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PMID: 183208 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Direct biochemical mapping of eukaryotic viral DNA by means of a linked transcription-translation cell-free system.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 73 ·No. 8 ·1976-08-00 ·Pages 2747-51

Rozenblatt S, Mulligan RC, Gorecki M, Roberts BE, Rich A

Abstract

A method is described for mapping regions of eukaryotic viral DNA coding for specific proteins, utilizing a linked transcription-translation cell-free system primed with DNA fragments generated by restriction endonucleases. Three simian virus 40 (SV40) DNA fragments derived from that region of the DNA expressed late in lytic infection were purified. They were: Hpa I-A (0.76-0.175 map units), Bgl I-EcoRI-B (0.672-0), and Hpa II-EcoRI-B (0.735-0). (Fragments are named from the cleaving restriction endonuclease and electrophoretic mobility. End positions on the conventional map are in clockwise order.) These fragments efficiently stimulated the incorporation of [3H]UTP and [35S]methionine into trichloroacetic-acid-insoluble material in the linkec system. The location of the region of DNA coding for the viral structural proteins VPI, VP2 and VP3 was determined from the spectrum of polypeptide synthesis directed by the individual intact fragments and their specific endonucleolytic digests. The polypeptides synthesized in the cell-free system were characterized on urea-sodium dodecyl sulfate polyacrylamide gradient gels and by two-dimensional tryptic peptide analysis. ..

MeSH Terms
Cell-Free System Chromosome Mapping DNA, Viral/analysis,metabolism Genes Peptides/analysis Protein Biosynthesis Simian virus 40/analysis,metabolism Transcription, Genetic Viral Proteins/analysis,biosynthesis
Chemicals
DNA, Viral Peptides Viral Proteins
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Rozenblatt S
Mulligan R C
Gorecki M
Roberts B E
Rich A
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29 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.73.8.2747
Published
1976-08-00
Pages
2747-51
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC430726
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.73.8.2747 PMC Full Text View journal details
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