Abstract
In eukaryotic cells, each chromosome is divided into several thousand tandemly arranged replicons, each synthesized at a characteristic time during the S period1,2. Although as yet no eukaryotic DNA sequence known for certain to be a eukaryotic chromosomal replication origin has been isolated, electron microscopic3,4 and biochemical5–7 evidence suggests that eukaryotic DNA synthesis is initiated at specific sites. We have attempted to establish a system in which functional chromosomal origins could be identified in vivo before their isolation by molecular cloning. For this purpose, we have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains 1,000 copies of a 135-kilobase (kb) early-replicating sequence8, and which includes the gene for dihydrofolate reductase (DHFR). We have shown that initiation of DNA synthesis within each repeated unit (amplicon) is restricted to a small subset of restriction fragments at the beginning of the S period9, suggesting that these fragments contain or flank origins of DNA synthesis. Here we report molecular cloning and restriction mapping experiments showing that all of these early-labelled fragments (ELFs) are derived from a single locus within each repeated unit. This result implies that synthesis of each amplicon initiates from a single origin of replication at the onset of S, and that an amplicon is formally equivalent to a replicon.
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Heintz, N., Milbrandt, J., Greisen, K. et al. Cloning of the initiation region of a mammalian chromosomal replicon. Nature 302, 439–441 (1983). https://doi.org/10.1038/302439a0
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DOI: https://doi.org/10.1038/302439a0
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