Cloning of the initiation region of a mammalian chromosomal replicon

N. H. Heintz, J. D. Milbrandt, K. S. Greisen, J. L. Hamlin

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Abstract

In eukaryotic cells, each chromosome is divided into several thousand tandemly arranged replicons, each synthesized at a characteristic time during the S period. Although as yet no eukaryotic DNA sequence known for certain to be a eukaryotic chromosomal replication origin has been isolated, electron microscopic and biochemical evidence suggests that eukaryotic DNA synthesis is initiated at specific sites. The authors 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, they have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains 1,000 copies of a 135-kilobase (kb) early-replicating sequence, and which includes the gene for dihydrofolate reductase (DHFR). The authors 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 period, suggesting that these fragments contain or flank origins of DNA synthesis. Here they 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.

Original languageEnglish
Pages (from-to)439-441
Number of pages3
JournalNature
Volume302
Issue number5907
DOIs
StatePublished - Jan 1 1983
Externally publishedYes

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