Advertisement

The Gene Cluster Instability (GCI) Assay for Recombination

  • Michael W. Killen
  • Dawn M. Stults
  • Andrew J. Pierce
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1105)

Abstract

A newly developed method for quantitatively detecting genomic restructuring in cultured human cell lines as the result of recombination is presented: the “gene cluster instability” (GCI) assay. The assay is physiological in that it detects spontaneous restructuring without the need for exogenous recombination-initiating treatments such as DNA damage. As an assay for genotoxicity, the GCI assay is complementary to well-established sister chromatid exchange (SCE) methods. Analysis of the U-2 OS osteosarcoma cell line is presented as an illustration of the method.

Key words

Genotoxicity Recombination Gene cluster Genomic instability 

References

  1. 1.
    Consortium, I. H. G. S (2004) Finishing the euchromatic sequence of the human genome. Nature 431:931–945CrossRefGoogle Scholar
  2. 2.
    Lupski JR, Stankiewicz P (2005) Genomic disorders: molecular mechanisms for rearrangements and conveyed phenotypes. PLoS Genet 1:e49PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Acilan C, Potter DM, Saunders WS (2007) DNA repair pathways involved in anaphase bridge formation. Genes Chromosomes Cancer 46:522–531PubMedCrossRefGoogle Scholar
  4. 4.
    McClintock B (1939) The behavior in successive nuclear divisions of a chromosome broken at meiosis. Proc Natl Acad Sci USA 25:405–416PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Tonon G, Wong KK, Maulik G, Brennan C, Feng B, Zhang Y, Khatry DB, Protopopov A, You MJ, Aguirre AJ, Martin ES, Yang Z, Ji H, Chin L, Depinho RA (2005) High-resolution genomic profiles of human lung cancer. Proc Natl Acad Sci USA 102:9625–9630PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Elliott B, Richardson C, Winderbaum J, Nickoloff JA, Jasin M (1998) Gene conversion tracts from double-strand break repair in mammalian cells. Mol Cell Biol 18:93–101PubMedCentralPubMedGoogle Scholar
  7. 7.
    Killen MW, Stults DM, Adachi N, Hanakahi L, Pierce AJ (2009) Loss of Bloom syndrome protein destabilizes human gene cluster architecture. Hum Mol Genet 18:3417–3428PubMedCrossRefGoogle Scholar
  8. 8.
    Stults DM, Killen MW, Williamson EP, Hourigan JS, Vargas HD, Arnold SM, Moscow JA, Pierce AJ (2009) Human ribosomal RNA gene clusters are recombinational hotspots in cancer. Cancer Res 69:9096–9104PubMedCrossRefGoogle Scholar
  9. 9.
    Stults DM, Killen MW, Pierce HH, Pierce AJ (2008) Genomic architecture and inheritance of human ribosomal RNA gene clusters. Genome Res 18:13–18PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Henderson AS, Warburton D, Atwood KC (1972) Location of ribosomal DNA in the human chromosome complement. Proc Natl Acad Sci USA 69:3394–3398PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Caburet S, Conti C, Schurra C, Lebofsky R, Edelstein SJ, Bensimon A (2005) Human ribosomal RNA gene arrays display a broad range of palindromic structures. Genome Res 15:1079–1085PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Worton RG, Sutherland J, Sylvester JE, Willard HF, Bodrug S, Dube I, Duff C, Kean V, Ray PN, Schmickel RD (1988) Human ribosomal RNA genes: orientation of the tandem array and conservation of the 5' end. Science 239:64–68PubMedCrossRefGoogle Scholar
  13. 13.
    Birren B, Lai E (1993) Pulsed field gel electrophoresis – a practical guide. Academic, San Diego, CAGoogle Scholar
  14. 14.
    Mertz LM, Rashtchian A (1994) Nucleotide imbalance and polymerase chain reaction: effects on DNA amplification and synthesis of high specific activity radiolabeled DNA probes. Anal Biochem 221:160–165PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2014

Authors and Affiliations

  • Michael W. Killen
    • 2
  • Dawn M. Stults
    • 3
  • Andrew J. Pierce
    • 1
  1. 1.Markey Cancer CenterUniversity of KentuckyLexingtonUSA
  2. 2.Markey Cancer CenterUniversity of KentuckyLexingtonUSA
  3. 3.Division of Hematology and Oncology, Department of MedicineVanderbilt UniversityNashvilleUSA

Personalised recommendations