THE CHINESE-HAMSTER DIHYDROFOLATE-REDUCTASE ORIGIN CONSISTS OF MULTIPLE POTENTIAL NASCENT-STRAND START SITES

Previous two dimensional gel replicon-mapping studies on the amplified dihydrofolate reductase (DHFR) domain in CHOC 400 cells suggested tha t replication can initiate at any of a large number of sites scattered throughout a 55-kb region lying between two convergently transcribed genes. It could be argued that this unusual distributive initiation mo de is unique to amplified chromosomal loci. In this paper, we report t he first application of the two-dimensional gel techniques to the anal ysis of a single-copy locus in mammalian cells. Results obtained with both synchronized and exponentially growing CHO cells suggest that (i) initiation can also occur at any of a large number of sites distribut ed throughout the intergenic region in the nonamplified DHFR locus, (i i) initiation is confined to the first 2 to 2.5 h of the S period, and (iii) initiation occurs only in a fraction of the DHFR loci in each c ell cycle.