Temporal order of replication of mouse ribosomal RNA genes during the cell cycle

The timing of replication of mouse ribosomal RNA (rRNA) genes was determined in cultured cells by using 5-bromodeoxyuridine labeling of DNA coupled with synchronization. Two subclasses of rRNA genes were characterized that differ in their temporal order of replication during S-phase. Approximately half of the rDNA repeat units replicated primarily during the first half of S-phase and the other 50% preferentially in the second half. This difference in replication timing was consistently observed for the approximately 400 rDNA repeat units of NIH3T3 fibroblasts, but not for plasmid DNA containing fragments of rRNA genes that had been stably transfected into the genome of these cells. The rDNA fragments inserted into these transfection vectors contained the recently mapped origin of bidirectional replication with or without amplification-promoting sequences, or none of the above. Since the plasmid DNA that was integrated into the host cell genome replicated randomly during S-phase we conclude that the integrated plasmid DNA is either replicated from a chromosomal origin in the neighborhood of its integration site or that inserts are replicated from their own origins and the timing of replication is determined by flanking sequences. Received: 7 July 1997; in revised form: 1 October 1997; Accepted: 1 October 1997