FIG. 1.

The affinity of Orc1 for chromatin changes during the M-to-G₁ phase transition. (A) Orc1 could be selectively eluted from chromatin in M-phase cells but not from that in G₁-phase cells. CHO C400 cells were synchronized at metaphase and then released into G₁ phase for 4 h. Aliquots of cells were then lysed either with SDS to measure total Orc1 or with CSK buffer to measure chromatin-bound and -unbound Orc1. Fractions were then subjected to SDS-PAGE; the upper portion of the gel was assayed for Orc1 protein, and the lower portion was stained for total proteins. Proteins were identified by their antibody specificities and their migration patterns during SDS-PAGE. The fraction of Orc1 was calculated by defining the amounts of total Orc1 in M- and G₁-phase cells each as 100%. The relative amount of Orc1 in each lane was normalized to the amount of either H1 or core histone present. (B) CHO C400 cells were lysed either in M-PER (shown here) or in CSK buffer containing 0.35 M NaCl (data not shown) in order to solubilize 90 to 95% of the Orc1 and Orc2 under conditions suitable for immunoprecipitation. S, soluble fraction; P, immunoprecipitate.