Analysis of methylation status of CaMV 35S promoter in genistein-treated
plants by restriction digestion of DNA fragments amplified with PCR from bisulfite-treated DNA. (A) Analysis of cytosine at position -288 (relative to the transcription initiation site) of the promoter using AluI. (B) Analysis of cytosines at positions -130, -119, and -79 of the promoter using MaeII. Note that treatments of PCR-amplified fragments with AluI and MaeII both resulted in lower levels of digestion when DNA isolated from genistein-treated plants was used for the analysis, indicating that genistein-treated plants have a lower frequency of cytosine methylation in the promoter. Sizes of DNA fragments (in bp) predicted by complete or partial digestions are indicated below the maps of the promoter. Arrows indicate primers for PCR. The position of the cis-acting as-1 element, to which binding of protein factor(s) is inhibited by cytosine methylation , is shown.