New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe.
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.
Sato
Masamitsu
M
Laboratory of Cell Regulation, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
Dhut
Susheela
S
Toda
Takashi
T
D024881:Drug Resistance, Bacterial / Q000235:genetics; D000939:Epitopes; D018390:Gene Targeting / Q000379:methods*; D005798:Genes, Bacterial; D005800:Genes, Fungal; D005819:Genetic Markers; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D006921:Hygromycin B / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000276:immunology*; D013309:Streptothricins / Q000494:pharmacology; D014170:Transformation, Genetic; D000097573:Red Fluorescent Protein
D000939:Epitopes; D005819:Genetic Markers; D005839:Gentamicins; D006921:Hygromycin B; D008164:Luminescent Proteins; D013309:Streptothricins; D000097573:Red Fluorescent Protein; C010680:antibiotic G 418