Fig. 1. Transiently transfected plasmids are transcribed in TDs or in a punctate pattern. (A) Plasmid maps of pHIV-ß, pHIV- and pHIV-ß. Boxes indicate promoters and exons, black arrows indicate transcriptional start sites, and `pA' indicates polyadenylation sites. The plasmids are based on the pUC18 backbone (not shown) and contain the HIV-LTR promoter upstream of the plasmid gene. (B) S1 nuclease analysis of cytoplasmic RNA from HeLa cells transiently transfected with pHIV-ß, pHIV-ß or pCMV-ß. The labelled probe was complementary to the third exon and 3' flank sequence of ß-globin, and mismatched with correctly processed mRNAs at the polyadenylation site. All plasmids generated correctly processed mRNAs corresponding to the band marked `ßpA' in lanes 1, 3 and 4. Transcription of pHIV-ß was Tat-dependent (compare lanes 2 and 3), whereas transcription of pCMV-ß was Tat-independent (lane 1). pHIV-ß was also co-transfected with plasmid pHIV-, which had no discernible effect on transcription levels (compare lanes 3 and 5). (C) RNA in situ hybridisation of HeLa cells transiently transfected with pHIV-ß. The majority of transfected nuclei displayed five to 20 discrete nascent RNA signals corresponding to plasmid TDs (left panel). A minority of cells displayed a punctate pattern of plasmid transcription in which many transcription sites were scattered throughout the nucleoplasm (right panel). Bars, 5 µm.