The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure.

Looney, J E; Ma, Chi; Leu, Tzong-Shyng; Flintoff, Wayne F.; Troutman, W B; Hamlin, Joyce L.

doi:10.1128/mcb.8.12.5268

Cited by 32 publications

(22 citation statements)

References 45 publications

(91 reference statements)

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“…There is one copy of DHFR gene per amplisome, and no large repetitive structure is detected. This is in sharp contrast with the observed structure and organization of the other cytogenetic anomalies such as DMs, episomes, or homogeneous staining regions that are also associated with amplified genes (Cowell 1982;Carroll et al 1987;Looney et al 1988;Ma et al 1988). Many groups have studied the size of the amplification units and the organization of the amplified genes on DMs, episomes, and HSRs.…”

Section: Discussioncontrasting

confidence: 46%

Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.

Wang

Shouse²,

Lipes³

et al. 1996

Genome Res.

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We have cloned and mapped a circular 630-kb human extrachromosomal structure {termed amplisome} using the bacterial artificial chromosome {BAC) cloning system. Twenty-one BACs were isolated from an amplisome-enriched library by colony hybridization. The insert sizes range from 25 to 143 kb, with an average size of 82 kb. The coverage of the amplisome in clones is -2.7-fold. To construct a physical map of the amplisome, we used three different but complementary methods: hybridization, STS content mapping, and fingerprinting. In addition, we compared the advantages and the drawbacks of these techniques in mapping the amplisomal BACs. The 21 BACs were grouped into two contigs and the two small gaps {3.5 and 26.5 kb) were filled by screening of a human genomic BAC library. The organization of the amplisome revealed by the BAC-based physical map is consistent with the long-range restriction map reported previously. Our results demonstrate that a 630-kb region can be rapidly cloned and mapped into contigs by use of the BAC system. Because of the low frequency {<0.1%) of chimerism and rearrangement, these BAC clones are ready for DNA sequencing and functional analysis.Human cell line HeLa-Bu25-10B3 was isolated by selection for growth in stepwise increases in methotrexate (MTX) concentration, and although it contains 300 copies of the dihydrofolate reductase (DHFR) gene, it lacks homogeneously staining regions (HSRs) and contains few (0-3 per cell) double minutes (DMs) (Masters et al. 1982). Further studies have shown that the DHFR gene is located on a 630-kb extrachromosomal element, termed an amplisome (Maurer et al. 1987). It has been found that the amplisome does not change in size and copy number during long-term culture with MTX selection, and its loss is much slower than that expected from simple dilution upon withdrawal of MTX (Pauletti et al. 1990). The amplisome was found to have one copy of the DHFR gene per molecule, and no repetitive structure was observed (Esnault et al. 1994). To characterize further the structure and organization of the DNA sequences on am- plisomes, we have cloned amplisomal DNA in a large insert vector system. Recently, bacterial vectors based on the bacteriophage P1 (Sternberg 1990) and on the Escherichia coli fertility plasmid IF-factor; bacterial artificial chromosomes (BAC)] (O'Conner et al. 1989;Hosoda et al. 1990;Shizuya et al. 1992) have been developed for the cloning of large DNA. These systems have a very low frequency of chimerism, and the inserts are very stable during propagation (Shizuya et al. 1992). BACs utilize the well-studied E. coli single-copy fertility plasmid and have been shown to be able to accept human DNA inserts as large as 300 kb. Very little or no rearrangement of the inserts has been observed even after >100 generations of serial growth (Shizuya et al. 1992). The transformation efficiency of the BAC can be as high as 107 clones/~g of DNA (Wang et al. 1994). Because of these advantages, we have used the BAC vector to construct an amplisome library and a p...

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Section: Discussioncontrasting

confidence: 46%

Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.

Wang

Shouse²,

Lipes³

et al. 1996

Genome Res.

