Fig. 6. High plasmid-copy numbers favour punctate plasmid transcription. (A) HeLa cells were transiently transfected with increasing concentrations of plasmid pHIV-ß and the plasmid transcription sites were labelled by RNA in situ hybridisation using probes of the ß-globin first intron. Higher DNA concentrations correlated with higher transfection efficiencies (dashed blue curve) and also with a greater percentage of transfected cells displaying punctate plasmid transcription signals (pink curve). (B) HeLa cells were transiently transfected with pHIV- and pHIV-ß at a concentration ratio of 9:1. The cell in this image displays pHIV-ß in TDs and pHIV- in punctate transcription sites indicating that high plasmid copy numbers do not inhibit TD formation by non-homologous plasmid in the same cell. Bar, 5 µm. (C) Plasmid pCMV-GFP-ß was transiently transfected into HeLa cells and the plasmid transcription sites were labelled by RNA in situ hybridisation using probes to the ß-globin first intron. RNA and GFP-ß-globin signal intensities were recorded for individual cells and plotted according to the plasmid transcription pattern. Results were normalised to the signal intensities observed in cells with TDs. Cells with punctate plasmid transcription showed significantly elevated levels of GFP staining relative to cells with TDs. A cell transfected with pCMV-GFP-ß displaying plasmid TDs (red) and GFP signals (green) is shown on the right. The overlapping GFP signals and TDs appear yellow. Bar, 5 µm.