Figure 4.

In vivo association of Cdc6 and MCM with the P. abyssi replication origin detected by formaldehyde cross-linking. (A) The locations of oriC and cdc6 in P. abyssi sequence coordinates, as well as five primer sets used for PCR amplification (a, 113,000; b, oriC (Left); c, oriC (Right); d, 133,000; control, 745,000) within a genome segment (110,000–135,000). (B) Specific association of Cdc6 and MCM proteins with chromatin was addressed with specific antisera and primer sets a–d, normalized against signals observed for the control locus 624 kb from the origin. 1/240, 1/960, and 1/3,840 of total precipitated DNA were used as PCR templates (loaded from left to right). Input and Mock refer to reactions performed with total sonicated input DNA and negative control reactions performed without antiserum, respectively. Analyses were performed with exponential-phase (with and without puromycin treatment) and stationary-phase cultures. (C) The relative abundance of each PCR product obtained under various conditions is indicated; 0.8 kb refers to data obtained with the use of an 800-nt-long oriC-specific PCR target (data not shown).