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Mol Cell Biol. 1990 Jun; 10(6): 3056–3066.
PMCID: PMC360670

Chromosomal destabilization during gene amplification.


Acentric extrachromosomal elements, such as submicroscopic autonomously replicating circular molecules (episomes) and double minute chromosomes, are common early, and in some cases initial, intermediates of gene amplification in many drug-resistant and tumor cell lines. In order to gain a more complete understanding of the amplification process, we investigated the molecular mechanisms by which such extrachromosomal elements are generated and we traced the fate of these amplification intermediates over time. The model system consists of a Chinese hamster cell line (L46) created by gene transfer in which the initial amplification product was shown previously to be an unstable extrachromosomal element containing an inverted duplication spanning more than 160 kilobases (J. C. Ruiz and G. M. Wahl, Mol. Cell. Biol. 8:4302-4313, 1988). In this study, we show that these molecules were formed by a process involving chromosomal deletion. Fluorescence in situ hybridization was performed at multiple time points on cells with amplified sequences. These studies reveal that the extrachromosomal molecules rapidly integrate into chromosomes, often near or at telomeres, and once integrated, the amplified sequences are themselves unstable. These data provide a molecular and cytogenetic chronology for gene amplification in this model system; an early event involves deletion to generate extrachromosomal elements, and subsequent integration of these elements precipitates a cascade of chromosome instability.

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  • Agard DA, Sedat JW. Three-dimensional architecture of a polytene nucleus. Nature. 1983 Apr 21;302(5910):676–681. [PubMed]
  • Arndt-Jovin DJ, Robert-Nicoud M, Zarling DA, Greider C, Weimer E, Jovin TM. Left-handed Z-DNA in bands of acid-fixed polytene chromosomes. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4344–4348. [PMC free article] [PubMed]
  • Blackburn EH. The molecular structure of centromeres and telomeres. Annu Rev Biochem. 1984;53:163–194. [PubMed]
  • Bostock CJ, Tyler-Smith C. Gene amplification in methotrexate-resistant mouse cells. II. Rearrangement and amplification of non-dihydrofolate reductase gene sequences accompany chromosomal changes. J Mol Biol. 1981 Dec 5;153(2):219–236. [PubMed]
  • Carroll SM, DeRose ML, Gaudray P, Moore CM, Needham-Vandevanter DR, Von Hoff DD, Wahl GM. Double minute chromosomes can be produced from precursors derived from a chromosomal deletion. Mol Cell Biol. 1988 Apr;8(4):1525–1533. [PMC free article] [PubMed]
  • Carroll SM, Gaudray P, De Rose ML, Emery JF, Meinkoth JL, Nakkim E, Subler M, Von Hoff DD, Wahl GM. Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin. Mol Cell Biol. 1987 May;7(5):1740–1750. [PMC free article] [PubMed]
  • Cherif D, Lavialle C, Modjtahedi N, Le Coniat M, Berger R, Brison O. Selection of cells with different chromosomal localizations of the amplified c-myc gene during in vivo and in vitro growth of the breast carcinoma cell line SW 613-S. Chromosoma. 1989 Jan;97(4):327–333. [PubMed]
  • Dancis BM, Holmquist GP. Telomere replication and fusion in eukaryotes. J Theor Biol. 1979 May 21;78(2):211–224. [PubMed]
  • DePamphilis ML. Transcriptional elements as components of eukaryotic origins of DNA replication. Cell. 1988 Mar 11;52(5):635–638. [PubMed]
  • Edenberg HJ, Huberman JA. Eukaryotic chromosome replication. Annu Rev Genet. 1975;9:245–284. [PubMed]
  • Feinberg AP, Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. [PubMed]
