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 (DHFR) locus containing the origin beta (ori-beta) initiation region 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 competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-beta region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-beta deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-beta region is sufficient to direct initiation and that specific DNA sequences in the ori-beta region are required for efficient initiation activity.
Altman
A L
AL
Department of Molecular Biology and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37232-6838, USA.
Fanning
E
E
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002875:Chromosomes / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D018741:Replication Origin; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D013762:Tetrahydrofolate Dehydrogenase