A ‘marker switch’ approach for targeted mutagenesis of genes in Schizosaccharomyces pombe

Abstract

The completion of the Schizosaccharomyces pombe genome sequencing project has led to a dramatic acceleration of gene characterization in this system. Once a gene has been identified, the challenge then comes in using reverse genetics to generate a range of mutants in this gene of interest so that the powerful genetics and wealth of genetic backgrounds available in Sz. pombe can be exploited to study the function of the newly identified molecule. Beyond simple PCR-tagging approaches, the high frequency with which illegitimate recombination occurs in Sz. pombe has made the manipulation of some loci complex, time consuming and a process of trial and error. Here we describe a simple 'marker switch' approach that enables the rapid selection of integration events at the locus of interest from an excessive background of integration at heterologous sites. We use the generation of temperature-sensitive mutations in the plo1 + gene to validate this approach.

Myosin V spatially regulates microtubule dynamics and promotes the ubiquitin-dependent degradation of the fission yeast CLIP-170 homologue, Tip1

“…Cells were cultured in rich (YES) or minimal (EMM2) supplemented media at 25°C. Temperature-sensitive (ts) myo52 alleles were isolated using the previously described marker switch technique (MacIver et al, 2003). Full-length ts myo52 alleles were sequenced to identify changes to the polypeptide sequence.…”

S. pombe CLASP needs dynein, not EB1 or CLIP170, to induce microtubule instability and slows polymerization rates at cell tips in a dynein-dependent manner

“…peg1 + was deleted by the same PCR approach using pFA6a-hphMX6 (Hentges et al 2005). The marker switch approach of MacIver et al (2003a), in which the ura4 + marker was inserted 936 base pairs (bp) upstream of the peg1 + ATG, was used to generate fusions in which the tag was located at the N terminus of Peg1. For twohybrid analysis, peg1 + , tip1 + , tea1 + , and mal3 + cDNAs were cloned into pMM5, pMM6 (Pereira et al 1999) as BamHI fragments and processed according to Pereira et al (1999).…”

A vector system for efficient and economical switching of C‐terminal epitope tags in Saccharomyces cerevisiae

“…The epitope switching method shares the advantages of the 'marker switch' method MacIver et al, 2003;Sato et al, 2005). First, it is easy and economical.…”

“…pombe, a 'marker switch' approach has been introduced that enables rapid selection of integration events at the locus of interest from an excessive background of integration at heterologous sites. Using this approach, a temperature-sensitive mutant for the plo1 + gene was generated (MacIver et al, 2003). The marker switch method has also been successfully applied to gene deletion and C-terminal tagging; the kanMX6 marker was efficiently switched to the natMX6, hphMX6 or bleMX6 marker, and vice versa, with using the 'universal' oligonucleotide primers Sato et al, 2005).…”