DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.

Abstract

A cell-free system for studying the synthesis of mRNA in mammalian cells was developed. The system consists of a dialyzed and concentrated whole-cell extract derived from [human cervical carcinoma] HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependnet upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The RNA synthesized in vitro contains the same 5'' capped RNase Tl undecanucleotide as does the in vivo transcript. RNA synthesis also initiates accurately at both an early and an intermediate adenovirus promoter site.