New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using 'split-marker' recombination.
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.
Fairhead
C
C
Unité de Génétique Moléculaire des Levures (UFR 927 Université P. & M. Curie, URA 1149 CNRS), Institut Pasteur, Paris, France.
Llorente
B
B
Denis
F
F
Soler
M
M
Dujon
B
B
D015825:Chromosomes, Fungal / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004260:DNA Repair; D005819:Genetic Markers; D005822:Genetic Vectors; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014170:Transformation, Genetic; D015003:Yeasts / Q000235:genetics*
D017931:DNA Primers; D005819:Genetic Markers