Sets of integrating plasmids and gene disruption cassettes containing improved counter-selection markers designed for repeated use in budding yeast.
Counter-selection is a useful gene manipulation technique for repeated gene disruptions, gene shufflings and gene replacements in yeasts. We developed a novel counter-selection system using a galactose-inducible growth inhibitory sequence (Kawahata et al.1999. Yeast 15: 1-10). This counter-selection marker, named GAL10p-GIN11, has several advantages over previous counter-selection markers, i.e. use of an inexpensive galactose medium for counter-selection, combined use with any transformation markers for gene introduction, and no requirement of specific mutations in the host strains. The GIN11 sequence, which is a part of an X-element of the subtelomeric regions, contained a conserved autonomously replicating sequence, causing the possibility of inefficient chromosomal integration. We isolated GIN11 mutants that lost the replication activity but retained the growth-inhibitory effect when overexpressed. A mutant GIN11M86 sequence was selected and fused to the CUP1 promoter for the counter-selection on a copper-containing medium. The GALp-GIN11M86 and the CUPp-GIN11M86 were used for constructing sets of integrating plasmids containing auxotrophic markers involving HIS3, TRP1, LEU2, URA3 or ADE2, or a drug-resistant marker PGKp-YAP1. In addition, a set of gene disruption cassettes that contained each of the auxotrophic markers and the GALp-GIN11M86, which were flanked by direct repeats of a hisG sequence, were constructed. The counter-selectable integrating plasmids and the gene disruption cassettes can allow the markers to be used repeatedly for yeast gene manipulations.
Akada
Rinji
R
Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan. rinji@po.cc.yamaguchi-u.ac.jp
Hirosawa
Isao
I
Kawahata
Miho
M
Hoshida
Hisashi
H
Nishizawa
Yoshinori
Y
D004271:DNA, Fungal / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics / Q000502:physiology; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000379:methods*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D012641:Selection, Genetic
D004271:DNA, Fungal; D005819:Genetic Markers