Insertion of a Kanamycin-resistance Gene in Bacteriophage fd

Abstract

Gene structures involved in replication of filamentous phages have been well characterized, and various derivatives, including chimeric phages, have been shown to form infectious filamentous particles (Grandis and Webster 1973; Griffith and Kornberg 1974; Enea and Zinder 1975; Herrmann et al., this volume). Thus, we can now construct appropriate vectors for cloning DNA and transducing phages using these filamentous phages. Our first interest was in isolating composite phages from wild-type fd and pML21, a plasmid containing the replication origin of ColE1 and a gene specifying kanamycin resistance (kan) derived from plasmid R6-5 (Hershfield et al. 1976). Phage fd was grown repeatedly on cells carrying pML21 from which kan-transducing phages were selected. All the kan-transducing phages isolated independently (fd-kan) contained a unique segment of the same size (3100 base pairs), but the sites of insertion apparently were different for each chimeric phage. This type of insertion is characteristic of transposable genetic elements (transposons).

All of the isolated fd-kan phages could transduce the kan marker without helper, but they could not form plaques. Upon transformation of fd-kan DNA into F⁻ cells, fd-kan replicative-form (RF) DNA was maintained in the cells like a plasmid, without creation of fd phage. However, F⁺Kan^r cells prepared by either transduction with fd-kan or transformation with fd-kan RF DNA produced not only fd-kan but also wild-type fd phage, suggesting that the excision of the kan segment from fd-kan DNA is promoted in F⁺ cells at the precise site where the insertion has occurred.

Isolation of kan-transducing Phage fd...