FIG. 2.

EBNA1 binding sites at DS must be exactly 21 bp apart, center to center, to support plasmid replication. (A) At 60 h after transfection of EBNA1-positive 143B cells, plasmids were isolated, digested with BamHI to linearize them and with DpnI to test for replication (loss of bacterial adenine methylation), and detected by Southern analysis. Arrow, full-length, DpnI-resistant plasmid. Plasmids carried oriP with DS intact (WT), with only EBNA1 sites 2 and 4 active (2&4), with only EBNA1 sites 3 and 4 present (3&4), or with 3&4 with altered spacing (−1, −2, and +2). 3&4c and its derivatives, −2c and +2c, also contain the T-to-A consensus mutation at nt 9046 of site 4 (38). The average signals for the replicated plasmid relative to that for the wild type are shown above the blot image. Duplicate transfections were analyzed except for −1 (nt 9050) in lane 10. For lane 1, 4 ng of plasmid was mixed with DNA from nontransfected cells and treated similarly, as a control for DpnI digestion. (B) Test for maintenance of the same set of plasmids in cells grown under selection for 3 to 4 weeks. The Southern analysis of uncut plasmids extracted from 5 × 10⁶ cells is shown. In the last two lanes, 50 and 250 pg of pHEBo was loaded, corresponding to 1.5 and 15 copies per cell. The supercoiled (S) and relaxed circular (RC) forms of the plasmid are indicated at the left. (C) Assay for replication of plasmids containing only EBNA1 sites 1 and 2 at DS during 50 h following transfection, as for panel A, but with only the most relevant part of the blot image shown. 1&2c contains consensus mutations in sites 1 and 2, as does the +1 mutant, for which duplicate transfections of two different plasmid preparations are shown (lanes 7 to 10). 1&2m has 1 bp deleted from the center of site 2. (D) Mutations at DS do not affect the inefficient replication of plasmids that can be detected in the absence of EBNA1. At 47 h after transfection of 293 cells, plasmids were extracted and analyzed as for panel A. Forty-five percent of the DNA was digested with BamHI and DpnI for the upper blot; for the lower blot, 5% of each was digested with BamHI to measure DNA uptake by cells. The average replication of each relative to that of pHEBo (WT), normalized for DNA uptake, is indicated above the upper image. The vector lacking oriP was tested for lanes 1 and 2.