Sizes of bacteriophage T4 early mRNA's separated by preparative polyacrylamide gel electrophoresis and identified by in vitro translation and by hybridization to recombinant T4 plasmids

Abstract

We determined the sized of specific T4 prereplicative nRNA's by preparative polyacrylamide gel electrophoresis, and we used the following two techniques to identify specific gene transcripts; cell-free protein synthesis accompanied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to distinguish T4 polypeptides and hybridization to recombinant plasmids containing T4 DNA of known genetic composition. In our first analysis, the use of nonsense and in-phase deletion mutants allowed unambiguous identification of the functional transcripts that encoded genes 32, rIIB, and rIIA. In addition, we identified the functional transcript that encoded genes 43, 45, 30, 39, and 52, the beta-glucosyl transferase gene, and the deletion 293 region. A single peak of mRNA activity that coded for gp43, gp39, gprIIA, beta-glucosyl transferase, and the polypeptide encoded in the deletion 293 region was present; the other polypeptides were encoded in multiple mRNA species, gp46 and gp32 were encoded by two mRNA's and gp52 and gprIIB were encoded by three nRNA's. By hybridizing fractionated, pulse-labeled early RNA to cloned restriction fragments of T4 DNA, we identified the same specific transcripts for genes 43, 52, and rIIB. In addition, a lower-molecular-weight RNA (presumably degraded nRNA) was present even in pulse-labeled RNA preparations. The distribution of pulse-labeled RNAs that hybridized to gene 39, gene 30, gene rIIA, gene 40 plus gene 41, and gene 42 plus the beta-glucosyl transferase gene indicated extensive degradation. We detected cotranscription of genes rIIA and rIIB by rehybridization of RNA first annealed to an rIIB plasmid and then eluted and annealed to an rIIA plasmid. The size distributions of normal and chloramphenicol-treated RNAs that hybridized to plasmids containing T4 immediate early gene 30, gene 39, gene 40 plus gene 41, and gene 42 plus the beta-glucosyl transferase gene were not significantly different.