Figure 2.

Replicating minichromosomes are concentrated in few foci. Cells were cotransfected with II^CMV,DsRed,pA (so transfection efficiencies could be monitored by FACS using DsRed fluorescence) and another encoding EGFP as indicated; after 8 h, cells were replated (to wash away input) and regrown for 4, 16, and 28 h. (A) The DNA copy number of II^CMV,EGFP,pA per transfected cell—determined by reference to known amounts of pure plasmid DNA (by blotting using a “Hirt” extract and qPCR using total DNA)—increases above the maximum possible background due to input (upper limit of gray area). Numbers (24 h) obtained in analogous experiments for I^45S,EGFP,pA, III^7SK,EGFP,pT, and II^CMV,EGFP_V,pA were similar (i.e., 23,000 ± 2,000, 18,000 ± 3,000, and 21,000 ± 3,000, respectively; one-way ANOVA, P > 0.05). (B) Promoters and introns affect the RNA copy number per transfected cell (determined using qRT-PCR by reference to known amounts of pure RNA). (C) DNA FISH shows that minichromosomes are concentrated in ∼20 foci. (inset) Fluorescent bead used for normalization. Bar, 2.5 μm. (D) Mean intensities (+SD) of pixels in or outside foci in 100 images like those in C were normalized relative to the beads; most signal is in the foci, and this increases with time.