In Vivo Robustness Analysis of Cell Division Cycle Genes in Saccharomyces cerevisiae

Abstract

Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named “genetic tug-of-war” (gTOW) that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes ( CDC genes). The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested.

Synopsis

Robustness is a property of a system that attempts to maintain its functions against internal and external perturbations. It is one of the fundamental and ubiquitously observed system-level properties of biological systems. Understanding the cellular robustness is important, not only to gain insights in biology, but also to identify potential therapeutic targets. Robustness is estimated by measuring how much parameters can be perturbed without disrupting essential functions; comprehensive, as well as quantitative perturbations of intracellular parameters, such as gene expression, are essential for solid robustness analysis. However, the lack of experimental methodology for the comprehensive quantification and defined perturbation of parameters has prevented experimental analyses of cellular robustness. The authors developed a novel genetic screening method named “genetic tug-of-war” (gTOW) that allows systematic measurement of upper limit gene copy number. gTOW applied for the robustness analysis of cell division cycle system in the model eukaryote, Saccharomyces cerevisiae, and revealed the point of fragility in the system. The gTOW method is particularly suitable for systems biology research and demonstrates the value of comprehensive and quantitative perturbation experiment to uncover system-level properties of the cellular system.