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. 2008 May 19;181(4):615–623. doi: 10.1083/jcb.200710053

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© 2008 Xu and Cook

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 5. — ChIP-3C shows that similar genes on mini- and host chromosomes lie together. Cells were transfected, grown for 24 (rows 1–7) or 8 h (rows 8–13), and treated with or without cross-linker. Active and inactive chromatin was separated by ChIP (using an antibody against trimethyl K4 in H3) and the proximity between selected genes was assessed by 3C. Images illustrate gels containing amplimers, whereas rows 7 and 13 give the relative cross-linking frequency (the amounts of 3C amplimers in bands were determined by reference to equivalent weights of DNA and normalized relative to ALDOA–ALDOA levels and the PCR control for amplification efficiency). ALDOA is an active housekeeping gene on the host chromosome. (A) Minichromosome–minichromosome. Cotransfection with II^CMV,DsRed,pA + II^CMV,EGFP,pA (II + II) or II^CMV,DsRed,pA + III^7SK,EGFP,pT (II + III). DsRed–EGFP 3C products were obtained from both supernatant and pellet (rows 1 and 3), which is consistent with naked input and/or active minichromosomes in the supernatant, and they were seen only after cross-linking (X-link; rows 1 and 4). More are seen in the pellet when promoters are identical (row 3, compare left and right lanes; row 7, mean of four experiments); therefore, templates lie closer together in the II + II combination than in the II + III combination. ALDOA–ALDOA 3C products are seen only in the pellet (rows 2 and 5) and in similar amounts in both combinations (row 5). Amplimers are obtained in roughly equal amounts using the same primers and synthetic 3C templates made by ligating equimolar amounts of pure DNA from relevant genes (row 6). (B) Minichromosome–host chromosome. Transfection with II^U2,U2G,3′ box (II^U2) or II^CMV,EGFP,pA (II^CMV). More plasmid–host 3C products (detected using primers targeting the plasmid backbone and sequences flanking host U2 genes) are seen with II^U2 than with II^CMV (row 1, compare left lane with the right; row 6, mean of four experiments); therefore, II^U2 lies closer to host U2 units than II^CMV. In contrast, amplimers from ALDOA–ALDOA, synthetic 3C templates, and the Neo^r gene on the backbone (which reflect plasmid copy number) are all obtained in roughly equal amounts (rows 3–5).