Skip to main content
SpringerLink
Log in

Telomere Length Analysis and In Vitro Telomerase Assay

  • Protocol
Mammalian Artificial Chromosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 240))

  • 360 Accesses

Abstract

Barring exceptional instances, the DNA contained in eukaryotic chromosomes is linear. Linearity of the chromosomal DNA and the compartmentalized architecture of the eukaryotic cell are the two principle features that distinguish the prokaryotes from the eukaryotes and that have facilitated the evolution of totally different strategies for interaction with other species and the environment. Major changes in strategy that were made possible by linearity of chromosomes were an enormous increase in the information content of the genome and the development of sexuality as a means for efficient exchange of genetic information (1). But linear chromosomes have ends, and the presence of ends produced two major biological problems. One problem was identified early by Muller (2,3) and by McClintock (4), as the requirement to protect natural ends of chromosomes from fusion and recombination with other chromosomes and from exonucleolytic erosion. It was thus recognized that the ends of chromosomes must have specialized structural and functional features required for chromosome stability. We know today that another potential cause of chromosomal instability that must be dealt with is that unless it is protected, a DNA end will be recognized as damaged DNA that the cell will attempt to heal with ensuing loss of chromosome integrity and cell viability.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ishikawa, F. and Naito, T. (1999) Why do we have linear chromosomes? A matter of Adam and Eve. Mutat. Res. 434, 99–107.

    PubMed  CAS  Google Scholar 

  2. Muller, H. J. (1938) The remaking of chromosomes. Collect. Net. 8, 182–195.

    Google Scholar 

  3. Muller, H. J. (1940) An analysis of the process of structural change in the chromosomes of Drosophila. J. Genet. 40, 1–66.

    Article  Google Scholar 

  4. McClintock, B. (1939) The behavior in successive nuclear divisions of a chromosome broken at meiosis. Proc. Natl. Acad. Sci. USA 25, 405–416.

    Article  PubMed  CAS  Google Scholar 

  5. Wright, J. H., Gottschling, D. E., and Zakian, V. A. (1992) Saccharomyces telomeres assume a nonnucleosomal chromatin structure. Genes. Dev. 6, 197–210.

    Article  PubMed  CAS  Google Scholar 

  6. Blackburn, E. H. and Gall, J. G. (1978) A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J. Mol. Biol. 120, 33–53.

    Article  PubMed  CAS  Google Scholar 

  7. Klobutcher, L. A., Swanton, M. T., Donini, P., and Prescott, D. M. (1981) All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3′ terminus. Proc. Natl. Acad. Sci. USA 78, 3015–3019.

    Article  PubMed  CAS  Google Scholar 

  8. Lundblad, V. and Blackburn, H. (1993) An alternative pathway for yeast telomere maintenance rescues est1-senescence. Cell 73, 347–360.

    Article  PubMed  CAS  Google Scholar 

  9. McEachern, M. J. and Blackburn, H. (1996) Cap-prevented recombination between terminal telomeric repeat arrays (telomere CPR) maintains telomeres in Kluyveromyces lactis lacking telomerase. Genes Dev. 10, 1822–1834.

    Article  PubMed  CAS  Google Scholar 

  10. Yeager, T. R., Neumann, A. A., Englezou, A., Huschtscha, L. I., Noble, J. R. et al. (1999) Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body. Cancer Res. 59, 4175–4179.

    PubMed  CAS  Google Scholar 

  11. Saiga, H. and Edstrom, J. E. (1985) Long tandem arrays of complex repeat units in Chironomus telomeres. EMBO J. 79, 315–328.

    Google Scholar 

  12. Roth, C. W., Kobeski, F., Walter, M. F., and Biessmann, H. (1997) Chromosome end elongation by recombination in the mosquito Anopheles gambiae. Mol. Cell. Biol. 17, 5176–5183.

    PubMed  CAS  Google Scholar 

  13. Gottschling, D. E. and Cech, T. R. (1984) Chromatin structure of the molecular ends of Oxytricha macronuclear DNA: phased nucleosomes and a telomeric complex. Cell 38, 501–510.

    Article  PubMed  CAS  Google Scholar 

  14. Gottschling, D. E. and Zakian, V. A. (1986) Telomere proteins: Specific recognition and protection of the natural termini of Oxytricha macronuclear DNA. Cell 47, 195–205.

