Abstract #43

A genetic screening that evaluates the upper limit gene copy number in S. cerevisiae; application to the system level analysis of cell division cycle related genes. Hisao Moriya, Yuki Shimizu-Yoshida, Hiroaki Kitano. The Systems Biology Institute, Shibuya-ku, Japan.
   Intracellular biochemical parameters such as the expression level of gene products are considered to be optimized so that a biological system involving the parameters functions properly and effectively. Recent studies with computational modeling for biological systems have predicted that those parameters 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 Saccharomyces cerevisiae and applied for 30 cell cycle related genes (CDC genes). This method uses a trend that the copy number of 2 micron-based plasmids with the leu2d gene becomes more than 100 under selectable conditions. When the copy number of a target gene cloned on this plasmid has an upper limit of less than 100, the plasmid copy number under the selectable conditions is expected to become close to the upper limit copy number of the target gene. The copy numbers of CDC genes determined in this gTOW experiment were diverse from less than 2 to more than 100. A group of genes with lowest copy numbers constructed a sub-system that regulates B-type CDK activity with three positive feedback loops. Thus, the experiment provided unique quantitative data which could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data was used to evaluate the current computational cell cycle mode, and refinements to the model ware suggested.

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