FIG. 6.

Integrity of oriP affects copy number. (A) The different mini-EBV mutants were analyzed by the Gardella gel technique in order to confirm the episomal status of the viral genomes 6 months posttransfection. Quantitative PCR and Southern blotting determined the copy numbers of the different mini-EBV variants. For Gardella gel analysis, the indicated amount of cells was lysed for the individual lanes (×10⁵ cells). Two different amounts of Raji cells were included for standardization, resulting in 10 × 10⁶ and 30 × 10⁶ EBV genomes, respectively (51). We used an oriP-bearing plasmid as a hybridization probe that recognizes the prokaryotic backbone of the mini-EBV genomes as well as oriP fragments. The copy numbers of the different mutants were determined using the AIDA Image Analyser software (Raytest) (third row). The resulting absolute copy number is given in row two. In parallel, the copy number was revealed by quantitative PCR generating a standard curve from a series of 10-fold dilutions from purified mini-EBV genomes (row four). The mean values and standard deviations of results of three independent experiments are shown in row five (copies/cell; Q-PCR). (B) Examples of FISH experiments that indicate different populations in the 2908wt-oriP and 2910ΔDS cell lines. 3D stacks of fluorescence images were projected according to the maximum intensity of the acquired volume pixels along the z axis. 2908wt-oriP cells showed an average of 5.5 and 12.4 signals per cell. 2910ΔDS cells illustrated an average of 1.8 and 5.5 signals per cell. Scale bar = 2 μm. The data are summarized in Table 3.