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Yeast. 1996 Nov;12(14):1439-57.

New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using 'split-marker' recombination.

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1
Unité de Génétique Moléculaire des Levures (UFR 927 Université P. & M. Curie, URA 1149 CNRS), Institut Pasteur, Paris, France.

Abstract

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-Scel 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.

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