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Updated: Aug 10, 2026

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization
Published on: December 10, 2012
The Chinese hamster dihydrofolate reductase replication origin beta is active at multiple ectopic chromosomal
1Department of Molecular Biology and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37232-6838, USA.
Researchers identified key DNA sequences controlling mammalian DNA replication. A specific fragment from the dihydrofolate reductase (DHFR) origin beta (ori-beta) region directs DNA replication initiation.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Mammalian chromosomal DNA replication requires specific cis-acting genetic elements.
- The dihydrofolate reductase (DHFR) locus contains a characterized initiation region, ori-beta.
Purpose of the Study:
- To identify essential cis-acting genetic elements for mammalian chromosomal DNA replication.
- To determine the sufficiency of the DHFR ori-beta region in directing DNA replication initiation.
Main Methods:
- Stable transfection of a 5.8-kb DHFR ori-beta fragment into hamster cells lacking the endogenous DHFR locus.
- Competitive PCR-based nascent strand abundance assay to measure initiation activity at ectopic sites.
- Analysis of deletion mutants to pinpoint critical DNA sequences.
Main Results:
- Ectopic ori-beta initiated DNA replication similarly to the endogenous locus.
- Deletion mutants showed reduced or enhanced initiation activity, indicating sequence-specific requirements.
- A 5.8-kb fragment of the DHFR ori-beta region was sufficient for directing initiation.
Conclusions:
- The 5.8-kb DHFR ori-beta fragment contains the necessary elements for replication initiation.
- Specific DNA sequences within the ori-beta region are crucial for efficient initiation activity.
- This study elucidates key regulatory elements governing mammalian DNA replication.
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