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Integration of DNA Fragments Coding for Antibiotic Resistance into the Genome of Phage fd In Vivo and In Vitro

Richard Herrmann, Kristina Neugebauer, Heinz Schaller, Hanswalter Zentgraf


To test the possibility of using filamentous bacteriophages as vectors for cloning and analysis of foreign DNA, a variety of derivatives were constructed from phage fd. DNA segments of different origins and sizes were integrated into the phage genome by in vivo transposition of a transposon or by in vitro recombination between fd replicative-form (RF) DNA and DNA fragments from plasmids coding for antibiotic resistance.

The in vivo studies were carried out with transposon 5 (Tn5), which is known to integrate into many different sites of a DNA genome (Berg et al. 1975; Berg 1976). Phage fd grown on a host carrying Tn5 acquired the ability to transduce kanamycin (Km) resistance, and a variety of phage clones could be isolated and analyzed (Table 1) (Herrmann et al. 1978). Integration of the intact transposon into fd DNA was accompanied by an 80% increase in the size of the DNA and phage as measured by gel electrophoresis and electron microscopy. Electron micrographs also showed the typical stem-loop structure of Tn5 attached to the viral single-stranded (SS) DNA circle (Fig. 1). In nondefective phages the sites of integration of Tn5 were in the intergenic region. Defective transducing phages were characterized by insertions of Tn5 into a phage gene, or by partial deletions or duplications of phage and transposon DNA, or by both. The size of the transducing phage from different defective clones varied from 0.6 μm to 3.0 μm and was directly proportional to the DNA content. These results demonstrate that filamentous bacteriophage...

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