FIG. 2.

Cap-defective mutants efficiently copy a single inserted foreign gene. (A) A schematic of the VSV genome containing an autonomous transcription unit containing all the necessary signals for initiation and termination inserted at the leader-N gene junction. Note that for the template rVSV-CAT-GFP, two I genes containing the conserved VSV gene start and gene end sequences were inserted prior to N. (B) In vitro transcription reactions were reconstituted with 1.0 μg of N-RNA template, 0.5 μg of purified P, and 1.0 μg of the indicated L protein in the presence of [α-³²P]GTP as described in Materials and Methods. Purified RNA was analyzed on acid-agarose gels and detected using a phosphorimager. The identity of the template is shown at the bottom of each panel, and the polymerase molecules are shown at the top. The most-abundant products of premature termination synthesized by the cap-defective polymerases are marked on the left-hand side by asterisks. Note that for the wild-type reactions reconstituted with wild-type L, only half of the fraction was loaded on the gel.