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

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.