The Netherlands Gap Repair Transforma - tion in Fission Yeast to Exchange Plasmid - Selectable Markers

The fission yeast Schizosaccharomyces pombe is an attractive model organism for studying eukaryotic biology due to the ease of genetic and molecular manipulations (3). In recent years, several selectable markers and promoters have been developed to enhance our ability to manipulate gene expression in this yeast. Recently, Invitrogen (Carlsbad, CA, USA) has developed the SpECTRA S. pombe Expression System that consists of three TOPO® cloning vectors that allow for rapid cloning of PCR products, with the option to epitope-tag the gene product (adding a carboxy-terminal V5-6his tag). Transcription of cloned open reading frames is under the control of the thiamine-repressible promoters nmt1, nmt41, and nmt81. (Promoters nmt41 and nmt81 are TATA box mutant derivatives of nmt1 that allow for different levels of transcription.) However, as all three plasmids utilize the same selectable marker (simian virus 40-driven Saccharomyces cerevisiae LEU2, which complements an S. pombe leu1 mutant allele), use of these vectors limits the choice of host strains and the possibility to transform strains with more than one plasmid construct. To enhance the use of the SpECTRA vectors by allowing for the transformation of Leu+ strains or for the introduction of two distinct plasmids into a host strain, we have developed a protocol that facilitates the exchange of the LEU2 marker for another selectable marker. This protocol takes advantage of the robust homologous recombination machinery found in yeasts, which has been used in the past to rescue chromosomal mutations onto plasmids by gap repair (8) or to screen mutant alleles created by PCR by co-transformation of the PCR product with a linearized plasmid (5,6). In both situations, the plasmid DNA is linearized before transformation and is repaired and recircularized by DNA that bears homology to the ends of the linearized plasmid. Using a protocol described here, we can readily replace the LEU2-selectable Benchmarks