A plasmid cloning system utilizing replication and packaging functions of the filamentous bacteriophage fd

Abstract

DNA cloning vectors were developed which utilize the replication origin ( ori) of bacteriophage fd for their propagation. These vectors depend on the expression of viral gene 2 that was inserted into phage λ, which in turn was integrated into the host genome. The constitutive expression of gene 2 in the host cells is sufficient for the propagation of at least 100 pfd plasmids per cell. In addition to the fd ori, the pfd vectors carry various antibiotic-resistance genes and unique restriction sites. Some of these vectors have no homologies to commonly used pBR plasmids or to A DNA. The nucleotide sequence of the vectors can be deduced from published sequences. Large DNA inserts can be stably propagated in pfd vectors; these are more stable than similar DNA fragments cloned in intact genomes of filamentous bacteriophage. Inclusion of phage sequences required for efficient phage packaging and infection with a helper phage resulted in formation of phage particles containing single-stranded plasmid genomes. Growth at 42°C without selective pressure results in loss of pfd plasmids.