A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae

Abstract

Gene tagging can be achieved by homologous recombination in yeast. The kan^r marker gene plays an important role in PCR-mediated gene disruption and PCR-mediated epitope tagging experiments. In this paper, new modules containing a tag-loxP-kanMX-loxP cassette are described that allow tagging of different genes by using the kan^r marker repeatedly.

Introduction

Epitope tagging is a powerful technique for the analysis of proteins in yeast and other organisms. In Saccharomyces cerevisiae, it is facilitated by the high efficiency of homologous recombination. PCR-based methods exist which allow single-step deletion or tagging of chromosomal genes (Goldstein et al., 1999, Güldener et al., 1996, Knop et al., 1999, Longtine et al., 1998, Lorenz et al., 1995, Schneider et al., 1995, Wach et al., 1997, Wach et al., 1994). These methods rely on the amplification by PCR of a selectable module using two primers with tail sequences which are homologous to the desired target sequence into which the cassette is to be integrated, followed by transformation and homologous recombination into the yeast genome. The chief advantages of this method are its speed and general applicability; practically any desired ORF can be tagged since the entire S. cerevisiae genome sequence is known and publicly available.

In 1994, Wach et al. fused the kan^r gene from the Escherichia coli transposon Tn903 with the TEF promoter and terminator from Ashbya gossypii, generating the kanMX expression module; yeast strains after integration of kanMX module into their genome are rendered resistant to geneticin (G418) (Wach et al., 1994). Güldener et al. (1996) created a disruption kanMX cassette flanked by loxP loci that could be used repeatedly in yeast. The Cre-loxP recombination system of bacteriophage P1 can facilitate efficient recombination between two loxP sites flanking a marker gene in yeast, resulting in the excision of the marker gene (Sauer, 1987).

Here we describe new plasmids containing novel modules for repeated C-terminal epitope tagging. The plasmids pU6H2MYC, pU6H3HA and pU6H3VSV (EMBL accession Nos. AJ132965, AJ132966 and AJ132967, respectively) contain either the 6His-2MYC-loxP-kanMX-loxP, the 6His-3HA-loxP-kanMX-loxP or the 6His-3VSV-loxP-kanMX-loxP cassette.