Genetic and physical mapping of the late region of bacteriophage T7 DNA by use of cloned fragments of T7 DNA

Abstract

Specific fragments of bacteriophage T7 DNA that account for about 99% of the total molecule have been cloned in the plasmid pBR322. This set of plasmids was used to map individual point mutations of T7, by measuring recombination between T7 mutants and cloned fragments of wild-type T7 DNA. Cloned fragments that complement mutants defective in one or more of genes 2, 3.5, 8, 9, 10, 11, 13, 14 and 18 were also obtained. All but one of the plasmids that provide T7 functions carry a promoter for T7 RNA polymerase, a feature that is probably needed for efficient expression from the plasmid during infection. However, the promoter need not be immediately ahead of the gene; the polymerase can apparently transcribe around the entire plasmid DNA before transcribing the T7 gene. The major protein of the T7 phage head is among those that can be provided from a plasmid, indicating that substantial amounts of plasmid-specified proteins can be made. Using a combination of nucleotide sequence and cloning information the locations of 41 known or potential genes in T7 DNA have now been identified, at least 34 of which are known to specify a protein. T7 genes appear to be closely packed but essentially non-overlapping. The only places left in T7 DNA where undiscovered T7 genes are likely to lie are between genes 6 and 8, and to one or both sides of gene 19. The physical and genetic locations of the promoters and termination site for T7 RNA polymerase have also been defined. Certain fragments of T7 DNA cannot be cloned intact, and the lethality of at least some such fragments appears to be due to weak promoters for Escherichia coli RNA polymerase (in the T7 DNA) linked to T7 genes that are lethal if expressed. Separating the promoter from the lethal gene allows the intact gene to be cloned, but only in the silent orientation, where the predominant transcription from promoters in the pBR322 DNA crosses the inserted T7 DNA in the opposite direction from transcription in wild-type T7 DNA.