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

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Abstract

<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>et al</jats:italic>.<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="#bib21">1999</jats:ext-link>. <jats:italic>Yeast</jats:italic> <jats:bold>15</jats:bold>: 1–10). This counter‐selection marker, named <jats:italic>GAL10p–GIN11</jats:italic>, 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 <jats:italic>GIN11</jats:italic> 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 <jats:italic>GIN11</jats:italic> mutants that lost the replication activity but retained the growth‐inhibitory effect when overexpressed. A mutant <jats:italic>GIN11M86</jats:italic> sequence was selected and fused to the <jats:italic>CUP1</jats:italic> promoter for the counter‐selection on a copper‐containing medium. The <jats:italic>GALp–GIN11M86</jats:italic> and the <jats:italic>CUPp–GIN11M86</jats:italic> were used for constructing sets of integrating plasmids containing auxotrophic markers involving <jats:italic>HIS3</jats:italic>, <jats:italic>TRP1</jats:italic>, <jats:italic>LEU2</jats:italic>, <jats:italic>URA3</jats:italic> or <jats:italic>ADE2</jats:italic>, or a drug‐resistant marker <jats:italic>PGKp–YAP1</jats:italic>. In addition, a set of gene disruption cassettes that contained each of the auxotrophic markers and the <jats:italic>GALp–GIN11M86</jats:italic>, which were flanked by direct repeats of a <jats:italic>hisG</jats:italic> 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. Copyright © 2002 John Wiley & Sons, Ltd.</jats:p>

Journal

  • Yeast

    Yeast 19 (5), 393-402, 2002-02-12

    Wiley

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