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Molecular and Cellular Biology, June 1999, p. 4231-4240, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Active Role of a Human Genomic Insert in Replication of a Yeast Artificial Chromosome

Anja J. van Brabant,dagger Walton L. Fangman, and Bonita J. Brewer*

Department of Genetics, University of Washington, Seattle, Washington 98195-7360

Received 1 December 1998/Returned for modification 11 January 1999/Accepted 4 March 1999

Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.

* Corresponding author. Mailing address: University of Washington, Department of Genetics, Box 357360, Seattle, WA 98195-7360. Phone: (206) 685-4966. Fax: (206) 543-0754. E-mail: bbrewer{at}genetics.washington.edu.

dagger Present address: Laboratory of Cancer Susceptibility, Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Molecular and Cellular Biology, June 1999, p. 4231-4240, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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