Linked Life Data

12401231

Source:http://linkedlifedata.com/resource/pubmed/id/12401231

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2002-10-28
pubmed:abstractText
Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or instability of the cloned DNA, or toxicity of the encoded products. We therefore created two mutant libraries in the human pathogen Helicobacter pylori using a simple, direct mutagenesis technique, which does not require E. coli as intermediate. H. pylori total DNA was digested, circularized and digested again with a frequently cutting restriction enzyme, and the resulting fragments were ligated to a kanamycin antibiotic resistance cassette. Subsequently, the ligation mixture was transformed into the parental H. pylori strain 1061. Insertion of the kanamycin cassette by double homologous recombination into the genome of H. pylori 1061 resulted in approximately 2500 kanamycin resistant colonies. Heterogeneity of kanamycin cassette insertion was confirmed by Southern blotting. The isolation of two independent H. pylori mutants defective in production of urease from this library underlines the potential of this mutagenesis strategy.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0167-7012
pubmed:author
pubmed:issnType
Print
pubmed:volume
52
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
93-100
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Direct random insertion mutagenesis of Helicobacter pylori.
pubmed:affiliation
Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't