1 Supplementary Information for Overexpression limits of fission yeast cell-cycle regulators in vivo and in silico. Hisao Moriya, Ayako Chino, Orsolya Kapuy, Attila Csikász-Nagy, and Béla Novák Table of contents Supplementary Materials and Methods ................................................................................. 3 1. Plasmid vectors used in this study....................................................................................... 3 Table S1. Plasmid used in this study. .............................................................................................. 3 2. Construction of the plasmids ............................................................................................... 3 Table S2. Oligo DNA primers used for the construction of plasmid vectors. ................................ 4 3. Construction of plasmid vectors .......................................................................................... 5 3-1. Construction of pTOWsp series vectors................................................................................... 5 Figure S1. Structure of the pTOW vectors. ..................................................................................... 5 3-2. Construction of pA6R ................................................................................................................ 6 Figure S2. Structure of pA6R. ......................................................................................................... 6 4. Construction of plasmids with S. pombe cdc genes ............................................................ 7 Figure S3. Design of the primers to amplify and clone the target genes. ....................................... 7 Figure S4. Design of the primers to construct frame-shift mutant genes. ....................................... 8 Table S3. Oligo DNA primers to amplify and clone cdc genes in S. pombe. ................................. 8 5. References for Supplementary Materials and Methods ................................................... 9 Supplementary Data ............................................................................................................... 10 Table S4. Copy numbers of S. pombe gTOW vectors. ................................................................. 10 Table S5. Comparison of the vectors used in gTOW experiment in yeasts. ................................. 10 Figure S5. Examples of gTOW experiments with cdc genes in S. pombe (enlarged part of Figure 2).................................................................................................................................................... 11 Figure S6. Frameshift mutants of rum1 and wee1 are leaky. ........................................................ 12 Table S6. Plasmid copy number determined in gTOW experiment. ............................................ 13 Table S7. Mean GFP fluorescence in gTOW experiment. ............................................................ 14 Table S8. Comparison of the copy number limits of orthologs in S. pombe and S. cerevisiae. ... 15 Figure S7. Increased copy numbers of cyclin genes do not give cdc phenotype. ......................... 16 Table S9. Copy number limits of cell-cycle regulators in the wild type (WT) and cell cycle mutant strains. ............................................................................................................................... 17 Supplementary model description and simulation results .................................................. 20 6. Mathematical models of fission yeast cell cycle ............................................................... 20 6.1 A model for DNA replication regulation (“basic model”) ..................................................... 20 6.2 Models to investigate gTOW experiments............................................................................... 20 7. Description of the regulatory interactions of cell cycle control network....................... 22 Figure S8. Regulatory interactions among cell cycle regulators in the fission yeast cell cycle control network. ............................................................................................................................ 22 7.1. Methods to construct a mathematical model of fission yeast cell cycle ............................... 24 7.2 Equations and parameters of the models ................................................................................ 24 8. The “basic model” (core model) ........................................................................................ 25 Figure S9. Different forms of DNA replication origins in the model. .......................................... 25 Figure S10. Simulation of Hermand & Nurse experiments with the core model. ........................ 27