Fidelity of chromatin transcription in vitro.

Abstract

Chromatin and DNA from Schneider's Drosophila melanogaster cell line 2 were transcribed in vitro with Escherichia coli RNA polymerase. Using mercurated UTP as precursor, the newly synthesized RNA could be separated from DNA and endogenous RNA by affinity chromatography on sulfhydryl-Sepharose 6B. Characterization of the transcription products with complementary DNA (cDNA) made from polyadenylated nuclear RNA and with fractionated cDNA probe demonstrated a fair quantitative fidelity in the in vitro transcript from chromatin which was not evident when DNA was transcribed. However, as shown by hybridization to total nuclear RNA, E. coli RNA polymerase transcribed both DNA strands from chromatin in vitro. We conclude that E. coli polymerase is able to distinguish sections of chromatin at which rapid synthesis of RNA occurs in the cell.