High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.
By the use of a highly sensitive mapping procedure allowing the identification 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. Biamonti, 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 investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.
Pelizon
C
C
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Diviacco
S
S
Falaschi
A
A
Giacca
M
M
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002874:Chromosome Mapping; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D004261:DNA Replication; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D018741:Replication Origin; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*
D017931:DNA Primers; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase