New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe

Masamitsu Sato, Susheela Dhut, Takashi Toda

Research output: Contribution to journalArticle

184 Citations (Scopus)

Abstract

We describe new heterologous modules for PCR-based gene targeting in the fission yeast Schizosaccharomyces pombe. Two bacterial genes, hph and nat, which display dominant drug-resistance phenotypes, are used as new selectable markers in these modules. Both genes have been used successfully in the budding yeast Saccharomyces cerevisiae, in which hph confers resistance to hygromycin B, while nat confers nourseothricin resistance (Goldstein and McCusker, 1999). Vector modules for gene disruption and C-terminal tagging with 3HA, 13Myc and GFP(S65T) are constructed using previously constructed pFA6a-MX6-derived plasmids (Bähler et al., 1998; Wach et al., 1997). In combination with the existing systems that are based upon the G418-resistance gene (kan), triple gene deletions or tags could be constructed. In addition a vector for one-step integration of a monomeric RFP (mRFP) to the C-terminus of proteins of interest is developed. Finally, oligonucleotides that allow a simple marker switch from kan to hph or nat, and vice versa, are described. The new constructs developed here should facilitate post-genomic molecular analysis of protein functions in fission yeast.

Original languageEnglish
Pages (from-to)583-591
Number of pages9
JournalYeast
Volume22
Issue number7
DOIs
Publication statusPublished - 2005 May 1
Externally publishedYes

Keywords

  • Fission yeast
  • Hygromycin B
  • Marker switch
  • Monomeric RFP
  • Nourseothricin
  • Oligonucleotide
  • PCR

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Genetics

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