Figure 2. VSV RNA polymerase incorporates BrUTP during transcription in vitro and in vivo.

(A) Incorporation of BrUTP into RNA synthesized in vitro. An autoradiograph of an acid agarose-urea gel is shown, depicting RNA transcribed by T7 RNA polymerase from a plasmid encoding VSV N (lanes 1–5) or synthesized by detergent activated virus in vitro (lanes 6–10) in the presence of increasing concentrations (0, 0.1, 0.5 or 1 mM) of BrUTP. The products of the reactions are indicated alongside the gel. (B) The samples of panel A were immune precipitated using an antibody raised against bromodeoxyuridine prior to acid-agarose gel electrophoresis. (C) The samples of panel A were isolated by oligo-dT chromatography prior to acid-agarose gel electrophoresis. (D) BSR-T7 cells were infected with wild-type VSV and, where indicated (+), transfected 4 hpi with BrUTP (5mM final concentration). Cells were exposed to [³H]-uridine for 5 hours and RNA was isolated prior to acid-agarose gel electrophoresis. Where indicated (IP) the RNA was immunoprecipitated as in panel (B).