    Article  PubMed  CAS  Google Scholar 

  15. Pennock, E., Buckley, K. and Lundblad, V. (2001) Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell 104, 387–396.

    Article  PubMed  CAS  Google Scholar 

  16. Grandin, N., Damon, C. and Charbonneau, M. (2001) Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13. EMBO J. 20, 1173–1183.

    Article  PubMed  CAS  Google Scholar 

  17. Baumann, P. and Cech, T. R. (2001) Pot1, the putative telomere end-binding protein in fission yeast and humans. Science 292, 1171–1175.

    Article  PubMed  CAS  Google Scholar 

  18. Berman, J. and Tachibana, C. Y. (1986) Identification of a telomere-binding activity from yeast. Proc. Natl. Acad. Sci. USA 83, 3713–3717.

    Article  PubMed  CAS  Google Scholar 

  19. Larson, G. P., Castanotto, D., Rossi, J. J. and Malafa, M. P. (1994) Isolation and functional analysis of a Kluyveromyces lactis RAP1 homologue. Gene 150, 35–41.

    Article  PubMed  CAS  Google Scholar 

  20. Bilaud, T., Brun, C., Ancelin, K., Koering, C. E., Laroche, T. and Gilson, E. (1997) Telomeric localization of TRF2, a a novel human telobox protein. Nat. Genet. 17, 236–239.

    Article  PubMed  CAS  Google Scholar 

  21. Broccoli, D., Smogorzewska, A., Chong, L., and De Lange, T. (1997) Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nat. Genet. 17, 231–235.

    Article  PubMed  CAS  Google Scholar 

  22. Ferreira, M. and Cooper, J. (2001) The fission yeast Taz1 protein protects chromosomes from Ku-dependent end-to-end fusion. Mol. Cell 7, 55–63.

    Article  PubMed  CAS  Google Scholar 

  23. Griffith, J. D., Comeau L., Rosenfield, S., Stansel R. M., Bianchi A., Moss, H., et al. (1999) Mammalian telomeres end in a large duplex loop. Cell 97, 503–514.

    Article  PubMed  CAS  Google Scholar 

  24. Marcand, S., Wotton, D., Gilson, E. and Shore, D. (1997) Rap1p and telomere length regulation in yeast. Ciba Found. Symp. 211, 76–93.

    PubMed  CAS  Google Scholar 

  25. Smogorzewska, A., van Steensel, B., Bianchi, A., Oelmann, S., Schaefer, M. R., et al. (2000) Control of human telomere length by TRF1 and TRF2. Mol. Cell. Biol. 20, 1659–1668.

    Article  PubMed  CAS  Google Scholar 

  26. Lee, H. W., Blasco, M. A., Gottlieb, G. J., Horner, J. W. II, Greider, C. W. and Harley, C. B. (1998) Essential role of mouse telomerase in highly proliferative organs. Nature 362, 569–574.

    Google Scholar 

  27. Lustig, A. J. (1998) Mechanisms of silencing in Saccharomyces cerevisiae. Curr. Opin. Genet. Dev. 8, 233–239.

    Article  PubMed  CAS  Google Scholar 

  28. Galy, V., Olivo-Merin, J. C., Scherthan, H., Doye V., Rascalou, N. and Nehrbass, U. (2000) Nuclear pore complexes in the organization of silent telomeric chromatin. Nature 403, 108–112.

    Article  PubMed  CAS  Google Scholar 

  29. Feuerbach, F., Galy, V., Sticken, E. T., Fromont-Racine, M., Jacquier, A., Gilson, E., et al. (2002) Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast. Nat. Cell Biol. 4, 214–221.

    Article  PubMed  CAS  Google Scholar 

  30. Bass, H. W., Riera-Lizarazu, O., Ananiev, E. V., Bordoli, S. J., Rines, H. W., Phillips, R. L., et al. (2000) Evidence for the coincident initiation of homolog pairing and synapsis during the telomere clustering (bouquet) stage of meiotic prophase. J. Cell Sci. 113, 1033–1042.

