Linked Life Data

15137072

Source:http://linkedlifedata.com/resource/pubmed/id/15137072

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
2004-5-11
pubmed:abstractText
Random mutagenesis is a powerful tool for studying the effects of a large number of permutations of a particular DNA sequence and its encoded products. Here we describe a new strategy of conducting in vitro random mutagenesis using ethyl methane sulfonate (EMS). The Bacillus aprN18 gene, coding for a serine protease with fibrinolytic activity, was used as a target gene. To study the mutations of the coding region, rather than the whole plasmid, the 1.4 kb gene fragment was cut out from an expression plasmid and treated with 10 mM EMS at 37 degrees C for 1 h. The treated fragment was then ligated back into the original expression vector and a library of random mutants was constructed in a protease-deficient Bacillus subtilis strain. A plate assay-based screening method was used to select for mutant clones with altered enzyme activity, and the change of activity was then confirmed by a semi-quantitative enzyme assay using liquid culture supernatant. The inserts of five clones with altered enzyme activity were randomly chosen for sequencing analysis. Among the point mutations detected, GC --> AT transition accounts for 42.1%, AT --> GC transition 34.2% and GC/CG transversion 23.7%, respectively. To our knowledge this is the first application of EMS for in vitro mutagenesis of a defined DNA sequence.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0006-3592
pubmed:author
pubmed:copyrightInfo
Copyright 2004 Wiley Periodicals, Inc.
pubmed:issnType
Print
pubmed:day
20
pubmed:volume
86
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
622-7
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2004
pubmed:articleTitle
A new approach to random mutagenesis in vitro.
pubmed:affiliation
Department of Biology, School of Life Sciences, East China Normal University, Shanghai, China.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't