Linked Life Data

10627047

Source:http://linkedlifedata.com/resource/pubmed/id/10627047

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:dateCreated
2000-2-17
pubmed:abstractText
The ability to generate mutants of Mycobacterium tuberculosis will be important if we are to understand the biology of this major pathogen. However, allelic replacement methods have only recently achieved success. We have developed a reproducible method for generating defined mutants of M. tuberculosis using homologous recombination. The transforming DNA was used following pre-treatment either with UV light or alkali denaturation in order to stimulate homologous recombination and abolish illegitimate recombination. Suicide vectors carrying one of nine amino acid biosynthesis genes were electroporated into M. tuberculosis, and homologous recombinants were obtained in all nine genes; eight resulted from single-crossover events (SCOs) and one from a double-crossover event (DCO) (in the metB gene). The remaining colonies were spontaneous hygromycin-resistant mutants; no products of illegitimate recombination were observed. To more efficiently distinguish spontaneous mutants, the lacZ gene was cloned into five vectors (two containing genes not previously tested), and the transformations were repeated. SCO mutants were identified by screening for blue colonies on indicator plates. White transformants were tested for auxotrophy and trpD, hisD and proC auxotrophic mutants were obtained. Only blue SCOs were obtained for argF and glnE. Thus, using this methodology we have obtained homologous recombination in 11 genes, and DCOs in 4 genes, showing that it is possible to generate targeted mutants in a reproducible way.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1350-0872
pubmed:author
pubmed:issnType
Print
pubmed:volume
145 ( Pt 12)
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3497-503
pubmed:dateRevised
2010-8-25
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination.
pubmed:affiliation
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, UK.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't