to DNA. Black filled symbols, alp7C amplicon with competitor, from the experiment represented in panel C; black open symbols, alp7C amplicon without competitor, from the experiment represented in panel B; blue, alp7R amplicon with competitor, from the experiment represented in panel C; green, origin of replication amplicon with competitor, from the experiment represented in panel C.