Figure 4.

Generation of the precise viral 3′ terminus is important for launching. (A and B) Several nucleotides in the ribozyme [ribozyme (−)] or at the viral 3′ end of the launching plasmid were modified. RNAs from transformants with the modified or unmodified launching plasmids were analyzed with a positive (A) or negative (B) strand-specific probe for 23S RNA as described in the Fig. 3 legend. (C) A fragment containing the complete sequences of 23S RNA and ribozyme from the modified or unmodified launching plasmid was subcloned into the pBluescript vector downstream of the T7 promoter. Uniformly labeled, run-off T7 transcripts were made in vitro. The transcripts with or without incubation at 55°C in the presence of 5 mM Mg²⁺ were separated in a 5% acrylamide gel and detected by autoradiography. The 90-nt cleavage products containing the ribozyme sequence are indicated. (D) Diagram of the primary transcript from the launching plasmid and modifications introduced thereon. The modified nucleotides and cleavage site by the ribozyme are indicated with filled circles and vertical arrows, respectively.