Synchronization of Saccharomyces cerevisiae Cells in G1 Phase of the Cell Cycle.

Abstract

The baker's yeast, Saccharomyces cerevisiae is a widely used model organism in molecular biology because of the high homology it shares with human cells in many basic cellular processes such as DNA replication, repair, recombination, transcription, and because of the ease its genome can be manipulated. Other advantages of working with yeast are its fast production rate which is comparable to bacteria's, and its cheap maintenance. To examine certain phenomena, for example whether a mutation affects the passage through a cell cycle phase, it can be necessary to work with a yeast culture, in which all the cells are in the same phase of the cell cycle. Yeasts can be arrested and kept in different phases of the cell cycle. Here we describe the method that allows synchronizing and keeping yeast cells in the G1 phase of the cell cycle with the mating pheromone, α-factor. Only MATa cells can be synchronized with α-factor which is produced by MATα cells. It is highly recommended to use a MATa bar1 deletion strain. The BAR1 gene encodes for an extracellular protease that inactivates α-factor by cleaving it (MacKay et al., 1988). To counteract the Bar1 protease activity when using BAR1 cells, 100-1,000 times more α-factor is needed as compared to bar1 deletion cells (α-factor is quite expensive!), and still the synchrony will be transient. In contrast, bar1 deletion cells can be kept in G1 phase with α-factor for several hours, and the degree of synchronization is usually higher than using a BAR1 strain. Moreover, bar1 deletion cells can be synchronized even at high cell density, whereas BAR1 cells, due to the activity of the secreted Bar1 protease, only at low cell density.