FIG. 2.

Analysis of VSV polymerase slippage by an RNase H-oligo(dT) digestion assay. (A) To determine what length of A tract was cleaved by RNase H in the presence of oligo(dT), positive-strand RNAs were transcribed in BHK cells by T7 RNA polymerase from four plasmids encoding subgenomic templates with gene junction U tracts of 6, 7, 9, and 14 nucleotides. These templates were transcribed to yield positive-stranded RNAs having corresponding A tracts of 6, 7, 9, and 14 nucleotides. Metabolically labeled RNAs were annealed to oligo(dT), digested with RNase H, and then visualized by agarose-urea gel electrophoresis and fluorography. Digestion of the labeled T7 RNA polymerase transcripts with an A₆ or A₇ tract was insignificant, whereas an expanded tract of nine A's was subjected to extensive digestion, as shown by the presence of two faster-migrating cleavage products. RNAs having A₁₄ tracts were digested to completion. (B) Metabolically labeled RNAs transcribed by the VSV polymerase from three subgenomic templates were subjected to the RNase H-oligo(dT) cleavage assay and visualized by agarose-urea gel electrophoresis and fluorography. The tetranucleotide sequences of gene junctions of these templates are shown above the corresponding lanes, written 3′-5′; + and − denote presence and absence of oligo(dT). As described above, cleavage products following RNase H/oligo(dT) digestion were released only when the A tract contained more than seven residues. This assay demonstrated that VSV polymerase slippage was more prevalent on RNAs transcribed from the subgenomic template with the tetranucleotide sequence 3′-AAAA-5′ than on templates with tetranucleotide sequence 3′-AGAA-5′ or 3′-CCCG-5′. (C). RNAs transcribed from a total of 20 templates with altered tetranucleotide sequences were analyzed by the RNase H/oligo(dT) slippage assay, and the results were tabulated according to a qualitative assessment of whether digestion of the corresponding RNAs resembled the digestion pattern of RNAs transcribed from the three templates shown in panel 2B. AAAA-like corresponds to abundant digestion, CCCG-like corresponds to no detectable digestion, and AGAA-like corresponds to an intermediate level of digestion.