FIG. 3.

Zta(m22/26,74/75) is unable to replicate an oriLyt plasmid. Ethidium bromide-stained gel of electrophoretically separated PCR-amplified fragments of the non-EBV backbone sequences of the oriLyt plasmid pEF52. Vero cells were transfected with pEF52, expression plasmids for the six core replication genes, Mta and Rta plus either control vector DNA or expression plasmids for wild-type Zta or Zta(m22/26,74/75). DNA was isolated from the transfected cells, and segments of the oriLyt plasmid backbone were amplified by PCR either before or after digestion of the DNA with the methylation-sensitive restriction enzyme DpnI. The test primers amplify a 510-bp fragment that contains seven DpnI sites, while the control primers amplify a 330-bp fragment that does not contain any DpnI sites. After DpnI digestion of the transfected cell DNA, the 510-bp fragment should only be amplifiable if the input DNA has been replicated in the transfected cells. Lane 1, marker DNA ladder; lanes 2 to 8, DpnI-digested DNA amplified with the test primers; lanes 9 to 12, undigested DNA amplified with the test primers; lanes 13 to 17, DpnI-digested DNA amplified with the control primers. Plasmid DNA, the PCR reaction was performed directly on the input plasmid DNA; water, no DNA added.