FIGURE 6.

In vitro methylation of wild type and ΔlaeA extracts of A. nidulans at vegetative, asexual, and sexual growth phases do not reveal LaeA methyl-accepting substrates. Whole cell extracts from wild type or ΔlaeA strains in vegetative (A), asexual (B), or sexual (C) growth stages were prepared as described under “Experimental Procedures.” Samples analyzed were as follows: lane 1, 300 μg of wild type whole cell extract; lane 2, 300 μg of ΔlaeA whole cell extract; lane 3, 300 μg of ΔlaeA whole cell extract mixed with 10 μg of purified MBP-LaeA, and lane 4, 300 μg of ΔlaeA whole cell extract mixed with 10 μg of purified LaeA^Δ1–86. Each sample was incubated with 100 nm [³H]AdoMet for 3 h at 37 °C, separated by 10% Tris glycine polyacrylamide gels with Tris glycine running buffer, and stained with Coomassie. The gels were treated with EN³HANCE, and the dried gels were exposed to film for 1 month at −80 °C. The positions of Bio-Rad broad range marker proteins (M) are shown on the left. Red asterisks denote the position of MBP-LaeA or LaeA^Δ1–86 in the Coomassie-stained gel and in the fluorograph.