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    Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12

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    Authors
    Poteete, Anthony R.
    Fenton, Anita C.
    Nadkarni, Ashwini
    UMass Chan Affiliations
    Department of Molecular Genetics and Microbiology
    Document Type
    Journal Article
    Publication Date
    2004-12-15
    Keywords
    Bacteriophage lambda
    Base Sequence
    Chromosomes, Bacterial
    DNA Primers
    DNA, Bacterial
    Escherichia coli K12
    *Gene Duplication
    Polymerase Chain Reaction
    Recombination, Genetic
    Microbiology
    Molecular Biology
    Molecular Genetics
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    Abstract
    BACKGROUND: An Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage lambda Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating lambda vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants. RESULTS: A number of observations concerning recombination events between the chromosome and linear DNAs were made: (1) Formation of Lac+ and Lac- recombinants depended upon the same recombination functions. (2) High multiplicity and high chromosome copy number favored Lac+ recombinant formation. (3) The Lac+ recombinants were unstable, segregating Lac- progeny. (4) A tetracycline-resistance marker in a site of the phage chromosome distant from cat was not frequently co-inherited with cat. (5) Recombination between phage sequences in the linear DNA and cryptic prophages in the chromosome was responsible for most of the observed Lac+ recombinants. In addition, observations were made concerning recombination events between the chromosome and circular DNAs: (6) Formation of recombinants depended upon both RecA and, to a lesser extent, Red. (7) The linked tetracycline-resistance marker was frequently co-inherited in this case. CONCLUSIONS: The Lac+ recombinants arise from events in which homologous recombination between the incoming linear DNA and both lac and cryptic prophage sequences in the chromosome generates a partial duplication of the bacterial chromosome. When the incoming DNA species is circular rather than linear, cointegrates are the most frequent type of recombinant.
    Source
    BMC Mol Biol. 2004 Dec 13;5(1):22. Link to article on publisher's site
    DOI
    10.1186/1471-2199-5-22
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/40351
    PubMed ID
    15596011
    Related Resources
    Link to article in PubMed
    ae974a485f413a2113503eed53cd6c53
    10.1186/1471-2199-5-22
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