Sets of integrating plasmids and gene disruption cassettes containing improved counter-selection markers designed for repeated use in budding yeast

Abstract

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.

The intron of tRNA-TrpCCA is dispensable for growth and translation of Saccharomyces cerevisiae

“…For this purpose, we adopted marker cassettes devised by Akada et al (2002). In these cassettes, an auxotrophic selection marker is accompanied by a negative selection marker, GAL1pTGIN11M86, which encodes a toxic protein under the control of the GAL1 promoter.…”

“…Yeast strains used and constructed in this study are summarized in Supplemental Table SI. Primers and plasmids are in Supplemental Tables SII and SIII, respectively. To construct pTYE379 and pTYE380, a 4.95 kb BglII-NotI fragment from pGG216 and a 6.03 kb BglII-NotI fragment from pGG217 (Akada et al 2002) were inserted into the BamHIPspOMI sites of a pDNR-1r (Clontech) derivative with a modified multicloning site, respectively.…”

“…To make an intronless version of a tRNA-Trp CCA gene, we applied overlap extension mutagenesis with a set of four primers, tRNA-Trp CCA gene specific forward and reverse primers, and tRNA-Trp CCA exon primers (tWccaG2_ex1_3 and tWccaG2_ex2_5) that provided a joint of the two exons. A marker cassette derived from pTYE379 or pTYE380, which harbors an auxotrophic marker (URA3 or ADE2) and a negative marker (GAL1pTGIN11M86) sandwiched with two hisG fragments (Akada et al 2002), and Cam r , was introduced For integration of the intronless tRNA-Trp CCA gene to the yeast genome, the plasmid with the intronless gene was digested with appropriate restriction enzymes, and the resulting DNA fragments were introduced to an appropriate yeast strain. Transformants with an auxotrophic marker were selected on an SCD medium [0.67% w/v Difco Yeast Nitrogen Base without Amino Acids (BD Biosciences), 0.5% w/v Vitamin Assay Casamino Acids (BD Biosciences), and 2% w/v glucose] with appropriate supplements.…”

Copper Response Element and Crr1-Dependent Ni 2+ -Responsive Promoter for Induced, Reversible Gene Expression in Chlamydomonas reinhardtii

“…Metal-responsive promoters, developed initially for mammalian cells (26,56,57), are favored tools for the design of inducible promoter systems and have been exploited in various model systems, including cyanobacteria, fungi, and plants (1,5,63). The extraordinarily tight transcriptional regulation of the Cyc6 gene immediately suggested its utility for such a purpose when the promoter was shown to be sufficient for conferring copper responsiveness to a heterologous gene (24,47).…”

New plasmids for the disruption and repeated use of selection markers in <i>Saccharomyces cerevisiae</i>

“…Therefore, to expand utility, we also constructed the pNK-4 plasmid (Fig. 1A) similarly to pNK-3, except that pGG215 (Akada et al, 2002) was used instead of pGG216. pNK-4 has the AgTEF1p-hisG-LEU2-GAL1pGIN11-M86-hisG-AgTEF1t sequence as the Table 2.…”