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Molecular and Cellular Biology, February 2001, p. 1098-1110, Vol. 21, No. 4
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.4.1098-1110.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The Chinese Hamster Dihydrofolate Reductase Replication Origin Beta Is Active at Multiple Ectopic Chromosomal Locations and Requires Specific DNA Sequence Elements for Activity

Amy L. Altman and Ellen Fanning*

Department of Molecular Biology and Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37232-6838

Received 24 August 2000/Returned for modification 11 October 2000/Accepted 16 November 2000

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.

* Corresponding author. Mailing address: Department of Molecular Biology, Vanderbilt University, 2325 Stevenson Center, 1161 21st Ave. South, Nashville, TN 37232. Phone: (615) 343-5677. Fax: (615) 343-6707. E-mail: fannine{at}ctrvax.vanderbilt.edu.

Molecular and Cellular Biology, February 2001, p. 1098-1110, Vol. 21, No. 4
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.4.1098-1110.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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