FIG. 8.

(A) Translation of mutant RNAs in Vero cell extracts. RNA transcripts were subjected to in vitro translation reaction in the presence of [³⁵S]methionine-cysteine for 4 h at 32°C. The translation products were electrophoresed on an SDS-polyacrylamide gel, and then the gel was dried. Radioactive signals were detected with a phosphorimager. (B) Positive- and negative-strand RNA synthesis in the cell-free reactions. The product RNA was analyzed by native agarose gel electrophoresis. Radioactive signals were detected with a phosphorimager. The positions of RF and ssRNA are indicated. (C) Cleavage by the hammerhead ribozyme. In vitro transcripts synthesized from pU5′Eco-5′rzm, pU5′Eco-mut9-5′rzm, and pU5′Eco-112-118-5′rzm were electrophoresed on a 3.5% polyacrylamide-7 M urea gel, and then the gel was stained with ethidium bromide. (D) Positive- and negative-strand RNA synthesis in cell-free reactions of mut9 and mut112-118 harboring the 5′ hammerhead ribozyme. The ratios of positive-strand to negative-strand RNA synthesis are indicated.