The complete 30‐base‐pair origin region of bacteriophage φ X174 in a plasmid is both required and sufficient for in vivo rolling‐circle DNA replication and packaging

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Abstract
The origin of replication of the isometric single-stranded DNA bacteriophages is located in a specific sequence of 30 nucleotides, the origin region, which is highly conserved in these phage genomes. Plasmids harboring this origin region are subject to rolling-circle DNA replication and packaging of single-stranded (ss) plasmid DNA into phage coats in .PHI.X174 or G4-phage-infected cells. This system was used to study the nucleotide sequence requirements for rolling-circle DNA replication and DNA packaging employing plasmids which contain the first 24, 25, 26, 27, 28 and the complete 30-base-pair (bp) origin region of .PHI.X174. No difference in plasmid ssDNA packaging was observed for plasmids carrying only the 30-bp origin region and plasmids carrying the 30-bp origin region plus surrounding sequences (i.e., plasmids carrying the HaeIII restriction fragment Z6B of .PHI.X174 replicative-form DNA). This indicates that all signals for DNA replication and phage morphogenesis are contained in the 30-bp origin region and that no contribution is made by sequences which immediately surround the origin region in the .PHI.X174 genome. The efficiency of packaging of plasmid ssDNA for plasmids containing deletions in the right part of the origin region decreases drastically when compared with the plasmid containing the complete 30-bp origin region (for a plasmid carrying the first 28 bp of the origin region to .apprx. 5% and 0.5% in the .PHI.X174 and G4 systems, respectively). Previous studies have shown that the presence of the first 27 bp of the origin region is necessary and sufficient for cleavage of the viral strand in the origin region by .PHI.X174 gene A protein. Brown et al. have shown that omission of the last 2 bp of the origin region does not interfere with .PHI.X174 rolling-circle DNA replication in vitro. These results therefore suggest that for optimal phage development in vivo, signals in the origin region are utilized which were not yet noticed by the in vitro systems for .PHI.X174 phage DNA replication and morphogenesis.