FIG. 3.

The efficiency of the initiation of DNA synthesis from engineered origins containing TRF2-half-binding sites correlates with the affinity of EBNA1 for its origin-binding sites. Equal masses of the control plasmid (p3567) and of one of the test plasmids containing wtDS (p3487), the MCS-only (p3488), an origin with the flanking TRF2-half-binding sites (p3512 to p3515), or the S-W-W-S origin (p3516) were electroporated into Raji cells. After 4 days, the extrachromosomal DNA was extracted from 10⁷ cells by alkaline lysis. A total of 90% of the samples was digested with both MluI and DpnI, and 10% was digested with MluI only to linearize the DNA. To test the completeness of DpnI digestion, 5 ng of pPur plasmid was added as “spiked DNA” during extraction. For the analysis of this DNA, a Southern blot using a radiolabeled probe produced by random priming of the pPUR vector backbone was hybridized to the electrophoresed and transferred DNAs. The identity of the test plasmid in each lane is given above it. As standards, 50 and 500 pg of linearized vector backbone were loaded on the left of the blot. This is a representative blot from an experiment conducted in triplicate.