Figure 3

Southern blot analysis of de novo methylation on plasmid DNA with various types of pre-existing cytosine methylation. A) Southern hybridization of HhaI-digested plasmid DNA harvested eight days after transfection into human cells. Plasmid DNA with no Dcm methylation (p220.2-Dcm) and with Dcm methylation (p220.2+Dcm) showed no detectable difference in the amount of methylation acquired at HhaI sites. When p220.2-Dcm DNA is methylated in vitro with HpaII methylase (me-HpaII p220.2), HaeIII methylase (me-HaeIII p220.2), or MspI methylase (me-MspI p220.2), and then used in the transfection experiment, no detectable difference due to either p220.2+Dcm or p220.2-Dcm was observed. B) Southern hybridization of HpaII-digested plasmid DNA harvested eight days after transfection into human cells. No detectable difference was observed between p220.2-Dcm, p220.2+Dcm, me-HaeIII p220.2, me-MspI p220.2 and plasmid in vitro methylated with HhaI methylase (me-HhaI p220.2).