In vivo transcription on oligomeric simian virus 40 (SV40) DNA

Abstract

A simian virus 40 nucleoprotein complex containing an endogenous RNA polymerase activity was extracted from infected BSC-1 nuclei with Triton X-100 in low ionic strength buffer. About 30% of both SV40 DNA and total SV40 transcriptional activity in isolated nuclei are recovered by this procedure. When the nucleoprotein complexes are labeled in vitro with [ 32P] UTP followed by sedimentation in neutral sucrose gradients containing Sarkosyl, labeled RNA is detected as a broad band extending from 21 S to more than 50 S with a major peak at 26 S. After treatment of the labeled complexes with pancreatic ribonuclease, 5 to 8% of the labeled RNA remains associated with the template DNA. The majority of the ribonuclease-resistant material sediments slightly faster than SV40 DNA I. The partially relaxed SV40 DNA I associated with this material represents the predominant template DNA for late SV40 transcription (Birkenmeier et al., 1977). The remainder of the ribonuclease-resistant material sediments in sucrose gradients in a series of faster-sedimenting peaks. The identification of these structures as closed circular oligomeric SV40 DNA molecules that are serving as templates for transcription is based on their sedimentation rates, the time of their accumulation during the lytic cycle, their migration in agarose gels, and their appearance in the electron microscope. At late times after infection at least 10% of the total SV40 template DNA is in the forms of oligomers. The possible correlation between those findings and the presence of high molecular weight virus-specific RNA is discussed.