Folding and unfolding cellular phenotype

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DESCRIPTION (provided by applicant): The maintenance of a stable phenotype requires a genome wide transcriptional oscillation with clusters of transcripts poised around the steady-state - this is the dynamic architecture of phenotype. We propose to demonstrate that changes in protein levels and mRNA expression occur through a folding or unfolding of the surface described by this circle of transcripts and suggest that the path from this 40-minute oscillation to the cell cycle and circadian rhythms takes place through a series of period doubling bifurcations. Cell to cell signaling in continuous cultures of the budding yeast S. cerevisiae leads to mutual entrainment or synchronization that is manifested as an oscillation in redox state and a genome -wide oscillation in transcription. In turn, this transcriptional redox attractor cycle (TRAC) times, or gates DNA replication and other cell cycle events. Using fluorescently tagged proteins we expect to take advantage of the detailed knowledge of the patterns of expression of all of the transcripts in our system to efficiently choose proteins to be GFP tagged. Having a well characterized subset of proteins we will map these proteins into the hypothesized attractor surface and explore the phase relationships between a transcript and its protein. Using this system it will be possible to sort out the relationship between noise and oscillatory signal in cells. Using strains with Cyan, Green and tdTomato-FP tagged proteins will make it possible to test the hypothesis that oscillations are ubiquitous by looking at the phase relationships and amplitudes of these selected proteins in unsynchronized cultures and cultures in early log phase. Early on, the widely used S288C strain will be put in the continuous culture system, with the expectation that use of this strain will make it possible to efficiently examine large numbers of GFP tagged proteins. The most important aim is to map the path taken by cells during the period doubling process to see which genes settle into which basins of attraction. This effort is worthwhile because of the conceptual benefits that arise from an understanding of how the folding and unfolding of an attractor surface can make readily testable predictions regarding stem cell development, cancer cell heterogeneity and tumor progression The goal would be to use this information to design experiments in a stem cell model system.