Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12
UMass Chan Affiliations
Department of Molecular Genetics and MicrobiologyDocument Type
Journal ArticlePublication Date
2004-12-15Keywords
Bacteriophage lambdaBase Sequence
Chromosomes, Bacterial
DNA Primers
DNA, Bacterial
Escherichia coli K12
*Gene Duplication
Polymerase Chain Reaction
Recombination, Genetic
Microbiology
Molecular Biology
Molecular Genetics
Metadata
Show full item recordAbstract
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 siteDOI
10.1186/1471-2199-5-22Permanent Link to this Item
http://hdl.handle.net/20.500.14038/40351PubMed ID
15596011Related Resources
Link to article in PubMedae974a485f413a2113503eed53cd6c53
10.1186/1471-2199-5-22