Fig. 3.

Raji ori's and Raji middle's support of DNA synthesis after establishment is similar to their support of DNA synthesis in endogenous EBV. (A) To measure colony formation, the cells harboring Raji ori plasmids with the DS cassette and Raji ori plasmids after deletion of the DS cassette were cultured in the absence of selection. At different times, the cells were plated into selective media, and their efficiency of colony formation was measured. Before the deletion of the DS cassette, Raji ori plasmids were lost at a rate of 4.4 ± 1.8% per cell generation (indicated by the solid regression line). After the deletion of the DS cassette, Raji ori plasmids were lost at the same rate of 4.4 ± 0.1% per cell generation (indicated by the dashed regression line). (B) Similar experiments to those described in A were performed with cells harboring the Raji middle plasmids before and after deletion of the DS cassette. Before deletion of the DS cassette, Raji middle plasmids were lost at a rate of 2.4 ± 2.2% per cell generation (indicated by the solid regression line). After the deletion of the DS cassette, Raji middle plasmids were lost at a higher rate of 6.5 ± 3.1% per cell generation (indicated by the dashed regression line). (C) To measure rates of proliferation, the cells harboring the Raji ori plasmids with the DS cassette and the Raji ori plasmids after deletion of the DS cassette were cultured in the presence of puromycin for selection. The cells were initially plated at 5 × 10⁴ per milliliter. The cells harboring Raji ori plasmids in the presence of the DS cassette doubled in 26.5 ± 0.4 h and doubled in 25.4 ± 0.5 h after deletion of the DS cassette. (D) In experiments parallel to those in C, cells harboring the Raji middle plasmids with the DS cassette and Raji middle plasmids after deletion of DS cassette were cultured in the presence of puromycin for selection. The cells were initially plated at 1 × 10⁴ per milliliter and measured over time. The cells harboring Raji middle plasmids in the presence of DS cassette doubled in 28.8 ± 2.2 h, whereas those lacking DS doubled more slowly, in 32.8 ± 2.5 h. The data were analyzed with the Wilcoxon rank-sum test; P value (two-sided) = 0.04.