Cell-free transcription of mammalian chromatin. Quantitative measurement of newly synthesized globin messenger RNA sequences.

Abstract

Transcription of globin mRNA sequences from rabbit marrow chromatin was detected by hybridization with globin complementary DNA (cDNA). The presence of newly synthesized RNA in cDNA-globin mRNA hybrids isolated by Cs2SO4 density equilibrium centrifugation required the addition of both rabbit marrow chromatin and DNA-dependent RNA polymerase (Escherichia coli) to the transcription reaction. No globin mRNA sequences were detected in RNA transcribed from rabbit liver chromatin or from rabbit marrow DNA. Selective transcription of globin mRNA sequences was therefore tissue-specific and dependent on the presence of chromosomal proteins. Globin mRNA sequences synthesized by E. coli RNA polymerase were distinguished from those synthesized by chromatin-bound (endogenous) RNA polymerases by the use of alpha-amanitin. A typical reaction with rabbit marrow chromatin yielded 100 mug of purified RNA which contained approximately 5 ng (0.005%) of globin mRNA sequences synthesized by E. coli RNA polymerase, 1 ng (0.001%) of globin mRNA seqeences synthesized by endogenous RNA polymerases, and 4 ng (0.004%) of globin mRNA sequences derived from chromatin-associated (endogenous) RNA. Forty per cent of the globin mRNA sequences derived from endogenous RNA could be removed by poly(U)-Sepharose chromatography. The accurate measurement of globin mRNA sequences required improved conditions for the purification and hybridization of RNA transcribed from chromatin.