Figure 4

Sodium bisulfite sequencing of three different regions of the EBNA1 gene from transfected plasmids. A) Fully unmethylated p220.2, B) HhaI methylated p220.2, and C) HpaII methylated p220.2 were harvested eight days after co-transfection into human cells and sodium bisufite sequenced. Regions 2, 4, and 6 of the EBNA1 gene as designated previously [14] were sequenced. There is a single HhaI site in region 2 and region 6, and there are three HpaII sites in region 4 as indicated on the top of the CpG sites and designated with a box. The percentage of methylation was calculated by dividing the total number of methylation sites detected (excluding methylation found in the HhaI and HpaII sites) by the total number of CpG sites examined (also excluding the HhaI and the HpaII sites). The white rectangular box indicates the premethylated HhaI and HpaII sites on the plasmids. D) Sorting and statistical analysis of the two groups of sequenced molecules from each region. The degrees of freedom (df) are equal to 2. Column A represents molecules from unmethylated p220.2, column B represents molecules from HhaI-methylated p220.2, and column C represents molecules from HpaII-methylated p220.2.