Tools and methods for genetic analysis of Saccharomyces castellii.

The budding yeast species Saccharomyces castellii has provided important new insights into molecular evolution when incorporated in comparative genomics studies and studies of mitochondrial inheritage. Although it shows some diversity in the specific molecular details, several analyses have shown that it contains many genetic pathways similar to those of S. cerevisiae. Here we have investigated the possibility of performing genetic analyses in S. castellii. We optimized the LiAc transformation protocol to achieve 200-300 transformants/microg plasmid DNA. We found that the commonly used plasmids for S. cerevisiae are stably maintained in S. castellii under selective conditions. Surprisingly, both 2micro and CEN/ARS plasmids are kept at a high copy number. Moreover, the kanMX cassette can be used as a resistance marker against the selective drug geneticin (G418). Finally, we determined that the S. cerevisiae GAL1 promoter can be used for the activation of transcription in S. castellii, thus enabling the controlled overexpression of genes when galactose is present in the medium. The availability of these tools provides the possibility of performing genetic analyses in S. castellii, and makes it a promising new model system in which hypotheses derived from bioinformatics studies can be experimentally tested.