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. 1973 Dec;70(12 Pt 1-2):3711–3715. doi: 10.1073/pnas.70.12.3711

Transcription of Simian Virus 40. III. Mapping of “Early” and “Late” Species of RNA

Joe Sambrook 1, Bill Sugden 1, Walter Keller 1, Phillip A Sharp 1
PMCID: PMC427312  PMID: 4129925

Abstract

To determine the orientation of transcription of the E and L strands of DNA from simian virus 40 (SV40), we used linear DNA prepared by cleavage of superhelical viral DNA by endonuclease R·R1 from Escherichia coli as a primer·template for DNA polymerase. The resulting molecules, which were labeled only at the 3′ end of each DNA strand, were then cleaved with Hemophilus parainfluenzae endonuclease Hpa I. The ensuing four DNA fragments, whose locations on the viral genome are known, were separated by electrophoresis, denatured, and hybridized to asymmetric SV40 complementary RNA. From the pattern of hybridization of the fragments containing the labeled 3′ ends, we conclude that transcription of SV40 proceeds in a clockwise direction on the L strand and in a counterclockwise direction on the E strand as drawn on the conventional SV40 map.

To map the “early” and “late” regions of the viral genome, we extracted RNA from lytically infected cells and hybridized it to the separated strands of the four fragments of 32P-labeled SV40 DNA. Early after infection, RNA complementary to part of the E strand of the contiguous fragments A and C was detected. Late polysomal RNA was complementary to part of the L strand sequences of fragments A and C and to the total L strand sequence of fragments B and D.

Keywords: restriction enzymes, RNA-dependent DNA polymerase, strand separation

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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