Introduction

The yeast Saccharomyces cerevisiae is a widely used experimental system for basic research in cell biology. Numerous researchers use it because of the ease of genetic manipulations. Two different techniques are often used for genetic manipulations. One is the use of vectors such as the integrative, centromeric, and episomal plasmids of the pRS series (1,2) for gene cloning and the second is the use of drug resistance markers for the deletion or tagging of genes (3,4,5,6,7,8,9,10,11,12,13). The latter technique has the advantage of being independent of the presence of auxotrophic markers in the yeast background. The common vectors used for yeast manipulation lack this advantage. They rely on the presence of an appropriate auxotrophic marker. This led to the construction of so-called designer deletion strains to combine several auxotrophic markers into one single yeast strain (1,14,15). Due to experimental conditions, however, it is not always possible to use one of these yeast strains, or the experiment may require growth on rich media in which plasmid selection is not possible. To circumvent these problems, we constructed a complete set of integrative, centromeric, and episomal plasmids based on the pRS series of plasmids containing the three different dominant drug resistance markers Geneticin® (kanMX4), nourseothricin (natNT2), and hygromycin B (hphNT1). The centromeric and episomal plasmids can be used similarly to conventional plasmids. YPD (yeast extract, peptone, dextrose) medium supplemented with the appropriate drug is used for plasmid selection, instead of synthetic medium lacking the selectable marker.

The pRS30x series of integrative plasmids has some drawbacks. To prevent multiple integrations, which could lead to overexpression of the inserted gene, we changed the integration mechanism. The new plasmids do not create a tandem repeat of the auxotrophic marker with the integrated DNA sequence in the middle (3). Instead, they replace part of the auxotrophic marker with the drug resistance marker (kanMX4, natNT2, or hphNT1) and the DNA sequence of choice.

The plasmids and sequence files can be obtained by contacting the authors (e-mails: taxis@embl.de or knop@embl.de) or by sending a letter with a self-addressed envelope to Dr. Michael Knop, EMBL Cell Biology and Biophysics Unit, Meyerhofstr. 1, D-69117 Heidelberg, Germany. Standard mail is preferred.

Materials and Methods

Construction of Plasmids

Standard techniques were used for DNA manipulations (16). The integrative plasmids are based on pRS303, pRS305, orpRS306 (1). First, the multicloning sites (MCS) of these plasmids were destroyed, followed by insertion of the drug resistance marker in the HIS3, LEU2, or URA3 gene. The centromeric and episomal plasmids were derived from plasmids pRS416 and pRS426, respectively (1,2). The URA3 gene and the flanking promoter and terminator regions in these plasmids were replaced by the three different dominant selection markers. A detailed description of the plasmid constructions is provided as supplementary material, which is available online at www.BioTechniques.com .

Yeast Strains and Growth Conditions

Yeast strain ESM356 (17) was used for in-yeast ligation by homologous recombination. YPD and synthetic dropout media were prepared as previously described (18). The following drug concentrations were added to standard YPD plates or liquid YPD medium after autoclaving and cooling to 60°C: 200 mg/L Geneticin Selective Reagent (G418; Invitrogen, La Jolla, CA, USA) (4) were added to select for kanMX4; 300 mg/L hygromycin B (InvivoGen, Toulouse, France) were added to select for hphNT1; and 100 mg/L nourseothricin (ClonNAT; Werner BioAgents, Jena-Cospeda, Germany) were added to select for natNT2. A sterile filtered stock solution was used in the case of ClonNAT, whereas Geneticin and hygromycin B were used as provided by the manufacturer.

The following media composition was used for the double selection of an auxotrophic marker plasmid together with a dominant drug resistance plasmid. The recipe was adapted from the composition reported in the supplementary information (SGA Analysis: Media) to Reference 19,. The main change compared with standard synthetic complete (SC) medium was the replacement of ammonium sulfate with monosodium glutamate as a nitrogen source. The medium contains 20 g/L glucose, 1.7 g/L yeast nitrogen base without ammonium sulfate and amino acids (Difco™ BD, Franklin Lakes, NJ, USA), 1 g/L monosodium glutamic acid, and 2 g/L amino acid drop-out powder (18) lacking the appropriate auxotrophic marker. The appropriate dominant drug should be added after autoclaving of the medium.