FIG. 7.

Replication precedes demethylation of the HpaII sites in the oriP region. A Southern blot of DNA harvested 7, 15, 19.5, 24, 40, and 66 h after transfection and digested with DpnI (D) or HpaII (H) is shown. The same probe used in the experiment in Fig. 3 that contains only the oriP region is used here. (A) DNA from cells transfected with unmethylated pCLH22; (B) DNA from cells transfected with SssI-methylated pCLH22 (me-pCLH22). The thin and thick lines above the time designation in panel B represent the possible methylation states within oriP on molecules replicated zero, one, or two times. Thick lines represent methylated DNA strands, and thin lines represent unmethylated DNA strands. The open circle on one end of the line indicates DNA that retains the bacterial dam methylation (at A of GATC). The DpnI-resistant DNA is detected in the DNA harvested at 40 and 66 h after transfection but not at any earlier time points. Plasmid DNA was not linearized in this experiment; therefore, the uncut DNA appears as nicked and supercoiled bands as indicated. The pCLH22 DNA can be digested by HpaII because it is not methylated. A 916-bp band and the smaller 396- and 326-bp, bands which appear as one band, are detected in the HpaII-digested pCLH22 DNA. The methylated pCLH22 DNA is not digestable by HpaII unless demethylation occurs. In the methylated pCLH22 DNA harvested at or before 40 h after transfection, the DNA remains uncut by HpaII. A 1.3-kb band is clearly detected in the methylated pCLH22 DNA harvested at 66 h after transfection. There are two DpnI-MboI sites within the oriP region that generate three complete digest bands at 2.8 kb, 1.0 kb, and 763 bp. Loss of dam methylation at specific sites on some unreplicated molecules, for unknown reasons, generates a strong 3.5-kb band and a weak 1.4-kb band in the transfected DNA (both methylated and unmethylated) in addition to the three bands described above when probed with the oriP fragment.