Transposition of bacteriophage Mu: properties of lambda phages containing both ends of Mu.
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Biomedical subjects
Publications and source records attributed to M M Howe.
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A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.
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We have cleaved phage Mu DNA with restriction endonucleases EcoRI and BamHI and have cloned three specific DNA fragments from the middle of the Mu genome into vector plasmid pBR322. By marker rescue experiments, we have determined that the two BamHI cleavage sites in Mu DNA occur within cistrons E and F.
Seve lambda dnif specialized transducing bacteriophages were isolated from Escherichia coli strains containing plasmids carrying the his-nif region of Klebsiella pneumoniae. These phages collectively carry deoxyribonucleic acid for all of the genes in the nif regulon and adjacent deoxyribonucleic acid of K. pneumoniae. The phages were isolated by using Mu insertions in the nif region to direct the integration of lambda pMu phages in nif via formation of lambda pMu-Mu dilysogens which, upon induction, yielded lambda dnif phages. This procedure should be generally applicable for isolating lambda specialized transducing phages carrying genes from E. coli or other bacteria.
A general method for constructing lambda specialized transducing phages is described. The method, which is potentially applicable to any gene of Escherichia coli, is based on using Mu DNA homology to direct the integration of a lambda pMu phage near the genes whose transduction is desired. With this method we isolated a lambda transducing phage carrying all 10 genes in the che gene cluster (map location, 41.5 to 42.5 min). The products of the cheA and tar genes were identified by using transducing phages with amber mutations in these genes. It was established that tar codes for methyl-accepting chemotaxis protein II (molecular weight, 62,000) and that cheA codes for two polypeptides (molecular weights, 76,000 and 66,000). Possible origins of the two cheA polypeptides are discussed.
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Five plasmids with insertions of a heat-inducible Mu prophage in a Mu-sensitive and P1-sensitive derivative of plasmid pRD1, a recombinant R factor containing the his-nif region of Klebsiella pneumoniae, were isolated and characterized. In one plasmid containing the Mu prophage integrated at the his-distal end of nif, selection for heat resistance resulted in the generation of deletions extending from the Mu prophage into the nif region. Thirty of these deltions were used to map 26 point mutations in nif.
Klebsiella pneumoniae M5a1 is naturally resistant to infection by bacteriophage Mu. Mutants of K. pneumoniae sensitive to Mu infection were isolated and found to support both lytic and lysogenic development of Mu. K. pneumoniae lysogens containing a heat-inducible Mu prophage integrated in his were isolated. Strains carrying deletions extending from his into nif were obtained after heat treatment of these lysogens. Such deletions should be useful for determining the map order and cistronic organization of the nif genes.
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