Skip to main content
SpringerLink
Log in

Non-reciprocal chromosomal bridge-induced translocation (BIT) by targeted DNA integration in yeast

  • Research Article
  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

Several experimental in vivo systems exist that generate reciprocal translocations between engineered chromosomal loci of yeast or Drosophila, but not without previous genome modifications. Here we report the successful induction of chromosome translocations in unmodified yeast cells via targeted DNA integration of the KANR selectable marker flanked by sequences homologous to two chromosomal loci randomly chosen on the genome. Using this bridge-induced translocation system, 2% of the integrants showed targeted translocations between chromosomes V-VIII and VIII-XV in two wild-type Saccharomyces cerevisiae strains. All the translocation events studied were found to be non-reciprocal and the fate of their chromosomal fragments that were not included in the translocated chromosome was followed. The recovery of discrete-sized fragments suggested multiple pathway repair of their free DNA ends. We propose that centromere-distal chromosome fragments may be processed by a break-induced replication mechanism ensuing in partial trisomy. The experimental feasibility of inducing chromosomal translocations between any two desired genetic loci in a eukaryotic model system will be instrumental in elucidating the molecular mechanism underlying genome rearrangements generated by DNA integration and the gross chromosomal rearrangements characteristic of many types of cancer.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1a,b
Fig. 2a,b
Fig. 3
Fig. 4
Fig. 5a–c
Table 3a,b

Similar content being viewed by others

References

  • Aguilera A (2001) Double-strand break repair: are Rad51/RecA-DNA joints barriers to DNA replication? Trends Genet 17:318–321

    Google Scholar 

  • Aylon Y, Kupiec M (2004) New insights into the mechanism of homologous recombination in yeast. Mutat Res 566:231–248

    Google Scholar 

  • Barr FG (1998) Translocations, cancer and the puzzle of specificity. Nat Genet 19:121–124

    Google Scholar 

  • Blomquist P, Belikov S, Wrange O (1999) Increased nuclear factor 1 binding to its nucleosomal site mediated by sequence-dependent DNA structure. Nucleic Acids Res 27:517–525

    Google Scholar 

  • Bosco G, Haber JE (1998) Chromosome break-induced DNA replication leads to nonreciprocal translocations and telomere capture. Genetics 150:1037–1047

    Google Scholar 

  • Bradbury JM, Jackson SP (2003) The complex matter of DNA double-strand break detection. Biochem Soc Trans 31:40–44

    Google Scholar 

  • Bruschi CV, Howe GA (1988) High frequency FLP-independent homologous DNA recombination of 2μ plasmid in the yeast Saccharomyces cerevisiae. Curr Genet 14:191–199

    Google Scholar 

  • Davis AP, Symington LS (2004) RAD51-dependent break-induced replication in yeast. Mol Cell Biol 24:2344–2351

    Google Scholar 

  • Dellaire G, Yan J, Little KC, Drouin R, Chartrand P (2002) Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells. Chromosoma 111:304–312

    Google Scholar 

  • Delneri D, Colson I, Grammenoudi S, Roberts IN, Louis EJ, Oliver SG (2003) Engineering evolution to study speciation in yeasts. Nature 422:68–72

    Google Scholar 

  • Difilippantonio MJ, Zhu J, Chen HT, Meffre E, Nussenzweig MC, Max EE, Ried T, Nussenzweig A (2000) DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature 404:510–514

    Google Scholar 

  • Difilippantonio MJ, Petersen S, Chen HT, Johnson R, Jasin M, Kanaar R, Ried T, Nussenzweig A (2002) Evidence for replicative repair of DNA double-strand breaks leading to oncogenic translocation and gene amplification. J Exp Med 196:469–480

    Google Scholar 

  • Egli D, Hafen E, Schaffner W (2004) An efficient method to generate chromosomal rearrangements by targeted DNA double-strand breaks in Drosophila melanogaster. Genome Res 14:1382–1393

    Google Scholar 

  • Fasullo M, Bennett T, AhChing P, Koudelik J (1998) The Saccharomyces cerevisiae RAD9 checkpoint reduces the DNA damage-associated stimulation of directed translocations. Mol Cell Biol 18:1190–1200

    Google Scholar 

  • Gabrielian A, Pongor S (1996) Correlation of intrinsic DNA curvature with DNA property periodicity. FEBS Lett 393:65–68

    Google Scholar 

  • Galgoczy DJ, Toczyski DP (2001) Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast. Mol Cell Biol 21:1710–1718

    Google Scholar 

  • Gjuracic K, Pivetta E, Bruschi CV (2004) Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots. Mol Genet Genomics 271:437–446

    Google Scholar 

  • Han J, Colditz GA, Samson LD, Hunter DJ (2004) Polymorphisms in DNA double-strand break repair genes and skin cancer risk. Cancer Res 64:3009–3013

