FIG. 6.

The last 10 nt of Le (nt 34 to 44) are not necessary for correct transcription initiation at the NS1 GS signal. Primer extensions were carried out by using templates of total RNA derived from transfections with minigenome A36 (lane 2) or D36 (lane 3) or oligo(dT)-purified RNA derived from transfections with minigenome A36 (lanes 4 and 5), A57 (lane 6), D36 (lane 7), or D57 (lane 8). Lane 4 is a negative control in which the transfection reaction mixture did not contain L plasmid. Lane 9 is a positive control using miniantigenomic RNA synthesized from C4 plasmid in vitro by T7 RNA polymerase. The size of this primer extension product is 1 nt longer than that of A36, consistent with its predicted structure. Lane 1 is a ddC sequencing reaction carried out with minigenome C41 as a template; the position of the first 4 nt (CCCC) of the NS1 GS signal is indicated. Solid arrowheads indicate the positions of cDNAs generated from RNAs initiated at the minigenome 3′ terminus, which are barely detectable in lanes 5 to 8, and a large open arrowhead indicates the position of cDNAs generated from RNAs initiated at the NS1 GS signal.