Characterization of the RNA transcribed in vitro from native mammalian DNA by Escherichia coli RNA polymerase.

High-molecular-weight native mouse DNA was transcribed with Escherichia coli RNA polymerase under low salt conditions, and the nature of the DNA sequences transcribed determined by molecular hybridization. The results indicated that E. coli RNA polymerase does not transcribe the sequences in native mouse DNA randomly under these conditions. First, hybridization with a large excess of mouse DNA showed that no more than 5% of the RNA synthesized had been transcribed from repeated sequences in the DNA. Second, hybridization with tracer amounts of labelled non-repeated mouse DNA indicated that the bulk of the RNA had been transcribed from less than 1% of the non-repeated sequences and only about 10% had been transcribed from a further 25% of these sequences; the remaining non-repeated sequences in the DNA, amounting to 50% of the genome, were not represented in the RNA synthesized in vitro to any detectable extent. Third, the proportion (40%) of complementary DNA transcribed from mouse-liver nuclear polyadenylated RNA which hybridized with the RNA synthesized in vitro was significantly greater than would have been expected if transcription had been random. The data have also been interpreted as indicating the presence of two types of initiation site for E. coli RNA polymerase in the non-repeated sequences in mouse DNA. The frequencies of their occurrence have been calculated to be one per 150 000 base-pairs and one per 500 base-pairs, respectively.