Figure 3

The 16% PAGE autoradiograms of the segment 8-derived cDNA fragments produced after reverse transcription of RNPs and RNPs lacking the polymerase complex (RNPs-pol) modified with DMS (A), kethoxal (B) or RNase T1 (C). The reactive bases are indicated on the right. Lane C is an incubation control of unmodified RNPs. Lanes 1–3 are incubations of RNPs with increasing amounts of modifying reagents. Lanes A, G, C and U are vRNA dideoxy sequencing reactions of segment 8 vRNA. Base reactivity at the 5' end of the genomic RNA is only obtained after removal of the polymerase. In (A), lanes 1–3 are incubations with 0.1, 0.2 and 0.6 l DMS. The right panel in (B) is another gel of the same RNPs-pol experiment, which shows the reactive G2 residue more clearly. For RNPs, lanes 1–3 result from incubation with kethoxal for 5, 10 and 20 min, for RNPs-pol, lanes 1 and 2 are 20 and 60 min incubations. In (C), lanes 1–3 are digestions of RNPs with 0.1, 0.5 and 1 U of RNase T1 respectively.