Cycloheximide (Actidione) is an antibiotic which inhibits eukaryotic protein synthesis. Although many yeasts including Saccharomyces cerevisiae are sensitive to cycloheximide, some yeast strains are resistant to this drug.
Among the resistant strains, Candida maltosa IAM12247 has an inducible resistance mechanism, as described in our previous paper. Results of in vitro protein synthesis with ribosomal and S-100 fractions prepared from cycloheximide-treated and nontreated C. maltosa cells indicated that ribosomes are modified in the cells during cultivation in the presence of cycloheximide. It was suggested that there is a gene (designated RIM-C for ribosome modification by cycloheximide) which functions in the presence of cycloheximide in such a way as to modify ribosomes so that protein synthesis in the cells is no longer inhibited by cycloheximide. In this investigation, we attempted to clone this presumptive gene by using a host-vector system of S. cerevisiae. We believe that the cloning of this gene will not only help to analyze the mechanism of cycloheximide resistance related to the RIMC gene in C. maltosa, but also will provide a convenient dominant vector marker for recombinant DNA technology with S. cerevisiae.
To determine whether ribosome modification occurred in S. cerevisiae(pRIM-C2), as it did in C. maltosa, in vitro protein synthesis was done. When ribosomes from cycloheximide-treated S. cerevisiae(pRIM-C2) were used in combination with fraction S-100 from either cycloheximide-treated S. cerevisiae(pRIM-C2) (Fig. 2a) or nontreated S. cerevisiae IF01256 (data not shown), the incorporation activity was scarcely influenced by the presence of cycloheximide at 2.5 or 25 ,ug/ml. In contrast, in the systems containing ribosomes from the control S. cerevisiae, the incorporation activity was inhibited by cycloheximide irrespective of the origin of the S-100 [S-100 from cycloheximide-treated S. cerevisiae(pRIM-C2), (Fig. 2b) or S-100 from the control S. cerevisiae (data not shown)]. From these results, we concluded that the RIM-C gene cloned in the pRIM-C2 plasmid modifies ribosomes so that they become resistant to cycloheximide.
Several dominant vector markers have been recognized in S. cerevisiae (3, 6, 7, 10, 11). To use the RIM-C gene as a selectable marker of plasmids in S. cerevisiae, we recommend the following procedure. S. cerevisiae AH22 is transformed with pRIM-C2 (or pRIM-C3) by the spheroplast method. The spheroplasts are mixed with about 20 ml of YPD containing 3% agar and 1.2 M sorbitol, poured into a petri dish, and incubated overnight at 30°C. The next morning, about 10 ml of YPD containing 0.6% agar and 5 ,ug of cycloheximide per ml is poured onto the dish, which is then incubated at 30°C. After 3 to 5 days, only those cells carrying pRIM-C2 (or pRIM-C3) make colonies. By this procedure, it was confirmed that S. cerevisiae AH22 carrying either of these plasmids can be isolated without using LEU2' as a selectable marker. The numbers of LEU2+ colonies and Cyhr colonies were almost the same when these colonies were selected and counted separately. The RIM-C gene may be useful as a dominant vector marker for recombinant DNA technology of many cycloheximide-sensitive yeasts that have no selectable marker.
SOURCES: M Takagi, S Kawai, I Shibuya, M Miyazaki and K Yano