Cell-free translation of early and late mRNAs selected by hybridization to cloned DNA fragments derived from the left 14 million to 72 million daltons of the vaccinia virus genome

Abstract

A preliminary translational map of the left 14 million to 72 million daltons of the vaccinia virus genome was constructed. To facilitate this study, the EcoRI D fragment of HindIII C and the successive HindIII N, M, K, F, E, O, I, G, L, J, H, and D fragments were cloned in the plasmid pBR322. The recombinant DNAs were immobilized on nitrocellulose filters and used to select immediate early viral RNA made in the presence of cycloheximide, early viral RNA made in the presence of cytosine arabinoside, and late viral RNA made in the absence of inhibitors. The selected mRNAs were translated in the reticulocyte cell-free system and the [ 35S]methionine-labeled products were analyzed by polyacrylamide gel electrophoresis. Approximately 75 polypeptides were detected among the translation products of early mRNAs selected to this portion of the genome. Most of these polypeptides were also made with immediate early mRNAs. Specific early mRNAs were selected by hybridization to each HindIII fragment; however, no immediate early mRNA hybridized to HindIII L. When selected late mRNAs were translated, more than 40 distinct new polypeptides were detected and synthesis of early polypeptides was greatly diminished. Late mRNAs were clustered near the center of the genome, particularly in HindIII fragments G, L, J, H, and D. No late polypeptides were detected by translation of mRNA hybridizing to fragments C′, N, M, and K and few were detected using mRNA that hybridized to fragments F, E, and O.