FIG. 2.
A short region of methylated DNA severely affects tk expression. (A) Schematic representation of the regionally methylated 14-aprt-tkCAT and 11-aprt-tkCAT plasmids. The methylated sites are depicted by lollipops. (B) Methylated (CH3; lanes 2, 4, 6, and 8) or mock-methylated (mock; lanes 1, 3, 5, and 7) plasmids 14-aprt-tkCAT (lanes 1, 2, 5, and 6) and 11-aprt-tkCAT (lanes 3, 4, 7, and 8) were injected into oocyte nuclei, and transcription from the HSV tk promoter was assayed by primer extension at 2 h (lanes 1 to 4) and 16 h (lanes 5 to 8) after injection. Coinjection of pCMVCAT served as an internal standard. The positions of correctly initiated transcripts from the tk promoter (tk) and the CMV promoter (CMV) are indicated. (C) Total DNA was isolated from oocytes 16 h after injection. To evaluate if comparable amounts of mock-methylated (lanes 1 and 3) or patch-methylated (lanes 2 and 4) DNAs were injected, purified DNA was analyzed by Southern blot using the tk promoter fragment as a probe. Positions of supercoiled (sc), linear (lin), and relaxed (rel) DNA are indicated. (D) Methylated or mock-methylated 14-aprt-tkCAT or 11-aprt-tkCAT DNAs were injected together with the CMV internal standard, and primer extension was performed 16 h later. Transcription signals were quantitated with a PhosphorImager, and tkCAT transcription was standardized against the internal standard. tkCAT inhibition is expressed as a percentage of the activity of the 100% active mock-methylated control and is indicated as the mean value calculated from three independent experiments. Vertical bars represent standard deviations.