Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells.

Looney, J E; Hamlin, Joyce L.

doi:10.1128/mcb.7.2.569

Cited by 104 publications

(113 citation statements)

References 31 publications

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“…The major repeating unit (amplicon) in this cell line is 240 kb in length (6). Results of intrinsic labeling studies on synchronized CHOC 400 cells were consistent with the presence of one (7,8), or possibly two (9,10), fixed initiation sites lying in the intergenic region between the DHFR and 2BE2121 genes [termed ori-f and ori-' (10); Fig.…”

Section: Introductionmentioning

confidence: 70%

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: Introductionmentioning

confidence: 70%

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 many cases characterized, these amplified regions contain large ''head-to-head'' duplications (palindromes) of sequences that can be tens to hundreds of kb in size (3)(4)(5)(6)(7)(8). Cytogenetic studies have further revealed that large inverted duplications of a chromosomal region are formed at early stages of gene amplification (9)(10)(11).…”

mentioning

confidence: 99%

Short inverted repeats initiate gene amplification through the formation of a large DNA palindrome in mammalian cells

Tanaka

Tapscott

Trask

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

105

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Gene amplification is a common form of genomic instability in a wide variety of organisms and is often associated with tumor progression in mammals. One striking feature of many amplified genes is their organization as large inverted duplications (palindromes). Here, we describe a molecular mechanism for palindrome formation in mammalian cells that is also conserved in protists. We introduced a short (79 or 229 bp) inverted repeat into the genome of Chinese hamster ovary cells and showed that it promoted the formation of a large DNA palindrome after an adjacent DNA double-strand break. This finding suggests that short inverted repeats in the mammalian genome can have a critical role in the initiation of gene amplification. This specific mechanism may provide a novel target for cancer therapies.

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“…Thus, during the amplification process, pieces of DNA from different parts of the genome are joined by recombination to form novel structures and novel joints (new restriction fragments). In some highly resistant cell lines, novel structures are amplified enough to be cloned by differential screening or to be detected by direct visualization or in gel renaturation, and highly redundant novel joints have been detected (2,9,22,32,37). In these cases, most of the amplified units seem to be derived from a single rearranged structure, formed early in * Corresponding author.…”

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confidence: 99%

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

Debatisse¹,

Saito²,

Buttin³

et al. 1988

Mol. Cell. Biol.

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In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of anl four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second-and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.In a number of systems, drug resistance is mediated by gene amplification, and in all cases studied, a sequence much larger than the selected gene is amplified. The first estimates of the average sizes of amplified units-defined as extra DNA sequences containing one copy of the selected geneindicate that they range from about 200 to 3,000 kilobases (kb) in highly resistant cells which have experienced several independent amplification events (for reviews, see references 18, 35, and 39). More recently, a study of the structures formed in the first step of amplification of the CAD gene in Syrian hamster cells has led to the hypothesis that, in this case, the average size of the amplified unit may be 10,000 kb or more (15).Several different techniques have been used to study the structure of the coamplified DNA: cloning of amplified sequences by differential screening (2,4,15) or from purified double minutes (5,14) or homogeneously staining regions (19,43), chromosome walking from the cloned selected genes (11,22), direct visualization of ethidium bromidestained amplified restriction fragments in agarose gels (24,40) and in gel renaturation (12, 31). In most cases, the results point to remarkable differences between the sequences coamplified in independent highly amplified mutants: the amplified units include a region comprising the selected gene and sequences immediately surrounding it in the wild-type line, but also include sequences which are amplified uniquely in each cell line (2,5,12, 34,36,43). Thus, during the amplification process, pieces of DNA from different parts of the genome are joined by recombination to form novel structures and novel join...

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Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells.

Cited by 104 publications

References 31 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

Short inverted repeats initiate gene amplification through the formation of a large DNA palindrome in mammalian cells

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

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