Open AccessDNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.
Author(s) -
James L. Manley,
Andrew Fire,
Amparo Cano,
Phillip A. Sharp,
Malcolm L. Gefter
Publication year - 1980
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.77.7.3855
Subject(s) - transcription (linguistics) , biology , microbiology and biotechnology , dna , rna , rnase p , rna polymerase ii , rnase h , promoter , gene , gene expression , biochemistry , philosophy , linguistics
We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent 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 T1 undecanucleotide as does the in vivo transcript. RNA synthesis also initiates accurately at both an early and an intermediate adenovirus promoter site.