Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Human DNA replication initiation factors, ORC and MCM, associate with oriP of Epstein-Barr virus
Figure Lengend Snippet: Detection of Mcm2 at DS of oriP by ChIP. ( A ) Diagram of p818, a 12.6-kb plasmid carrying oriP and flanking sequences (light gray), the EBNA1 gene and flanking sequences (black), and hygromycin B-resistance cassette (hph, dark gray). The regions that were amplified by PCR for ChIP are indicated inside the circle. The distal end of the control region, E1 3′, is 560 bp from the edge of FR and 2.1 kb from DS, whereas E1 5′ is 2.1 kb farther away from oriP . hph is about 6 kb from oriP. ( B ) ChIP of chromatin from 293 cells carrying p818. Chromatin (30 μg) was precipitated by using antibodies to Orc2, Mcm2, or EBNA1 or by using nonimmune rabbit IgG, as indicated. Either 1/10th (lanes 2, 4, and 6) or 1/50th (lanes 1, 3, and 5) of the recovered DNA was amplified by PCR to detect the four indicated regions of the plasmid. DNA from the chromatin preparation was tested in lanes 6–8, by using 6.4, 32, and 160 ng, respectively. PCR products were separated by agarose-gel electrophoresis and detected by staining with SYBR Green. A reverse image is shown. M, marker. The specific PCR products ranged from 184 bp (hph) to 435 bp (DS).
Article Snippet: A clone of the EBV-negative Burkitt's lymphoma cell line, DG75, that is close to tetraploid and carries p818 at about 50 copies per cell was obtained and maintained in RPMI medium supplemented with 9% FBS, penicillin and streptomycin, and hygromycin B at 300 μg/ml. p818 (Fig. A ) is a 12.6-kb plasmid similar to p201 ( ) but contains 4 kb of additional EBV DNA extending rightward from oriP to the Bam HI site at 13215.
Techniques: Chromatin Immunoprecipitation, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, SYBR Green Assay, Marker