The Filamentous Phage M13 as a Carrier DNA for Operon Fusions In Vitro

Abstract

The possibility of using the filamentous single-stranded DNA phages in transduction experiments has always been an attractive speculation, albeit an unlikely one considering that integration of host genes into phage DNA, as occurs with lambda (Hershey 1971), has never been observed (Ray 1977). In considering this possibility, however, the question arose whether there was any site in the filamentous phage DNA at which integration of foreign DNA (in vivo or in vitro) could occur without loss of viability. One way to identify a nonessential region of the phage genome would be to take advantage of the generality of the translocation of transposons that is known to occur in a rec-independent fashion at many different sites in suitable receptor molecules (Berg et al. 1975; So et al. 1975). Once a transposon was inserted into the filamentous phage DNA, it would then provide many potential sites into which a foreign DNA fragment could be cloned. One difficulty with this approach could be that the inserted transposon is itself unstable and is lost at a significant rate (Herrmann et al. 1978).

An alternate approach would be to focus on the detection of a restriction endonuclease cleavage site within a nonessential region of the phage genome. This would require (1) the generation of linear M13 replicative-form (RF) molecules of unit length with specifically located blunt ends, preferably in a nonessential region of the phage DNA, and (2) a blunt end-to-end ligation between the linear RF molecules and a restriction endonuclease cleavage fragment containing an...