Physical Mapping of Hybrid Bacteriophage T7/T3 RNA Polymerase Genes

Abstract

The late regions of the T7 and T3 bacteriophage genomes are transcribed by phage-specified RNA polymerases, the products of gene 1. Although these phage transcriptional systems share many characteristics and are obviously related, they have diverged to such an extent that neither of their respective RNA polymerases utilizes the promotor sites of the other phage at an appreciable rate. However, it is possible to construct viable T7/T3 hybrids which have hybrid gene 1 sequences; the resultant hybrid enzymes exhibit altered transcriptional patterns in that they are capable of transcribing both T7 and T3 DNA to various degrees. The aim of this study was to define more closely the region(s) of the gene 1 sequence which encodes the transcriptional selectivity determinant by correlating the genetic constitution of these hybrid gene 1 sequences with their transcriptional properties. The recombinant sites within the gene 1 regions of several T7/T3 hybrids were mapped by using restriction sites as genetic markers. The results indicated that forcing a crossover event within a particular region often results in the inadvertant selection of additional genetic rearrangements. Several of the hybrid gene 1 sequences were found to have resulted from multiple crossover events, even though only one was directly selected for. In some cases the predicted crossovers were not detected; instead, several hybrids contained recombination sites elsewhere in the gene 1 region. These findings suggest that only certain combinations of T7/T3 gene 1 sequences are compatible; it may be that active hybrid T7/T3 gene 1 sequences rarely result from single genetic rearrangements. Taken together, the results of this study suggest that more than one region of the gene 1 sequence is involved in transcriptional selectivity. More specifically, the region from approximately 25 to 59% (from the left of the gene), together with the carboxyl end, appears to play an important role.