  • Solomon E, Hiorns LR, Spurr N, Kurkinen M, Barlow D, Hogan BL, Dalgleish R. Chromosomal assignments of the genes coding for human types II, III, and IV collagen: a dispersed gene family. Proc Natl Acad Sci U S A. 1985 May;82(10):3330–3334. [PMC free article] [PubMed]
  • Ford M, Fried M. Large inverted duplications are associated with gene amplification. Cell. 1986 May 9;45(3):425–430. [PubMed]
  • Gaudray P, Trotter J, Wahl GM. Fluorescent methotrexate labeling and flow cytometric analysis of cells containing low levels of dihydrofolate reductase. J Biol Chem. 1986 May 15;261(14):6285–6292. [PubMed]
  • Giulotto E, Saito I, Stark GR. Structure of DNA formed in the first step of CAD gene amplification. EMBO J. 1986 Sep;5(9):2115–2121. [PMC free article] [PubMed]
  • Glanville N. Unstable expression and amplification of a transfected oncogene in confluent and subconfluent cells. Mol Cell Biol. 1985 Jun;5(6):1456–1464. [PMC free article] [PubMed]
  • Gudkov AV, Chernova OB, Kazarov AR, Kopnin BP. Cloning and characterization of DNA sequences amplified in multidrug-resistant Djungarian hamster and mouse cells. Somat Cell Mol Genet. 1987 Nov;13(6):609–619. [PubMed]
  • Hastie ND, Allshire RC. Human telomeres: fusion and interstitial sites. Trends Genet. 1989 Oct;5(10):326–331. [PubMed]
  • Heartlein MW, Latt SA. Amplified inverted duplications within and adjacent to heterologous selectable DNA. Nucleic Acids Res. 1989 Feb 25;17(4):1697–1716. [PMC free article] [PubMed]
  • Holmquist GP, Dancis B. Telomere replication, kinetochore organizers, and satellite DNA evolution. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4566–4570. [PMC free article] [PubMed]
  • Hyrien O, Debatisse M, Buttin G, de Saint Vincent BR. A hotspot for novel amplification joints in a mosaic of Alu-like repeats and palindromic A + T-rich DNA. EMBO J. 1987 Aug;6(8):2401–2408. [PMC free article] [PubMed]
  • Hyrien O, Debatisse M, Buttin G, de Saint Vincent BR. The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification. EMBO J. 1988 Feb;7(2):407–417. [PMC free article] [PubMed]
  • Kaufman RJ, Schimke RT. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line. Mol Cell Biol. 1981 Dec;1(12):1069–1076. [PMC free article] [PubMed]
  • Kaufman RJ, Sharp PA, Latt SA. Evolution of chromosomal regions containing transfected and amplified dihydrofolate reductase sequences. Mol Cell Biol. 1983 Apr;3(4):699–711. [PMC free article] [PubMed]
  • Kim HS, Smithies O. Recombinant fragment assay for gene targetting based on the polymerase chain reaction. Nucleic Acids Res. 1988 Sep 26;16(18):8887–8903. [PMC free article] [PubMed]
  • Lin CC, Alitalo K, Schwab M, George D, Varmus HE, Bishop JM. Evolution of karyotypic abnormalities and C-MYC oncogene amplification in human colonic carcinoma cell lines. Chromosoma. 1985;92(1):11–15. [PubMed]
  • Looney JE, Hamlin JL. Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells. Mol Cell Biol. 1987 Feb;7(2):569–577. [PMC free article] [PubMed]
  • McCLINTOCK B. Chromosome organization and genic expression. Cold Spring Harb Symp Quant Biol. 1951;16:13–47. [PubMed]
  • McClintock B. The significance of responses of the genome to challenge. Science. 1984 Nov 16;226(4676):792–801. [PubMed]
  • Meinkoth J, Killary AM, Fournier RE, Wahl GM. Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification. Mol Cell Biol. 1987 Apr;7(4):1415–1424. [PMC free article] [PubMed]
  • Melton DW, Konecki DS, Ledbetter DH, Hejtmancik JF, Caskey CT. In vitro translation of hypoxanthine/guanine phosphoribosyltransferase mRNA: characterization of a mouse neuroblastoma cell line that has elevated levels of hypoxanthine/guanine phosphoribosyltransferase protein. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6977–6980. [PMC free article] [PubMed]
