72] Template function of restriction enzyme fragments of phage M13 replicative form DNA

Abstract

This chapter discusses the template function of restriction enzyme fragments of phage M13 replicative form DNA. The F-specific filamentous coliphages, among which are included M13, fI, fd, and ZJ/2, are unique among E. coli phages as they do not kill or lyse their host cells. Their genome consists of a circular covalently closed single-stranded DNA molecule, which upon infection, is converted into a double-stranded replicative form molecule. In both in vivo and in vitro, only the nonviral strand of this duplex DNA molecule functions as a template for transcription. The techniques useful for the expression of restriction enzyme fragments of phage M13 replicative form DNA in a coupled transcription and translation system are described. The methods used for the identification and characterization of the in vitro synthesized polypeptides are reported.