Gene amplification in methotrexate-resistant mouse cells

Tyler-Smith, Christopher; Alderson, Thomas

doi:10.1016/0022-2836(81)90274-6

Cited by 35 publications

(9 citation statements)

References 45 publications

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“…The Southern blots of aphid DNA cannot be interpreted with the same certainty as similar data from cell cultures [18], because the aphid clones originated in the field and may have arisen independently, rather than i o 10--..…”

Section: Discussionmentioning

confidence: 94%

“…R 2 sequentially, as in the cell-culture-selection experiments. However, the 15-kb fragment in translocated aphids may be equivalent to the new bands appearing after drug selection of cell cultures and believed to result from the formation ofnovel joints during the amplification [14,18]. The 15-kb fragment in aphids could thus arise from the 10-kb fragment ofnon-translocated aphids.…”

Section: Al Mrmentioning

confidence: 99%

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Molecular evidence that insecticide resistance in peach-potato aphids (Myzus persicae Sulz.) results from amplification of an esterase gene

Field

Devonshire

Forde

1988

Biochemical Journal

226

116

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cDNA clones for the esterase (E4) responsible for broad insecticide resistance in peach-potato aphids (Myzus persicae Sulz.) were isolated and used to study the molecular basis of resistance. Increased esterase synthesis by resistant aphids was found to be associated with amplification of the structural gene for the esterase (E4 or its closely related variant, FE4), the degree of amplification being correlated with the activity of the esterase and the level of resistance. Hybridization of the cDNA clones to genomic Southern blots showed that only some of the esterase-related restriction fragments are amplified. Qualitative differences between restriction patterns in different clones of resistant aphids correlated with the presence or absence of a specific chromosome translocation and with production of E4 or FE4.

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Section: Discussionmentioning

confidence: 94%

Section: Al Mrmentioning

confidence: 99%

Molecular evidence that insecticide resistance in peach-potato aphids (Myzus persicae Sulz.) results from amplification of an esterase gene

Field

Devonshire

Forde

1988

Biochemical Journal

226

116

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“…Since S180, 3T6, and other cell lines are highly aneuploid, it is possible that frequent genomic rearrangements could alter the position of the target gene before or during the selection process. Consistent with this hypothesis are the observations that the frequency and stability of CAD gene (42) and v-src (17) amplification can be significantly influenced by genomic location and that gene translocation is frequently observed as an early step in the amplification process (2,36,37,41,42).…”

Section: Resultsmentioning

confidence: 64%

“…6) were isolated from a single parental single-cell clone. In previous work (22,41) the drug-resistant lines analyzed were derived from uncloned populations and therefore were not likely to be derived from the same parental clone. Second, we analyzed the stability of the amplified CAD genes at the first selective step and not after long periods of growth under selective conditions.…”

Section: Resultsmentioning

confidence: 99%

Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.

Meinkoth¹,

Killary²,

Fournier³

et al. 1987

Mol. Cell. Biol.

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We analyzed the amplification of the CAD gene in independently isolated N-(phosphonacetyl)-L-aspartate-resistant clones derived from single parental clones in two mouse cell lines. We report for the first time that the CAD gene is amplified unstably in mouse cells, that the degree of instability varies greatly between clones, and that minute chromosomes and highly unstable chromosomelike structures contain the amplified sequences. These data are most consistent with the idea that the amplified unit in each clone consists of different flanking DNA and that such differences engender amplified sequences with unequal stability. We also introduced the mouse chromosome containing the CAD gene into hamster cells by microcell-mediated chromosome transfer to determine whether the propensity for unstable extrachromosomal amplification of the mouse CAD gene would prevail in the hamster cell nuclear environment. We report that the mouse CAD gene was amplified stably in expanded chromosomal regions in each of seven hybrids that were analyzed. This observation is consistent with the idea that the nuclear environment influences whether mutants containing intra- or extrachromosomally amplified sequences will be isolated.

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“…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).…”

mentioning

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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Gene amplification in methotrexate-resistant mouse cells

Cited by 35 publications

References 45 publications

Molecular evidence that insecticide resistance in peach-potato aphids (Myzus persicae Sulz.) results from amplification of an esterase gene

Molecular evidence that insecticide resistance in peach-potato aphids (Myzus persicae Sulz.) results from amplification of an esterase gene

Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

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