New cassettes for single-step drug-resistance and prototrophic marker switching in fission yeast

Alexander Lorenz (Corresponding Author)

Research output: Contribution to journalArticle

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Abstract

Construction of multiply mutated strains for genetic interaction analysis and of strains carrying different epitope tags at multiple open reading frames for testing protein localization, abundance and protein-protein interactions is hampered by the availability of a sufficient number of different selectable markers. Moreover, strains with single gene deletions or tags often already exist in strain collections; for historical reasons these will mostly carry the ura4(+) gene or the G418-resistance kanMX as marker. Because it is rather cumbersome to produce multiply deleted or tagged strains using the same marker or to completely reconstruct a particular strain with a different marker, single-step exchange protocols of markers are a time-saving alternative. In recent years dominant drug resistance markers (DDRMs) against clonNAT, Hygromycin B, and Bleomycin have been adapted and successfully used in Schizosaccharomyces pombe. The corresponding DDRM cassettes - natMX, hphMX, and bleMX - all carry the TEF-promotor and -terminator sequences from Ashbya gossypii as kanMX, this provides flanking homologies to enable single-step marker swapping by homologous gene targeting. To expand this very useful toolset for single-step marker exchange I constructed MX-cassettes containing the nutritional markers arg3(+) , his3(+) , leu1(+) , and ura4(+) . Furthermore, a set of constructs was created to enable single-step exchange of ura4(+) to kanMX6, natMX4, and hphMX4. The functionality of the cassettes is demonstrated by successful single-step marker swapping at several loci. These constructs allow a straight-forward and rapid re-marking of existing ura4(+) - and MX-deleted and -tagged strains.

Original languageEnglish
Pages (from-to)703-710
Number of pages8
JournalYeast
Volume32
Issue number12
Early online date17 Sep 2015
DOIs
Publication statusPublished - Dec 2015

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Schizosaccharomyces
Drug Resistance
Yeast
Genetic Terminator Regions
Hygromycin B
Pharmaceutical Preparations
Proteins
Gene Targeting
Bleomycin
Gene Deletion
Genes
Open Reading Frames
Epitopes
Availability
Testing

Keywords

  • Schizosaccharomyces pombe
  • selectable marker
  • marker switch
  • plasmid
  • PCR

Cite this

New cassettes for single-step drug-resistance and prototrophic marker switching in fission yeast. / Lorenz, Alexander (Corresponding Author).

In: Yeast, Vol. 32, No. 12, 12.2015, p. 703-710.

Research output: Contribution to journalArticle

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