Cloning of bacteriophage fd gene 2 and construction of a plasmid dependent on fd gene 2 protein.

Bacteriophage fd gene 2 was cloned in plasmid pBR325. Cells carrying the hybrid plasmid produce about 200 times more enzymatically active fd gene 2 protein than did cells infected with phage fd wild type, as measured by replication of phage fd replicative form I in vitro. Cloned gene 2 supports replication of an artificial phage fd miniplasmid consisting of the origin of bacteriophage fd replication and a gene coding for kanamycin resistance. This plasmid occurs in high copy numbers and is viable only in cells carrying the cloned fd gene 2 or in cells infected with phage fd. Because the miniplasmid is not propagated in natural hosts, it can be considered a safe cloning vector. Its fusion with the gene 2 hybrid plasmid provides an autonomous replicon independent of the polA function of the host cell. fd gene 2 is the only phage-encoded trans-acting function required for replication of double-stranded fd DNA in vivo.