Fig. 6. Origin firing in response to Cdt1. (A) 2D gels of genomic DNA extracted from cdc25-22 nmt1-cdc18+ pREP4-cdt1+ cells probed for ars3001. Samples were collected every 2 h following the shift to 37°C in the absence of thiamine. (B) 2D gel of wild-type cells treated with HU (11 mM) for 3.5 h at 32°C, probed for ars3001. The arrow indicates the stronger bubble arc. (C) 2D gel of cdc25-22 nmt1-cdc18+ pREP4-cdt1+ cells shifted to 37°C in the absence of thiamine and treated with HU (24 mM) at 4 h. Samples were collected at 6 h at 37°C, after 2 h of treatment with HU. Gels were probed for ars3001. (D) 2D gels of cdc25-22 nmt1-cdc18+ pREP4 and cdc25-22 nmt1-cdc18+ pREP4-cdt1+ cells shifted to 37°C in the absence of thiamine, treated with HU (24 mM) at 7 h and collected 1 h later. Gels were probed for ars3001. (E) 2D gel of genomic DNA extracted from cdc25-22 nmt1-cdc18+ pREP4-cdt1+ cells and probed for a non-origin region downstream of ars3001. Samples were collected every 2 h following the shift to 37°C in the absence of thiamine. (F) 2D gel of cdc25-22 nmt1-cdc18+ pREP4-cdt1+ cells shifted to 37°C in the absence of thiamine and treated with HU (24 mM) at 4 h. Cells were collected at 10 h after 6 h of treatment with HU. The 2D gel was probed with the same fragment as described in (E).