Gene, 39 (1985) 55-59 Elsevier GENE 1391 Cloning of mini-Mu bacteriophage in cosmids: in vivo packaging (In vivo gene fusion; transposon-directed mutagenesis; 2 transduction; Diego de Mendoza* and Albert0 L. Rosa* 55 into pbage lambda heads transposition; recombinant DNA) Departarnento de Bioquimica de la Nutrickin, Institute Superior de Investigaciones Biokigicas (INSIBIO), CONICET - Univer- .sidad National de Tucumrin. Chacabuco 461 4000~San Miguel de Tucumrin (Reptiblica Argentina) Tel. (081)220671 (Received February 2nd, 1985) (Revision received May 29th, 1985) (Accepted May 30th, 1985) SUMMARY A technique has been developed that permits the packaging of mini-Mu-carrying cosmids into phage 2 heads. This procedure has several advantages over packaging into Mu helper capsids: (i) the amounts of DNA to be packaged can be increased, (ii) the packaging efficiency is improved, and (iii) the stability of transducing lysates is high. INTRODUCTION Temperate phage Mu-l can be considered as a transposable element; it integrates and transposes its DNA to random sites in the Escherichia zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA coli host genome (Toussaint and Resibois, 1983). Lysogeny results in Mu insertions into different sites of the * Present addresses: (D.d.M.) Departamento de Microbiologia e Inmunobiologia, Facultad de Ciencias Bioquimicas y Farmactu- ticas, Universidad National de Rosario, Suipacha 531 - 2000 Rosario (Republica Argentina) Tel. (041)390017; (A.L.R.) De- partamento de Quimica Biolbgica, Facultad de Ciencias Quimi- cas, Universidad National de Cbrdoba, 5000 - Cbrdoba (Repu- blica Argentina) Tel. (051)64955. Abbreviations: Ap, ampicillin; cos, cohesive ends of phage /1DNA; d. deletion; kb, kilobase pairs; Km, kanamycin; LB, see MATERIALS AND METHODS, section zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA a; m.o.i., multiplicity of infection; pfu, plaque-forming units; R, resistance; ‘, sensi- tivity; Tc, tetracycline; ts, temperature sensitive; ( ), designates prophage state; [ 1, designates plasmid-carrier state. 0378-l 119/85/$03.30 0 1985 Elsevier Science Publishers genome or into a plasmid if present in the cell (Toussaint and Resibois, 1983). Specific insertion of deleted Mu phage (a mini-Mu) into a plasmid can be obtained by a cointegrate transposition event, follow- ed by packaging of the cointegrate within Mu capsids provided by a nondefective helper phage (Castilho et al., 1984; Chaconas et al., 1980). Upon infection of a recA + cell with such plasmid-transducing phage particles, homologous recombination can occur between two Mu sequences with the same orien- tation leading to the formation of a plasmid with an insertion (Castilho et al., 1984). Mu transduction of a plasmid with mini-Mu insertion is a very useful procedure to mutate hybrid plasmids and to form in vivo lac gene fusions when the mini-bacteriophage contains the E. coli lac operon structural genes (Castilho et al., 1984). However, the size of the cointegrate to be transduced is limited by the amount of DNA that can be packaged inside the Mu head (approx. 38 kb; Castilho et al., 1984). In this paper we report an improvement in the size