FIG. 6.

In vitro transcription activities of nuclear extracts (NE), RNAP II holoenzymes (Holo), and pooled low-molecular-mass fractions (Low). In vitro transcription reactions were carried out using a linear DNA template containing the Ad MLP. Reactions were performed with nuclear extracts (46 μg) or 10 μl of concentrated fractions from gel filtration chromatography, adjusted to contain similar quantities of RNAP II. Transcription was template dependent and sensitive to 2 μg of α-amanitin/ml, indicating transcription by RNAP II. Arrow indicates runoff transcripts originating at the Ad MLP. Lanes 1 and 3, nuclear extracts prepared from infected and uninfected cells. Lanes 2 and 4, concentrated fractions from the ∼2-MDa peak of Sepharose CL-2B gel filtration chromatography (fractions indicated in Fig. 2 and 3). Transcription signals were at background levels in lane 4. Lanes 5 and 6, crude uninfected nuclear extracts and pooled concentrates from the ∼670-kDa peak of Sepharose CL-2B chromatography (fractions shown in Fig. 2 and 3). Lanes 7 and 8, crude infected nuclear extracts and pooled concentrates from the ∼670-kDa peak of Sepharose CL-2B chromatography (fractions indicated in Fig. 2 and 3).