FIG. 2.
Effect of extract protein/template DNA ratio on Alu, VAI transcription in vitro. Increasing amounts (10–156 μg protein) of transcription extract prepared from HeLa S3 cells were added to pol III-dependent in vitro transcription assays containing 10 ng mini-s-AluK and 1 ng mini-s-VAI template DNA. (A) Radiolabeled AluK and VAI transcripts were resolved by electrophoresis in a 6% polyacrylamide-urea gel and visualized by autoradiography. Incorporation of [α-32P]GTP into Alu and VAI transcripts was quantified by phosphorimaging and is expressed as the ratio of radiolabeled AluK/VAI transcripts in each lane. (B) Pol Ill-dependent in vitro transcription assays, as in (A), performed at either low (25 μg/assay) or high (150 μg/assay) extract protein/template ratios, using either uninfected HeLa cell extract (U) or extract prepared from HeLa cells 24 h after infection with adenovirus (Ad); autoradiography: 45 h.