In vitro transcription of Moloney leukemia virus genes in infected cell nuclei and chromatin: elongation of chromatin associated ribonucleic acid by Escherichia coli ribonucleic acid polymerase

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Abstract
The in vitro transcription of viral specific DNA sequences in nuclei and chromatin isolated from mouse [fibroblast NiH 3T3] cells chronically infected with Moloney murine leukemia virus (Mo-MuLV) was studied. The in vitro RNA synthesized by E. coli RNA polymerase was isolated by SH affinity column following reaction in the presence of 5-Hg-UTP. By comparison of the Crt [DNA-RNA hybridization rate] curves of the in vitro RNA with that of 70S viral RNA, the content of viral sequences is 1.3% in nuclei product and 0.24% in chromatin product, which is lower than the 2.5% found in chromatin associated RNA. This latter value is very close to the in vivo viral RNA content in pulse-labeled [3H]RNA of the infected cells. Unexpectedly, it is observed that > 20% of the chromatin associated RNA prelabeled in vivo with [5-3H]uridine is elongated and tagged with Hg atoms during RNA synthesis catalyzed by the exogenous E. coli RNA polymerase in the presence of Hg-UTP. The elongation reaction is dependent on the presence of all 4 nucleotide triphosphates and appears to be due to E. coli RNA polymerase per se. Most of the viral specific sequences observed in the in vitro RNA products are probably initiated and derived from the chromatin associated species. The implication of the present findings for in vitro RNA synthesis in nuclei and chromatin as related to regulation of gene expression is discussed.