Fig. 2. EBNA1 binds cell cycle independently at oriP. (A) Mini-EBV plasmid 1478.A used to immortalize human primary B cells. Some functional elements are shown on the outer circle. The plasmid backbone derived from the F-factor plasmid pMBO132 (arrows). EBNA1 is shown with some cis elements [OriP, oriLyt (the lytic origin of DNA replication), the terminal repeats and the W repeats]. The inner circle of the map indicates the locations of the fragments that were analysed by PCR amplification after immunoprecipitation. (B) Enlarged view of oriP (top). The locations and designation of the PCR fragments used to scan the binding sites of EBNA1, HsOrc and Mcm2-Mcm7 proteins are shown below the ruler (sc2-sc10, I3). Different cell cycle phases were separated by centrifugal elutriation and five cell cycle fractions were subjected to ChIP (G1, 40 ml minute^-1; G1/S, 50 ml minute^-1; S, 60 ml minute^-1; S/G2, 80 ml minute^-1; G2/M, 90 ml minute^-1). Cross-linked chromatin of 1x10⁷ cells was used for each immunoprecipitation. Co-precipitated DNA was isolated and 1/50 thereof was used for each PCR. The histogram shows the result of EBNA1-experiments. The difference between the crossing points of the EBNA1 immunoprecipitate and the isotype control is indicated on the logarithmic y axis (C_p). The threshold level marked by the reference I3 is indicated as dotted line. The graph shows the mean values and standard deviations from three independent experiments.

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