Analysis of the Budding Yeast Saccharomyces cerevisiae Cell Cycle by Morphological Criteria and Flow Cytometry

Abstract

The budding yeast Saccharomyces cerevisiae represents the eukaryotic model system in which the checkpoint concepts were initially developed (1). Whereas many arguments can be made in favor of the continued use of S. cerevisiae as a model organism for checkpoint studies, the ease of distinguishing cell cycle stages is usually not put forward. Indeed, peculiar features of the yeast cell cycle can be regarded as a disadvantage. For instance, there is no discernible G2 stage, and the intranuclear mitotic spindle is assembled already during S phase. Also, the degree of chromosome compaction during mitosis is insufficient to allow visualization of individual chromosomes or definitive detection of mitotic stages by conventional light microscopy. On the other hand, there are a number of morphological cell cycle landmarks available that are easily traceable with very little experience and experimental manipulation. Even such a complex methodology as fluorescence-activated cell sorting (FACS) can be readily adapted with additional calibration for the small cell size and the relatively low deoxyribonucleic acid (DNA) content of diploid or haploid yeast. In the following, we have compiled protocols for some of the most common methods of yeast cell cycle analysis. In general, we have assumed time-course experiments, following the activation of checkpoint arrest responses (e.g., by irradiation of synchronized cells). Individually, some of these protocols will only suffice for preliminary studies, and a combination of methods is highly recommended to monitor certain cell cycle transitions unequivocally.