(A) Transcription of ctp1+ is regulated by MBF. Wild type and nrm1Δ strains were synchronized in G2 by centrifugal elutriation. Samples were taken as cells underwent mitosis and septation. The septation index approximately coincides with S phase. Transcript levels from ctp1+ and cdc22+ were determined by real-time PCR, normalized to act1+ transcript levels and shown as relative transcript levels (%) to maximum wild type levels during the cell cycle (wild type maximum is 100%).
(B) MBF localizes at the ctp1+ promoter region. Association of Cdc10-HA, Res2-HA and Nrm1-HA with the ctp1+, cdc22+ and act1+ promoters was determined by ChIP using log phase cultures. Tagged constructs were expressed from the endogenous loci. Whole cell extract (WCE) from the “no tag” strain was used as a control. Immunoblots confirmed that Nrm1 protein is expressed in res2Δ cells (data not shown).
(C) Immunoblot of Ctp1-TAP in asynchronous, irradiated and HU-treated cells. Immunoblot of Cdc2 with PSTAIR antisera is the loading control.
(D) Immunoblot of Ctp1-TAP in asynchronous, nitrogen-starved or carbon-starved cells. Immunoblot of tubulin is the loading control.
(E) Immunoblot of Ctp1-TAP in cells released from an HU arrest. Septation index indicates completion of the cell cycle.