Show simple item record

dc.contributor.authorPoteete, Anthony R.
dc.contributor.authorFenton, Anita C.
dc.contributor.authorNadkarni, Ashwini
dc.date2022-08-11T08:09:47.000
dc.date.accessioned2022-08-23T16:43:42Z
dc.date.available2022-08-23T16:43:42Z
dc.date.issued2004-12-15
dc.date.submitted2008-04-14
dc.identifier.citationBMC Mol Biol. 2004 Dec 13;5(1):22. <a href="http://dx.doi.org/10.1186/1471-2199-5-22 ">Link to article on publisher's site</a>
dc.identifier.issn1471-2199 (Electronic)
dc.identifier.doi10.1186/1471-2199-5-22
dc.identifier.pmid15596011
dc.identifier.urihttp://hdl.handle.net/20.500.14038/40351
dc.description.abstractBACKGROUND: 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.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15596011&dopt=Abstract ">Link to article in PubMed</a>
dc.subjectBacteriophage lambda
dc.subjectBase Sequence
dc.subjectChromosomes, Bacterial
dc.subjectDNA Primers
dc.subjectDNA, Bacterial
dc.subjectEscherichia coli K12
dc.subject*Gene Duplication
dc.subjectPolymerase Chain Reaction
dc.subjectRecombination, Genetic
dc.subjectMicrobiology
dc.subjectMolecular Biology
dc.subjectMolecular Genetics
dc.titleChromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12
dc.typeJournal Article
dc.source.journaltitleBMC molecular biology
dc.source.volume5
dc.source.issue1
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1314&amp;context=oapubs&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/315
dc.identifier.contextkey489653
refterms.dateFOA2022-08-23T16:43:42Z
html.description.abstract<p>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.</p>
dc.identifier.submissionpathoapubs/315
dc.contributor.departmentDepartment of Molecular Genetics and Microbiology
dc.source.pages22


Files in this item

Thumbnail
Name:
15596011.pdf
Size:
452.4Kb
Format:
PDF

This item appears in the following Collection(s)

Show simple item record