Figure 4.

Functional investigations of the cloned P_DR2 construct. The sequence section of the pBR322 plasmid with homologies to the mammalian U6 promoter was subcloned into the multiple cloning site of pUC18 and subsequently analysed by transcription experiments in vitro. (A) Dependence of the P_DR2 clone on TBP-DR2. A transcription system, based on phosphocellulose fractions PCB and PCC (lanes 1–7), corresponding to the reconstituted transcription system shown in Figure 1, was supplemented either with TBPwt (lanes 2–4; 5, 10 and 20 ng) or TBP-DR2 (lanes 5–7; 5, 10 and 20 ng). Lane 1 shows the control sample without additional recombinant human TBP. The asterisk indicates the TBP-DR2 dependent vector transcript of ∼320 nt. The transcript resulting from the subcloned promoter is marked by P_DR2. (B) α-Amanitin characterisation of the P_DR2 transcription in vitro. Three identical transcription samples contained 10 µl PCB, 20 µl PCC, 10 ng recombinant human TBP-DR2 and 1 µg pUP_DR2 as template. The control sample without the inhibitor α-amanitin is shown in lane 1. The samples shown in lanes 2 and 3 contained α-amanitin at a concentration of 2 and 300 µg/ml respectively. The P_DR2 transcript is appropriately indicated. The signal at the bottom of the gel represents tRNA contained as a carrier.