JCS03434 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) Experimental strategy to generate a recombinant EBV expressing HA-tagged EBNA1 protein. (Top) The structures of EBNA1 protein and HA-tagged EBNA1 protein are shown. (Bottom) BAC engineering and transfection strategy. See Materials and methods for the detailed description. Hyg^R, hygromycin resistance gene; Cm^R, chloramphenicol resistance gene; Km^R, kanamycin resistance gene; W, wild-type (endogenous) EBV. (B) PCR primer sequences (77-mers) for targeting the EBNA1 gene. Both primers have a 57-mer oligonucleotide (indicated as bold characters) that is homologous to the target region (EBNA1 gene) of the EBV genome at their 5′ ends, and a 20-mer oligonucleotide homologous to the PCR template (pIPpoI-km) at their 3′ ends.

• Supplemental Figure S2 -

  Fig. S2. (A) Intact BAC clones were recovered from the cells harboring the recombinant EBV. The recovered BAC clones were analyzed by EcoRI digestion and 0.8% agarose gel electrophoresis. The results of eight independent BAC clones are shown. Note that the band pattern of these BAC clones is identical to that of control BACmid DNA that was generated in E. coli, with the only exception of the band of terminal repeats (indicated by arrowhead). (B) IB4 cells are known to have integrated copies of EBV genomes (Hurley et al., 1991b). IB4 cells were subjected to FISH analyses by using an EBV genome-derived FISH probe. Note that a pair of signals symmetrically localizes on a pair of sister chromatids in every metaphase spread (left and middle panels). In addition, the number of FISH signals was either one (unreplicated) or two (replicated) in interphase nuclei (right panel). (C) Cells harboring the recombinant EBV were treated with 50 μM of hydroxyurea for 5 weeks. The localization of remaining EBV genomes was examined by FISH analyses. Note that the signals are asymmetrical on sister chromatids, indicating that EBV genomes are extrachromosomal in these cells.

• Supplemental Figure S3 -

  Fig. S3. Cells were processed for immunofluorescence analyses with either anti-EBNA1 polyclonal antibody (Daikoku et al., 2004) or anti-HA monoclonal antibody as primary antibodies. Cy5-conjugated anti-rabbit IgG and Cy3-conjugated anti-mouse IgG were used as secondary antibodies.

• Supplemental Figure S4 -

  Fig. S4. Quantitative analysis to determine the frequency of symmetrical localization of EBNA1 on sister chromatids of G2-PCCs. The signals of EBNA1 (red) and CENP-C (green) are shown. Number of paired EBNA1 dots with paired CENP-C dots are assumed to be symmetrically localized EBNA1 dots (surrounded by a red rectangle). Number of symmetrically localized EBNA1 dots are indicated in each sub-panel. Additional EBNA1 double-dots that do not fit the above criteria are also shown. Question marks indicate obscure CENP-C staining.