FIGURE 5.

In vitro methylation assay using wild type, laeA overexpressing, and ΔlaeA nuclear extracts. Approximately 110 μg of crude nuclear protein from each strain was isolated as described under “Experimental Procedures” and incubated with 100 nm [³H]AdoMet for 4 h at 37 °C. When added, MBP-LaeA and LaeA^Δ1–86 were present at 4 μg of protein. The samples were electrophoresed on a 10% Tris glycine polyacrylamide gel with Tris glycine running buffer and Coomassie-stained. Fluorography was performed by treating the gel with EN³HANCE and exposing the dried gel to film for 9 months at −80 °C. The positions of Bio-Rad broad range marker proteins are shown on the left. Recomb, recombinant.