    Google Scholar 

  • Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168

    Google Scholar 

  • Kaiser C, Michaelis S, Mitchell A (1994) Preparation of chromosome-sized yeast DNA molecules in solid agarose. In: CSH (ed) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 179–180

    Google Scholar 

  • Kanaar R, Hoeijmakers JH, van Gent DC (1998) Molecular mechanisms of DNA double strand break repair. Trends Cell Biol 8:483–489

    Google Scholar 

  • Kolodner RD, Putnam CD, Myung K (2002) Maintenance of genome stability in Saccharomyces cerevisiae. Science 297:552–557

    Google Scholar 

  • Lin Y, Waldman AS (2001) Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA. Nucleic Acids Res 29:3975–3981

    Google Scholar 

  • Morrow DM, Connelly C, Hieter P (1997) “Break copy” duplication: a model for chromosome fragment formation in Saccharomyces cerevisiae. Genetics 147:371–382

    Google Scholar 

  • Myung K, Chen C, Kolodner RD (2001) Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae. Nature 411:1073–1076

    Google Scholar 

  • Pane F, Intrieri M, Quintarelli C, Izzo B, Muccioli GC, Salvatore F (2002) BCR/ABL genes and leukemic phenotype: from molecular mechanisms to clinical correlations. Oncogene 21:8652–8667

    Google Scholar 

  • Riley J, Butler R, Ogilvie D, Finniear R, Jenner D, Powell S, Anand R, Smith JC, Markham AF (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res 18:2887–2890

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Analysis of genomic DNA by southern hybridization. In: CSH (ed) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 9.31–9.58

    Google Scholar 

  • Shaw CJ, Lupski JR (2004) Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease. Hum Mol Genet 13:57–64

    Google Scholar 

  • Sherman F, Hicks J (1991) Micromanipulation and dissection of asci. In: Guthrie C, Fink GR (eds) Guide to yeast genetics and molecular biology: methods in enzymology. Academic Press, San Diego, CA, pp 21–37

    Google Scholar 

  • Shroff R, Arbel-Eden A, Pilch D, Ira G, Bonner WM, Petrini JH, Haber JE, Lichten M (2004) Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr Biol 14:1703–1711

    Google Scholar 

  • Storici F, Coglievina M, Bruschi CV (1999) A 2-micron DNA-based marker recycling system for multiple gene disruption in the yeast Saccharomyces cerevisiae. Yeast 15:271–283

    Google Scholar 

  • Tennyson RB, Ebran N, Herrera AE, Lindsley JE (2002) A novel selection system for chromosome translocations in Saccharomyces cerevisiae. Genetics 160:1363–1373

    Google Scholar 

  • Umezu K, Hiraoka M, Mori M, Maki H (2002) Structural analysis of aberrant chromosomes that occur spontaneously in diploid Saccharomyces cerevisiae: retrotransposon Ty1 plays a crucial role in chromosomal rearrangements. Genetics 160:97–110

    Google Scholar 

  • Wach A, Brachat A, Pohlmann R, Philippsen P (1999) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 410:1793–1808

    Google Scholar 

  • Waghmare SK, Caputo V, Radovic S, Bruschi CV (2003) Specific targeted integration of kanamycin resistance-associated nonselectable DNA in the genome of the yeast Saccharomyces cerevisiae. BioTechniques 34:1024–1028

    Google Scholar 

  • Yeh A, Wei M, Golub SB, Yamashiro DJ, Murty VV, Tycko B (2002) Chromosome arm 16q in Wilms tumors: unbalanced chromosomal translocations, loss of heterozygosity, and assessment of the CTCF gene. Genes Chromosomes Cancer 35:156–163

    Google Scholar 

  • Yu X, Gabriel A (1999) Patching broken chromosomes with extra-nuclear cellular DNA. Mol Cell 4:873–881

    Google Scholar 

  • Yu X, Gabriel A (2004) Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining. Genetics 166:741–751

    Google Scholar 

Download references

Acknowledgements

S.K.W. was a Ph.D. student supported by a fellowship from the International Centre for Genetic Engineering and Biotechnology.

Author information

Authors and Affiliations

  1. ICGEB Microbiology Laboratory, AREA Science Park, Padriciano 99, 34012, Trieste, Italy

    Valentina Tosato, Sanjeev K. Waghmare & Carlo V. Bruschi

Authors
  1. Valentina Tosato
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sanjeev K. Waghmare
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carlo V. Bruschi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carlo V. Bruschi.

Additional information

Communicated by J. Diffley

Electronic Supplementary Material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tosato, V., Waghmare, S.K. & Bruschi, C.V. Non-reciprocal chromosomal bridge-induced translocation (BIT) by targeted DNA integration in yeast. Chromosoma 114, 15–27 (2005). https://doi.org/10.1007/s00412-005-0332-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00412-005-0332-x

Keywords

Search

Navigation