Transcriptional mapping of the DNA polymerase gene of vaccinia virus

Abstract

Vaccinia virus DNA polymerase, a single-subunit enzyme of 110,000 molecular weight, is induced early after infection. Genetic analysis suggests that the gene encoding the enzyme maps within a 15-kilobase HindIII fragment located 45 kilobases from the left-hand end of the genome. We identified the in vitro translation product with these properties and mapped the transcript by hybrid selection, RNA filter hybridization, and S1 nuclease mapping. Two mRNAs from this region, 3.4 and 3.9 kilobases in size, could be translated in vitro to yield a 110K polypeptide. The two RNAs shared a common 5' terminus and had staggered 3' ends. Sequences mapping entirely within this gene were shown to be biologically active in rescuing mutants with temperature-sensitive or drug-resistant polymerase activity to the wild-type phenotype.

Characterization of a temperature-sensitive mutant of vaccinia virus reveals a novel function that prevents virus-induced breakdown of RNA

“…6) for a DNA fragment from the vaccinia virus DNA polymerase gene. This gene is transcribed early during infection into two mRNAs, 3.9 and 3.4 kb long (33). Early during WT or ts22 infection at 31 or 40°C (2 h postinfection), the probe hybridized to the 3.9-and 3.4-kb transcripts (33; data not shown).…”

“…The membrane was dried, baked at 80°C for 3 h, and prehybridized at 42°C for 12 h. The prehybridization buffer contained 50% formamide (deionized), 0.2% polyvinylpyrrolidone (molecular weight [MW] 40,000), 0.2% bovine serum albumin, 0.2% Ficoll (MW 400,000), 0.05 M Tris hydrochloride (pH 7.5), 1.0 M NaCl, 0.1% sodium pyrophosphate, 1.0% SDS, 10% dextran sulfate (MW 500,000), and denatured salmon sperm DNA (>100 ,ug/ml). A cloned 2.0-kilobase (kb) ClaI-BglII subfragment of the HindIII-E region of the vaccinia virus genome (obtained from P. Traktman), which contains half of the vaccinia virus DNA polymerase gene (33), was labeled by nick translation as described above and used as a hybridization probe. The probe was denatured by boiling at 100°C for 15 min, followed by quick cooling.…”

Molecular cloning of a 2',3'-cyclic nucleotide 3'-phosphodiesterase: mRNAs with different 5' ends encode the same set of proteins in nervous and lymphoid tissues

“…Dlasmid DNA containing. CNP cDNA inserts was covalently bound'tb activated paper (DBMS, and the hybridization selection was performed exactly as described by Traktman et al (1984). Selected mRNA was used to program in vitro translations in the rabbit reticulocyte translation system as recommended (Traktman et al, 1984).…”

Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication

“…plishes the faithful duplication and stable assembly of the viral chromosome. These investigations have identified the gene encoding the viral DNA polymerase (15,27,30,31) and a gene encoding a 90-kilodalton (kDa) protein (12,20,24) that is required continuously throughout DNA synthesis (E. Evans and P. Traktman, manuscript in preparation). In this report, we present the mapping and phenotypic analysis of a third complementation group of viral mutants that arrest at the stage of viral DNA replication (6, 7).…”