An approach for isolation of mutants defective in 35S ribosomal RNA synthesis in Saccharomyces cerevisiae

Abstract

We have developed a method to isolate mutants of Saccharomyces cerevisiae that are primarily defective in the transcription of 35S ribosomal RNA (rRNA) genes by RNA polymerase I. The method uses a system in which the 35S rRNA gene is fused to the GAL7 promoter and is transcribed by RNA polymerase II under control of the GAL regulatory system. Chromosomal mutations affecting components specifically involved in synthesis of 35S rRNA by RNA polymerase I can be suppressed by this hybrid gene in the presence of inducer (galactose) but not in its absence. We looked for mutants the growth of which depended on the presence of plasmid expressing the hybrid gene. For this purpose, we used a red/white-colony color assay as the initial screen followed by a test for galactose-dependent growth. We have thus isolated many mutants and identified at least nine genes (RRN1-RRN9) involved in 35S rRNA synthesis, two of which correspond to known RNA polymerase I subunit genes RPA190 and RPA135.