REPLICATION INITIATION SITES ARE DISTRIBUTED WIDELY IN THE AMPLIFIED CHO DIHYDROFOLATE-REDUCTASE DOMAIN

In previous studies, we utilized a neutral/neutral two-dimensional (2- D) gel replicon mapping method to analyze the pattern of DNA synthesis in the amplified dihydrofolate reductase (DHFR) domain of CHOC 400 ce lls. Replication forks appeared to initiate at any of a large number o f sites scattered throughout the 55 kb region lying between the DHFR a nd 2BE2121 genes, and subsequently to move outward through the two gen es. In the present study, we have analyzed this locus in detail by a c omplementary, neutral/alkaline 2-D gel technique that determines the d irection in which replication forks move through a region of interest. In the early S period, forks are observed to travel in both direction s through the intergenic region, but only outward through the DHFR gen e. Surprisingly, however, replication forks also move in both directio ns through the 2BE2121 gene. Furthermore, in early S phase, small numb ers of replication bubbles can be detected in the 2BE2121 gene on neut ral/neutral 2-D gels. In contrast, replication bubbles have never been detected in the DHFR gene. Thus, replication initiates not only in th e intergenic region, but also at a lower frequency in the 2BE2121 gene . We further show that only a small fraction of DHFR amplicons sustain s an active initiation event, with the rest being replicated passively by forks from distant amplicons. These findings are discussed in ligh t of other experimental approaches that suggest the presence of a much more narrowly circumscribed initiation zone within the intergenic reg ion.