Figure 3.

Dual-bait one-hybrid systems to eliminate background clones derived from methylation-insensitive DNA-binding proteins. (A) Schematic representation of a dual-bait reverse one-hybrid system. In this system, methylatable and unmethylatable versions of the same bait sequence precede ADE2/HIS3 and URA3, respectively. X, methylated DNA-binding protein; BTA, basic transcription apparatus. Filled circle shown on the bait, methylated CpG. Open circle shown on the bait, unmethylated CpG. (B) Discrimination between methylation-dependent and -insensitive DNA-binding by the dual-bait reverse one-hybrid system. In the absence of 5-FOA, both the methylation-dependent DNA-binding protein MBD2 and the methylation-insensitive protein KLF6 made the cell capable of growing in the absence of adenine and histidine (left panel). In the presence of 5-FOA, MBD2, but not KLF6, made the cell capable of growing in the absence of adenine and histidine (right panel). (C) Schematic representation of a dual-bait one-hybrid system using two unrelated methylatable bait sequences. The results of the screening using this system are summarized in Table 2. X, methylated DNA-binding protein; BTA, basic transcription apparatus. Filled circle shown on the bait, methylated CpG.