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New vectors for combinatorial deletions in yeast chromosomes and for gap‐repair cloning using ‘split‐marker’ recombination
Author(s) -
Fairhead Cécile,
Llorente Bertrand,
Denis Françoise,
Soler Maria,
Dujon Bernard
Publication year - 1996
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(199611)12:14<1439::aid-yea37>3.0.co;2-o
Subject(s) - ura3 , biology , genetics , plasmid , homologous recombination , cloning (programming) , flp frt recombination , genome , computational biology , gene , cloning vector , transformation (genetics) , multiple cloning site , recombination , recombinant dna , vector (molecular biology) , genetic recombination , computer science , programming language
New tools are needed for speedy and systematic study of the numerous genes revealed by the sequence of the yeast genome. We have developed a novel transformation strategy, based on ‘split‐marker’ recombination, which allows generation of chromosomal deletions and direct gene cloning. For this purpose, pairs of yeast vectors have been constructed which offer a number of advantages for large‐scale applications such as one‐step cloning of target sequence homologs and combinatorial use. Gene deletions or gap‐repair clonings are obtained by cotransformation of yeast by a pair of recombinant plasmids. Gap‐repair vectors are based on the URA3 marker. Deletion vectors include the URA3, LYS2 and kanMX selection markers flanked by I‐ Sce I sites, which allow their subsequent elimination from the transformant without the need for counter‐selection. The application of the ‘split‐marker’ vectors to the analysis of a few open reading frames of chromosome XI is described.