Abstract
A general approach for assaying the in vivo direction of replication for any DNA segment has been developed. This technique allows the scanning of genomic regions to detect bidirectional tail-to-tail replication, indicating the presence of a functional origin. By this criterion we identified the approximate positions of two origin sites downstream of the Chinese hamster DHFR gene. Further mapping revealed areas of head-to-head replication, signifying locations of replication termination and thus defining the landmarks of a complete animal cell replicon. Genetic proof for the existence of the DHFR origin was obtained by showing that this region serves as a bidirectional DNA synthesis initiation point following its integration into other sites in the genome by transfection. To show the general applicability of this methodology, we studied the APRT domain. Replication mapping together with the use of deletion mutants allowed the identification of an origin at a far-upstream locus.
| Original language | English |
|---|---|
| Pages (from-to) | 909-920 |
| Number of pages | 12 |
| Journal | Cell |
| Volume | 57 |
| Issue number | 6 |
| DOIs | |
| State | Published - 16 Jun 1989 |
Bibliographical note
Funding Information:We are greatly indebted to Joyce Hamlin for making available a full range of cosmid clones covering the Chinese hamster DHFR domain and for fruitful discussions on DNA replication. We also thank Bob Martin for providing the SV40-transformed cell line, and Larry Chasin for letting us use his cell line containing a transfected DHFR cosmid. Daniel Kitsberg and Marc Michaelson helped us with several technical aspects of the work, and Geoff Sargent was instrumental in analyzing the 5209 deletion mutant. We would also like to thank Caroline Gopin for handling the processing of this paper, and A. Razin and G. Glaser for their helpful comments on the manuscript. This research was supported by grants from the National Institutes of Health (to H. C.), the Israel Cancer Research Fund (to H. C.), and the Imperial Cancer Research Fund (to M. M.).