Figure 1.

(a) The pT7 plasmid system. In the plasmid pT7Qβ(+)500, a bacteriophage T7 promoter (dark arrow) is directed into 5′ Qβ(+) cDNA. Induction of E. coli BL21(DE3) host cells with IPTG results in RNA transcripts containing the entire 4217 nt Qβ(+) strand sequence, and a polyadenosine sequence (A₁₀₀) 3′ to the last Qβ nucleotide. Transcripts terminate within a 3′ bacteriophage T7 5S T1T2 terminator region. The map of Qβ RNA shows the nucleotide positions of the translational initiation sites for the maturation (mat), coat/readthrough (coat/rt) and replicase (rep) cistrons. (b) Agarose gel analysis showing total RNA isolated from host cells in which pT7Qβ(+)500 plasmid derivatives were used to generate variant Qβ RNA transcripts: lane 1, uninduced; lane 2, T-502 RNA; lane 3, T-504 RNA; lane 4, T-503 RNA; and lane 5, T-500 RNA. For each, the Qβ RNA product represented 25–30% of the total RNA isolated relative to 16S and 23S RNAs. In each case, Qβ RNA transcripts were isolated and confirmed by RT–PCR.