BACTERIOPHAGE-T7 DNA PACKAGING .2. ANALYSIS OF THE DNA-SEQUENCES REQUIRED FOR PACKAGING USING A PLASMID TRANSDUCTION ASSAY

Recombinant plasmds carrying a bacteriophage T7 origin of DNA replication and sequences from the T7 concatemer junction are efficiently packaged into transducing particles during phage infection. With some constructs, as many as 50 transducing particles are produced per infected cell. We have used this plasmid packaging system to determine which T7 DNA sequences are required for the processing and packaging of the plasmid concatemers and to investigate the effects of altering the spacing and orientation of the required sequences. An origin of T7 DNA replication is essential for high-efficiency transduction, presumably to form the plasmid concatemers that are the substrates of the packaging reaction. In addition, two short sequences from the concatemer junction are required, one flanking the site where the right end of T7 DNA is formed (pacR) and the other flanking the site for formation of the left end (pacL). The spacing between pacR and pacL is not important, but the sequences must be positioned in the same orientation on the plasmid. With certain deletions of pacL, the specificity of end formation is reduced but the efficiency of packaging is near normal. Plasmids that contain only one of the two pac sites are packaged at about 10% of the efficiency of those with both sites. The residual packaging of these plasmids results from regeneration of the other packaging site by recombination with T7 phage DNA. To function in plasmid packaging, the sequences from the concatemer junction must be positioned on the plasmid in the same orientation relative to the T7 replication origin as is found in T7 DNA. This apparently results from a requirement for transcription through these sequences in the rightward direction from the T7 promoter that is associated with the replication origin. Such transcription from another T7 promoter (Φ10), that is not itself a replication origin, allows packaging when the origin is in the opposite orientation. © 1990 Academic Press Limited.