期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1973
卷号：70
期号：12
页码：3711-3715
DOI：10.1073/pnas.70.12.3711
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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{middle dot}R1 from Escherichia coli as a primer{middle dot}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.
关键词：restriction enzymes ; RNA-dependent DNA polymerase ; strand separation
