Abstract
Purification, properties, and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase III from uninfected and adenovirus 2-infected KB cells
Highlights
Class III DNA-dependent RNA polymerases have been purified from human KB ceils and from KB cells infected with adenovirus 2 by chromatography on heparin-sepharose, DEAE-cellulose, DEAE-Sephadex, CM-Sephadex, and phosphocellulose and by sedimentation in sucrose density gradients
-The combined fractions from heparin-Sepharose chromatography were dialyzed against 10 volumes of Buffer B to a final concentration of 0.05 M ammonium sulfate
A similar purification is achieved from whole cell extracts of uninfected cells resulting in a Fraction F4 containing approximately 50% less protein and nucleic acid than an equivalent number of infected cells
Summary
Class III DNA-dependent RNA polymerases have been purified from human KB ceils and from KB cells infected with adenovirus 2 by chromatography on heparin-sepharose, DEAE-cellulose, DEAE-Sephadex, CM-Sephadex, and phosphocellulose and by sedimentation in sucrose density gradients. The subunit compositions of the purified RNA polymerases were analyzed by polyaerylamide gel electrophoresis under denaturing conditions. Comparative structural studies with class III enzymes fromXenopus Zueuis oocytes and the mouse plasmacytoma, MOPC 315, have revealed striking similarities in subunit composition as well as a few slight differences. Analysis of the transcripts by sucrose gradient sedimentation, polyacrylamide gel electrophoresis, and hybridization to separated strands and restriction endonuclease fragments of adenovirus 2 DNA has demonstrated that the in vitro transcription is random and completely symmetric. $ Recipient of a predoctoral fellowship from Sigma Chemical Co. The function and regulation of RNA polymerase III during infection by adenovirus 2 and the components necessary to effect selective transcription in reconstructed systems
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Published Version
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