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PMID: 6090698 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus.

Journal of virology ·Vol. 52 ·No. 1 ·1984-10-00 ·Pages 206-14

Morgan JR, Cohen LK, Roberts BE

Abstract

The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5' and 3' termini were precisely located by S1 nuclease analysis.

MeSH Terms
Animals DNA, Viral/genetics Genes Genes, Viral Guanosine Triphosphate/metabolism L Cells/enzymology Macromolecular Substances Methyltransferases/genetics,isolation & purification Mice Molecular Weight Multienzyme Complexes/genetics,isolation & purification Nucleic Acid Hybridization Nucleotidyltransferases/genetics,isolation & purification Peptide Fragments/analysis Phosphoric Monoester Hydrolases Phosphorus Radioisotopes Protein Biosynthesis RNA, Viral/isolation & purification Transcription, Genetic Vaccinia virus/enzymology,genetics Viral Proteins
Chemicals
DNA, Viral Macromolecular Substances Multienzyme Complexes Peptide Fragments Phosphorus Radioisotopes RNA, Viral Viral Proteins Guanosine Triphosphate Methyltransferases Nucleotidyltransferases Phosphoric Monoester Hydrolases capping enzyme, vaccinia virus
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Morgan J R
Cohen L K
Roberts B E
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45 references, click to expand
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.52.1.206-214.1984
Published
1984-10-00
Pages
206-14
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC254507
Subset
IM
Grants
NIAID NIH HHS · 1 R01 AI2030 · United States
NCI NIH HHS · CA 06611 · United States
NCI NIH HHS · T32 CA 09031 · United States
External Links
PubMed source DOI: 10.1128/JVI.52.1.206-214.1984 PMC Full Text View journal details
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