Figure 8.

Polyomavirus origin-dependent replication assay for replication enhancer activity. (A) A series of plasmids containing the wild type polyomavirus (Py) core origin of replication were used in transient replication assays to test RIP60 for replication enhancer activity. pPyOICAT contains the Py core origin and the native α-enhancer region. The negative control plasmid pPyOICAT lacks the α-enhancer, whereas the positive control plasmid pPy(AM)₆OICAT contains six AP-1 sites in place of the α-enhancer. Test plasmids pPy(DHFR-E)OICAT and pCH30 contained the DHFR-E fragment or five copies of the DSR in place of the α-enhancer, respectively. (B) Representative results of transient Py-origin dependent plasmid replication assays. The indicated reporter plasmids were cotransfected into NIH 3T3 cells with unmethylated pUC19 plasmid DNA, an expression vector for Py large T antigen, and the indicated expression vectors for c-fos, c-jun, or RIP60. After imaging by autoradiography, the signals in each band were quantified in a phosphoimager and the ratio of signal between the reporter and the unmethylated pUC19 control plasmid was calculated. Coexpression of Fos and Jun stimulated replication of pPy(AM)₆OICAT ~40-fold in this experiment.