The Chinese hamster dihydrofolate reductase replication origin beta is active at multiple ectopic chromosomal locations and requires specific DNA sequence elements for activity

To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb, fragment from the Chinese hamster dihydrofolate reductase (DIIFR) locus containing the origin beta (ori-beta) initiation r egion was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-beta in uncloned pools of transfected cells was measured using a compe titive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-beta region in Chinese hamster ovary K1 cells. Initiatio n activity of three ectopic ori-beta deletion mutants was reduced, while th e activity of another deletion mutant was enhanced. The results suggest tha t a 5.8-kb fragment of the DHFR ori-beta region is sufficient to direct ini tiation and that specific DNA sequences in the ori-p region are required fo r efficient initiation activity.