Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain

Vaughn, James M.; Dijkwel, Pieter A.; Hamlin, Joyce L.

doi:10.1016/0092-8674(90)90071-l

Cited by 258 publications

(287 citation statements)

References 34 publications

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“…Apparently, this variant, on average, replicates earlier in S than does the predominant amplicon type. In agreement with earlier observations (15,16), no trace of a bubble arc is detectable in the DHFR gene at any film exposure. In samples taken 180 min after release from mimosine, only single fork arcs can be detected in all four fragments (data not shown), in agreement with the experiment in Fig.…”

Section: Dhfr (Rh)supporting

confidence: 80%

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Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain

Dijkwel¹,

Vaughn

Hamlin

1994

Nucl Acids Res

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In previous studies, we utilized a neutral/neutral twodimensional (2-D) gel replicon mapping method to analyze the pattern of DNA synthesis in the amplified dihydrofolate reductase (DHFR) domain of CHOC 400 cells. Replication forks appeared to initiate at any of a large number of sites scattered throughout the 55 kb region lying between the DHFR and 2BE2121 genes, and subsequently to move outward through the two genes. In the present study, we have analyzed this locus in detail by a complementary, neutral/alkaline 2-D gel technique that determines the direction in which replication forks move through a region of interest. In the early S period, forks are observed to travel in both directions through the intergenic region, but only outward through the DHFR gene. Surprisingly, however, replication forks also move in both directions through the 2BE2121 gene. Furthermore, in early S phase, small numbers of replication bubbles can be detected in the 2BE2121 gene on neutral/neutral 2-D gels. In contrast, replication bubbles have never been detected in the DHFR gene. Thus, replication initiates not only in the intergenic region, but also at a lower frequency in the 2BE2121 gene. We further show that only a small fraction of DHFR amplicons sustains an active initiation event, with the rest being replicated passively by forks from distant amplicons. These findings are discussed in light of other experimental approaches that suggest the presence of a much more narrowly circumscribed initiation zone within the intergenic region.

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Section: Dhfr (Rh)supporting

confidence: 80%

“…However, 2-D gel studies on this locus, as described herein and previously (15,16), have painted a somewhat different picture. Instead of narrowing down the position of the preferred initiation locus to within a few hundred base pairs, which both techniques are capable of doing when applied to genetic replicators in yeast (e.g.…”

Section: Discussionmentioning

confidence: 92%

Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain

Dijkwel¹,

Vaughn

Hamlin

1994

Nucl Acids Res

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“…The replication efficiencies of various plasmids in 293S cells determined as described in Table 1 site of initiation might be located in the vicinity of the region required. In S. cerevisiae, 2-D gel electrophoresis techniques permit mapping of initiation sites of chromosomal replication to short stretches of DNA that coincide with ARS elements Linskens and Huberman, 1988 (Vaughn et al, 1990;Dijkwel and Hamlin, 1992). Interestingly, application of 2-D gel techniques to the ura4 region in S. pombe showed that initiation sites cannot be located more precisely than 3-5 kb (Zhu et al, 1992 (Haase and Calos, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…It has been also shown that replication is initiated within a 5-10-kb region near the hamster rhodopsin gene by analysis of temporal order in which neighboring segments replicate (Gale et al, 1992). On the other hand, 2-D gel electrophoresis analysis has shown that replication of the DHFR gene is initiated from a large number of locations scattered within a 55-kb zone rather than from one or two unique regions (Vaughn et al, 1990;Dijkwel and Hamlin, 1992). In addition, initiation of replication at multiple sites in the Drosophila histon gene cluster has been shown by 2-D gel techniques and also by another method Ina, 1991, 1993).…”

Section: Introductionmentioning

confidence: 99%

Autonomous replication of human chromosomal DNA fragments in human cells.

Masukata

Satoh

Obuse

et al. 1993

MBoC

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We have examined whether a human chromosome has distinct segments that can replicate autonomously as extrachromosomal elements. Human 293S cells were transfected with a set of human chromosomal DNA fragments of 8-15 kilobase pairs that were cloned on an Escherichia coli plasmid vector. The transfected cells were subsequently cultured in the presence of 5-bromodeoxyuridine during two cell generations, and several plasmid clones labeled in both of the daughter DNA strands were isolated. Efficiency of replication of these clones, as determined from the ratios of heavy-heavy and one-half of heavy-light molecules to total molecules recovered from density-labeled cells, was 9.4% per cell generation on the average. Replication efficiency of control clones excluded during the selection was about 2.2% and that of the vector plasmid alone was 0.3%. A representative clone plWl replicated in a semiconservative manner only one round during the S phase of the cell cycle. It replicated extrachromosomally without integration into chromosome. The human segment of the clone was composed of several subsegments that promoted autonomous replication at different efficiencies. Our results suggest that certain specific nucleotide sequences are involved in autonomous replication of human segments.

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“…, and a centered bubble-containing fragments (curve c). Panels B&C. 2-D gel patterns of CHO replication intermediates isolated in very early S-phase before (B) and after (C) trapping; transfers of the DNA were hybridized with a probe specific for the ori-β locus in the DHFR initiation zone (29). The characteristic composite pattern shown in Panel B indicates that initiation can occur at several sites within this fragment (including its center), resulting in a complete bubble arc; however, the fragment is usually replicated passively from forks emanating from other sites within the initiation zone (resulting in a prominent and complete single fork arc).…”

Section: Figure 1 Principle Of the Bubble Trapping Protocolmentioning

confidence: 99%

Isolation of Restriction Fragments Containing Origins of Replication from Complex Genomes

Mesner¹,

Hamlin²

2015

Methods in Molecular Biology

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The identification and isolation of origins of replication from mammalian genomes has been a demanding task owing to the great complexity of these genomes. However, two methods have been refined in recent years each of which allows significant enrichment of recently-activated origins of replication from asynchronous cell cultures. One of these involves the selective entrapment of bubblecontaining fragments in gelling agarose and their subsequent recovery and isolation by molecular cloning. Libraries prepared by this method from Chinese hamster and human cells have been shown to be extremely pure, and provide a renewable resource of origins that can be used as probes on microarrays or sequenced by high-throughput techniques to localize them within the genomic source. The bubble-trapping method is described here for asynchronous mammalian cells that grow with reasonable doubling times and from which nuclear matrices can be reliably prepared. The method for nuclear matrix preparation and enrichment of replication intermediates is described in an accompanying chapter entitled, "Purifying replication intermediates from mammalian cells by enrichment on the nuclear matrix and BND-cellulose chromatography" (Mesner, Dijkwel, and Hamlin, Chapter __).

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Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain

Cited by 258 publications

References 34 publications

Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain

Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain

Autonomous replication of human chromosomal DNA fragments in human cells.

Isolation of Restriction Fragments Containing Origins of Replication from Complex Genomes

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