AMV RNA transcription in cell-free systems and properties of in vitro chromatin-directed RNA synthesis.

In this report we have presented evidence that viral sequences in the genome of AMV-infected myeloblasts can be transcribed in vitro. The RNA products synthesized in either nuclei isolated from these cells or by eukaryotic RNA polymerase B from the isolated chromatin contained approximately 1% virus-specific sequences. This result, which is in agreement with the fraction of viral RNA in infected cells (Garapin et al. 1971), is higher than expected from a random transcription of the genome, and thus shows that a degree of selectivity in transcription is maintained in both systems. The inhibition of synthesis of viral sequences in nuclei by alpha-amanitin as well as the finding that RNA polymerase B catalyzed the synthesis of viral sequences from chromatin support the hypothesis that the expression of viral information is mediated by nucleoplasmic RNA polymerase. An investigation of the properties of the chromatin-directed products led to the suggestion that RNA synthesis in vitro was initiated on single-stranded or denatured regions of the template; a limiting factor in the synthesis of large molecular weight RNA from isolated chromatin appeared to be the extent of the denatured region available to the enzyme. These findings are consistent with the suggestion that gene activation in eukaryotic organisms results from the unwinding of segments of chromatin DNA (Crick 1971).