Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12.
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.
Poteete
Anthony R
AR
Dept, of Molecular Genetics & Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA. anthony.poteete@umassmed.edu
Fenton
Anita C
AC
Nadkarni
Ashwini
A
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D048168:Escherichia coli K12 / Q000235:genetics*; D020440:Gene Duplication; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial