FIG. 1.

(A) Schematic representation of the expression cassettes and oriP status of the tested plasmids. Plasmids were based on pCEP₄ (Invitrogen) deleted for the EBNA1-encoding sequence. The plasmids pTG11056, pTG11052, and pTG11182 carry the wild-type oriP sequences (positions 7334 to 9519 according to reference 3), indicated by oriP. The DS element (positions 8994 to 9134 according to reference 3) within oriP was deleted (pTG11155; ΔDS), or oriP was replaced by the DS element only (positions 9021 to 9133 according to reference 3) (pTG11157). The luciferase (luc) expression cassette contains the mouse HMG1 intron (intron) and the SV40 polyadenylation signal (pA) and was under the control of either the CMV IE1 promoter (CMV p) or the RSV promoter (RSV p; pTG11181 and pTG11182). pTG11052 carries an empty expression cassette. (B) Representation of the relative light units (RLU) obtained after quantification of luciferase activity in 293-EBNA1 cells (Invitrogen) transfected with the indicated plasmids: 3 × 10⁴ cells per well had been seeded on 48-well plates (Costar) and transfected with 30 ng of the indicated plasmid by using lipofectin (Gibco BRL) as described in the manufacturer’s protocol. Aphidicolin (Aph) treatment (1 μg/ml) of 293-EBNA1 cells before and during the transfection is indicated. Cells were harvested 16 h after transfection, and total cell protein was extracted (Promega lysis buffer). One-fifth of the material was used to quantify luciferase activity (Promega luciferase kit; Berthold Microlumat LB96P).