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Cdc7p over-expression in a sid4 mutant will result in septation.

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posted on 05.08.2015 by Anastasia Chasapi, Paulina Wachowicz, Anne Niknejad, Philippe Collin, Andrea Krapp, Elena Cano, Viesturs Simanis, Ioannis Xenarios

(A) Steady states of in silico, double mutation experiments. The model predicts that in the absence of SIN scaffold proteins (Cdc11p or Sid4p) and over-expression of Cdc7p, the cell will septate. (B) sid4-SA1 leu1-32 was transformed with a REP1-based plasmids [80] expressing cdc7; empty vector served as a control. Cells were grown to exponential phase in EMM2 medium at 25°C containing 2mM thiamine. Expression was induced by washing with EMM2 and growth for 16h at 25°C; cells were then shifted to 36°C for 5h, fixed, and stained with DAPI and Calcofluor as described [81]. Note that the cells carrying empty vector have become elongated and multinucleated, while 75% of cells expressing cdc7 have one or more septa. The scale bar represents 10 μm. (C) The strain leu1::pADH1-cdc7 was grown to exponential phase in YE medium at 19°C. A sample was taken and cells were fixed and stained with DAPI and Calcofluor. The remainder of the culture was incubated for 5h at 36°C before fixation. Note the elevated percentage of septated cells. The scale bar represents 10 μm. (D) The indicated strains were grown to exponential phase in YE medium, counted, and diluted to 106 ml-1. 10 μl of serial 5-fold dilutions were spotted on plates, allowed to dry and then incubated at the indicated temperature until the wild-type control had formed colonies.