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“…The sizes of such extrachromosomal amplicons could easily conform to the majority of amplicon sizes detected at the CHO DHFR locus (Looney et al 1988). However, since some amplicons are probably larger than one replicon (Borst et al 1987;Looney et al 1988;Jongsma et al 1989), their genesis would require simultaneous or sequential breaks in two or more adjacent replication intermediates.…”

Section: A Model For Gene Amplification Involving Chromosome Breakagementioning

confidence: 99%

“…Circles produced by four breaks/ligations will contain imperfect inverted repeats of the type typically found in mammalian amplicons (Ford and Fried 1986;Saito and Stark 1986;Looney and Hamlin 1987;Passananti et al 1987;Hyrien et al 1988;Ruiz and Wahl 1988). The sizes of such extrachromosomal amplicons could easily conform to the majority of amplicon sizes detected at the CHO DHFR locus (Looney et al 1988). However, since some amplicons are probably larger than one replicon (Borst et al 1987;Looney et al 1988;Jongsma et al 1989), their genesis would require simultaneous or sequential breaks in two or more adjacent replication intermediates.…”

Section: A Model For Gene Amplification Involving Chromosome Breakagementioning

confidence: 99%

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

Windle¹,

Draper²,

Yin³

et al. 1991

Genes Dev.

239

147

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A CHO cell line with a single copy of the DHFR locus on chromosome Z2 was used to analyze the structure of the amplification target and products subsequent to the initial amplification event. Dramatic diversity in the number and cytogenetic characteristics of DHFR amplicons was observed as soon as eight to nine cell doublings following the initial event. Two amplicon classes were noted at this early time: Small extrachromosomal elements and closely spaced chromosomal amplicons were detected in 30-40% of metaphases in six of nine clones, whereas three of nine clones contained huge amplicons spanning >50 megabases. In contrast, the incidence of metaphases containing extrachromosomal amplicons fell to 1-2% in cells analyzed at 30-35 cell doublings, and most amplicons localized to rearranged or broken derivatives of chromosome Z2 at this time. Breakage of the Z2 chromosome near the DHFR gene, and deletion of the DHFR gene and flanking DNA was also observed in cells that had undergone the amplification process. To account for these diverse cytogenetic and molecular consequences of gene amplification, we propose that chromosome breakage plays a central role in the amplification process by (1) generating intermediates that are initially acentric and lead to copy number increase primarily by unequal segregation, (2) creating atelomeric ends that are either incompletely replicated or resected by exonucleases to generate deletions, and (3) producing recombinogenic ends that provide preferred sites for amplicon relocalization.

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“…Although some information is available concerning the structure and disposition of amplicons late in the amplification process (see Ardeshir et al 1983;Federspiel et al 1984;Debatisse et al 1986;Guilotto et al 1986;Looney and Hamlin 1987;Looney et al 1988;Ma et al 1988), very little is known about the molecular mechanisms that initiate and perpetuate the amplification process in any mammalian system. The following models have been proposed (for review, see Schimke 1982;Wahl 1984i Hamlin et al 1991): (1) unequal sister chromatid exchange (USCE) could result in the juxtaposition of both copies of a locus on one chromosome arm, presumably followed by additional rounds of USCE (Fig.…”

mentioning

confidence: 99%

Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells.

Ma¹,

Martin²,

Trask³

et al. 1993

Genes Dev.

Self Cite

185

103

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We have utilized a dihydrofolate reductase (DHFR) probe in combination with selected probes from other positions along the 2q chromosome arm in a two-color fluorescence in situ hybridization analysis of early DHFR gene amplification events in CHO cells. These studies show clearly that the most frequent initiating event is the formation of a giant inverted duplication, resulting either from chromosome breakage and terminal fusion or a reverse unequal sister chromatid exchange. The dicentric chromosomes thus formed initiate bridge/breakage/fusion cycles that appear to mediate subsequent amplification steps to higher copy number.

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The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure.

Abstract: We have previously cloned and characterized two different dihydrofolate reductase amplicon types from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400

Cited by 32 publications

References 45 publications

Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.

Bacterial artificial chromosome cloning and mapping of a 630-kb human extrachromosomal structure.

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells.

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