Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903

Abstract

We have developed a new procedure for selecting yeast transformants without the need for complementing auxotrophic markers. The procedure is based on resistance to antibiotic G418 imparted to transformants by recombinant DNA vectors. We constructed several Escherichia coli-yeast shuttle vectors containing the kanamycin (G418)-resistance gene of Tn903, plus several yeast genes making dual selections possible. The efficiency for selecting G418-resistant transformants was dependent upon several factors including the composition of the growth medium and the time at which G418 selective pressure was administered. Media which contained levels of salts found in yeast nitrogen base rendered cells partially to completely resistant to G418 and could not be used for selecting G418-resistant transformants. On the other hand, untransformed cells remained sensitive to G418 when grown on YEPD medium thus allowing selection of G418-resistant transformants. A lag phase of 12 to 18 h, following growth at 30 degrees C, was required prior to administration of G418 to achieve maximal transformation frequency. Transformation frequencies ranged from 100 to 700 per micrograms of DNA and varied with the vector and strain used. The kanamycin gene imparted resistance to G418 in either the episomally or chromosomally integrated state. The gene was highly stable in the integrated state, even without selective pressure. The utility of the procedure was demonstrated by selecting transformants of four different strains of Saccharomyces cerevisiae and by cloning autonomous replication sequences (ARS) from the yeast Kluyveromyces lactis. We believe that this or related procedures could be used to develop transformation systems for many eukaryotic and prokaryotic cells for which no transformation procedure is available.