FIG. 4.

Nucleosomal assembly leads to transcriptional repression of the HPV-16 promoter. (A) Analyses of in vitro transcription from free pHPV-16-Luc DNA (lane 1) or the same plasmid assembled into nucleosomes by increasing amounts of Drosophila S190 extract (lanes 2 to 6). Lane M, 100-bp ladder serving as size standard. The specific transcript initiated at the HPV-16 E6 promoter is indicated by an arrow on the right. (B) The Drosophila S190 extract does not contain nonhistone components that might inhibit in vitro transcription, as shown by the addition of 20, 50, and 70 μl of S190 extract (lanes 2 to 4, respectively) at the beginning of an in vitro transcription reaction with nucleosome-free DNA (lane 1). As a further control, we ensured that the factors Sp1 and AP-1 are not limiting in the basic transcriptional system. Lanes 6 to 11 confirm that an excess of AP-1 and Sp1 does not superstimulate transcription of nonchromatin templates.