  • Nalbantoglu J, Meuth M. DNA amplification--deletion in a spontaneous mutation of the hamster aprt locus: structure and sequence of the novel joint. Nucleic Acids Res. 1986 Nov 11;14(21):8361–8371. [PMC free article] [PubMed]
  • Passananti C, Davies B, Ford M, Fried M. Structure of an inverted duplication formed as a first step in a gene amplification event: implications for a model of gene amplification. EMBO J. 1987 Jun;6(6):1697–1703. [PMC free article] [PubMed]
  • Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J. Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9138–9142. [PMC free article] [PubMed]
  • Ruiz JC, Choi KH, von Hoff DD, Roninson IB, Wahl GM. Autonomously replicating episomes contain mdr1 genes in a multidrug-resistant human cell line. Mol Cell Biol. 1989 Jan;9(1):109–115. [PMC free article] [PubMed]
  • Ruiz JC, Wahl GM. Formation of an inverted duplication can be an initial step in gene amplification. Mol Cell Biol. 1988 Oct;8(10):4302–4313. [PMC free article] [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Saito I, Groves R, Giulotto E, Rolfe M, Stark GR. Evolution and stability of chromosomal DNA coamplified with the CAD gene. Mol Cell Biol. 1989 Jun;9(6):2445–2452. [PMC free article] [PubMed]
  • Saito I, Stark GR. Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8664–8668. [PMC free article] [PubMed]
  • Schimke RT. Gene amplification in cultured cells. J Biol Chem. 1988 May 5;263(13):5989–5992. [PubMed]
  • Schimke RT, Sherwood SW, Hill AB, Johnston RN. Overreplication and recombination of DNA in higher eukaryotes: potential consequences and biological implications. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2157–2161. [PMC free article] [PubMed]
  • Sen S, Hittelman WN, Teeter LD, Kuo MT. Model for the formation of double minutes from prematurely condensed chromosomes of replicating micronuclei in drug-treated Chinese hamster ovary cells undergoing DNA amplification. Cancer Res. 1989 Dec 1;49(23):6731–6737. [PubMed]
  • Stark GR, Debatisse M, Giulotto E, Wahl GM. Recent progress in understanding mechanisms of mammalian DNA amplification. Cell. 1989 Jun 16;57(6):901–908. [PubMed]
  • Stark GR, Wahl GM. Gene amplification. Annu Rev Biochem. 1984;53:447–491. [PubMed]
  • Trent J, Meltzer P, Rosenblum M, Harsh G, Kinzler K, Mashal R, Feinberg A, Vogelstein B. Evidence for rearrangement, amplification, and expression of c-myc in a human glioblastoma. Proc Natl Acad Sci U S A. 1986 Jan;83(2):470–473. [PMC free article] [PubMed]
  • Urlaub G, Käs E, Carothers AM, Chasin LA. Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell. 1983 Jun;33(2):405–412. [PubMed]
  • van der Bliek AM, Lincke CR, Borst P. Circular DNA of 3T6R50 double minute chromosomes. Nucleic Acids Res. 1988 Jun 10;16(11):4841–4851. [PMC free article] [PubMed]
  • Von Hoff DD, Needham-VanDevanter DR, Yucel J, Windle BE, Wahl GM. Amplified human MYC oncogenes localized to replicating submicroscopic circular DNA molecules. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4804–4808. [PMC free article] [PubMed]
  • Wahl GM. The importance of circular DNA in mammalian gene amplification. Cancer Res. 1989 Mar 15;49(6):1333–1340. [PubMed]
  • Wahl GM, Robert de Saint Vincent B, DeRose ML. Effect of chromosomal position on amplification of transfected genes in animal cells. Nature. 1984 Feb 9;307(5951):516–520. [PubMed]
  • Wahl GM, Vitto L, Padgett RA, Stark GR. Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization. Mol Cell Biol. 1982 Mar;2(3):308–319. [PMC free article] [PubMed]

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