Analysis of Quiescent and Non-quiescent Cells in Stationary-phase Yeast Cultures

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The long-term goal of this research is to understand how yeast cells enter, survive in, and exit the quiescent state and, eventually, to use this information to understand this process in other organisms. Quiescence or G0 is the most common state of cells on earth and was surely one of the first states in the evolution of living cells. Paradoxically, this fundamentally important process has not been well studied, but the advent of genomics and the development of ancillary technologies have recently enabled significant progress in understanding this state. Leading up to the current project, this research group has studied the changes in gene expression as cells in stationary-phase cultures re-enter the mitotic cell cycle. They identified over 30 new genes required for long-term survival of cells in stationary-phase cultures. They recently developed a method that allows separation of quiescent and non-quiescent cells in stationary-phase cultures and developed a rapid sampler that allows them to reproducibly harvest cells in culture for genomic analysis at intervals as short as 10s. The group will now characterize quiescent and non-quiescent yeast cells from stationary-phase yeast cultures. GFP constructs and electron microscopy will be used to detect morphological differences between cell types. Various biochemical and genomic analyses will be carried out on both cell types as a function of time in culture. The non-quiescent cells will be evaluated using several assays to determine the percentage of cells that exhibit apoptotic or senescent characteristics. As necessary, modified separation methods will be used to increase the purity of populations from the non-quiescent-cell fraction. The intellectual merit of the work comes from the novel insight provided by being able to study, for the first time, pure populations of quiescent and non-quiescent cells and through the identification of genes and pathways critical for the differentiation of these cell types. This work has broad relevance, since, based on previous work with yeast, these differentiation processes are likely to be conserved in other microbes and eukaryotic organisms. The broader impacts of this work are in the involvement of students and faculty from underrepresented groups, the breadth of collaborations between biologists, statisticians and engineers, the commitment to the free distribution of newly developed technology, and sharing genomic reagents.