HIGH-RESOLUTION MAPPING OF THE ORIGIN OF DNA-REPLICATION IN THE HAMSTER DIHYDROFOLATE-REDUCTASE GENE DOMAIN BY COMPETITIVE PCR

Bg the use of a highly sensitive mapping procedure allowing the identi fication of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Bia monti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was inv estigated, The method entails the purification of short stretches of n ascent DNA issuing from DNA replication origin regions and quantificat ion, within this sample, of the abundance of different adjacent segmen ts by competitive PCR. Distribution of marker abundance peaks around t he site front which newly synthesized DNA had emanated, The results ob tained by analysis of the genomic region downstream of the DHFR single -copy gene in asynchronous cultures of hamster CHO K1 cells are consis tent with the presence of a single start site for DNA replication, loc ated approximately 17 kb downstream of the gene. This site is coincide nt with the one detected by other studies using different techniques i n CHO cell lines containing an amplified DHFR gene domain.