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. Author manuscript; available in PMC: 2008 Jan 1.
Published in final edited form as: Exp Cell Res. 2006 Sep 28;313(1):109–120. doi: 10.1016/j.yexcr.2006.09.020

Figure 4.

Figure 4

Initiation activity of mutant DNR ori-beta constructs at the specific chromosomal site in Hela cells. (A) Following transfection with ori-beta constructs and drug selection, genomic DNA from clonal cell lines was tested for site-specific integration by PCR. A PCR product using primers 1+2 indicates no integration at the FRT site, but a product using primers 1+3 indicates integration in the correct orientation. (B) Southern blots EcoRI-digested genomic DNA from clonal lines was hybridized to either the Hyg probe or Neo probe as described in the Materials and Methods. The Hyg probe hybridizes to a 2.3 kb fragment in the acceptor cell line and to a 5.6 kb fragment when ori-beta is integrated. The Neo probe hybridizes to a 5.6 kb fragment only in the integrated ori-beta. (C) Summary of the methodology used to prepare and analyze nascent DNA samples as previously described [10]. (D) Analysis of the initiation activity of ori-beta constructs at the specific integration site in Hela. 406, Hela acceptor cell line; WT, full-length ori-beta fragment; ΔDNR, DNR deletion construct; DNRrev, construct in which the DNR orientation was reversed; DNRspcr, replacement of DNR with a 235 bp SV40 intron fragment. “Initiation activity” is defined as the abundance of target sequences in nascent DNA-enriched fractions detected at primer sets pp2b (black bars) or ppHyg (white bars), divided by that at primer set ppGlobin. Brackets indicate SEM for WT.