Linked Life Data

8921867

Source:http://linkedlifedata.com/resource/pubmed/id/8921867

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1-2
pubmed:dateCreated
1996-12-26
pubmed:abstractText
For the assembly of a large human locus, overlapping regions on yeast artificial chromosomes (YACs) and cosmids were linked up using their regions of homology. By site-specific recombination a YAC of 410 kb was created accommodating the major part of the human lambda light chain locus in authentic configuration with 28 variable (V) genes, all joining (J) segments, all constant (C) genes and the downstream enhancer. A contiguous region was first constructed from three overlapping cosmids. Each of these was linearized at unique sites in the vectors and YAC arms were ligated to the 5' and 3' ends. After cells of Saccharomyces cerevisiae were transformed with the three cosmids, YACs of 120 kb were obtained which contained the reassembled 3' J-C region in authentic configuration. The assembled YAC was further extended by mitotic recombination with a YAC containing a 280-kb region of the C-proximal part of the V gene cluster with a 15-kb 3' overlap. For this, a simple three-way selection procedure was developed involving the integration of different selectable marker genes at specific sites in the left and right YAC arms. Rare recombination events between two overlapping YACs could be identified in yeast clones able to grow in lysine- and adenine-deficient medium in the presence of 5-fluoro-orotic acid which is toxic for yeast cells containing a YAC with a functional URA3 gene. This approach made it possible to assemble and extend large YACs from an unlimited number of smaller overlapping YACs by positive-negative selection.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0378-1119
pubmed:author
pubmed:issnType
Print
pubmed:day
24
pubmed:volume
177
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
195-201
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Assembly and extension of yeast artificial chromosomes to build up a large locus.
pubmed:affiliation
Department of Development and Genetics, Babraham Institute, Cambridge, UK. andrei.popov@bbsrc.ac.uk
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't