Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus
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-/sup 32/P)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 /sup 32/P-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 /sup 32/P-labeled large subunit and the (/sup 35/S)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.
- Research Organization:
- Harvard Medical School, Boston, MA
- OSTI ID:
- 6137576
- Journal Information:
- J. Virol.; (United States), Vol. 52:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
VACCINIA VIRUS
DNA SEQUENCING
DNA
ENZYMES
HYBRIDIZATION
LABELLED COMPOUNDS
MESSENGER-RNA
METHIONINE
PHOSPHORUS 32
POLYPEPTIDES
SULFUR 35
TRACER TECHNIQUES
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DRUGS
EVEN-ODD NUCLEI
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PARASITES
PEPTIDES
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
RNA
STRUCTURAL CHEMICAL ANALYSIS
SULFUR ISOTOPES
VIRUSES
550201* - Biochemistry- Tracer Techniques