Deletion analysis of a replication origin of human cytomegalovirus by a novel assay system with a combination of microinjection and polymerase chain reaction.

We have developed a sensitive assay for DNA replication in mammalian cells that enables us to detect replicated DNA fragments of human cytomegalovirus. A 1/100 portion of DNA extracted from 1000 microinjected cells was subjected to polymerase chain reaction after digestion with appropriate restriction enzymes to differentiate replicated from nonreplicated plasmids. A portion of the amplified DNA was electrophoresed to detect replicated DNA. Subfragments of the HindIII fragment A (24 kb, map units 0.37-0.47) of strain Towne, which contains a previously identified origin of DNA replication (oriLyt), were analyzed by the assay system. A 4.3-kb subfragment (AatII-SacI) replicated as efficiently as the HindIII fragment A. Efficient replication ability was lost with a 1.3-kb deletion from the AatII end or a 0.9-kb deletion from the SacI end. These results suggest that the boundaries of oriLyt of Human cytomegalovirus strain Towne lie within the 1.3- and the 0.9-kb regions of the 4.3-kb fragment.