    PubMed  CAS  Google Scholar 

  31. Loidl, J. (1990) The initiation of meiotic chromosome pairing: the cytological view. Genome 33, 759–778.

    PubMed  CAS  Google Scholar 

  32. Cooper, J. P. (2000) Telomere transitions in yeast: the end of the chromosome as we know it. Curr. Opin. Genet Dev. 10, 169–177.

    Article  PubMed  CAS  Google Scholar 

  33. Nimmo, E. R., Pidoux, A. L., Perry, P. E., and Allshire, R. C. (1998) Defective meiosis in telomere-silencing mutants of Schizosac-charomyces pombe. Nature 392, 825–828.

    Article  PubMed  CAS  Google Scholar 

  34. Lingner, J., Cech, T. R., Hughes, T. R. and Lundblad, V. (1997) Three ever shorter telomere (EST) genes are dispensable for in vitro yeast telomerase activity. Proc. Natl. Acad. Sci. USA 94, 11,190–11,195.

    Article  PubMed  CAS  Google Scholar 

  35. Nugent, C. I., Hughes, T. R., Lue, N. F., and Lundblad, V. (1996) Cdc13p: a single-strand telomeric DNA-binding protein with dual role in yeast telomere maintenance. Science 274, 249–252.

    Article  PubMed  CAS  Google Scholar 

  36. Chan, S. W., Chang, J., Prescott, J., and Blackburn, E. H. (2001) Altering telomere structure allows telomerase to act in yeast lacking ATM kinases. Curr Biol. 11, 1240–1250.

    Article  PubMed  CAS  Google Scholar 

  37. Kishi, K. and Lu, P. L. (2002) A critical role for Pin2/TRF1 in ATM-dependent regulation. J. Biol. Chem. 277, 7420–7429.

    Article  PubMed  CAS  Google Scholar 

  38. Forsyth, N. R., Wright, W. E and Shay, J. W. (2002) Telomerase and differentiation in multicelluar organisms: turn it off, turn it on, and turn it off again. Differentiation 69, 188–197.

    Article  PubMed  CAS  Google Scholar 

  39. Zhou, X. Z. and Lu, K. P. (2001) The Pin2/TRF1-interacting protein PinX1 is a potent telomerase inhibitor. Cell 107, 347–359.

    Article  PubMed  CAS  Google Scholar 

  40. Shen, Z.Y., Xu, L.Y., Li, C., Cai, W. J., Shen, J., Chen, J. Y., and Zeng, Y. (2001) A comparative study of telomerase activity and malignant phenotype in multistage carcinogenesis of esophageal epithelial cells induced by human papillomavirus. Int. J. Mol. Med. 8, 633–639.

    PubMed  CAS  Google Scholar 

  41. Muller, M. (2002) Telomerase: its clinical relevance in the diagnosis in bladder cancer. Oncogene 21, 650–655.

    Article  PubMed  CAS  Google Scholar 

  42. Corey, D. R. (2002) Telomerase inhibition, oligonucleotides, and clinical trials. Oncogene 21, 631–637.

    Article  PubMed  CAS  Google Scholar 

  43. Ito, H., Kio, S., Kanaya, T., Takakura, M., Inoue, M., and Namiki, M. (1998) Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer. Clin. Cancer. Res. 4, 1603–1608.

    PubMed  CAS  Google Scholar 

  44. de Kok, J. B., Schalken, J. A., Aalders, T. W., Ruers, T.J., Willelms, H. L., and Swinkels, D. W. (2000) Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas. Int. J. Cancer 87, 217–220.

    Article  PubMed  Google Scholar 

  45. Nakamura, T. M, Morin, G. B., Chapman, K. B., Weinrich, S. L., Andrews, W. H., Lingner, J., et al. (1997) Telomerase catalytic subunit homologs from fission yeast and human. Science 277, 955–959.

    Article  PubMed  CAS  Google Scholar 

  46. Prescot, J. and Blackburn, E. H. (1997) Functionally interacting telomerase RNAs in the yeast telomerase complex. Genes Dev. 11, 2790–800.

    Article  Google Scholar 

  47. Lee, M. S. and Blackburn, E. H. (1993) Sequence-specific DNA primer effects on telomerase polymarization activity. Mol. Cell. Biol. 13, 6586–6599.

    PubMed  CAS  Google Scholar 

  48. Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C.B., West, M. D., Ho, P. L., et al. (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011–2015.

    Article  PubMed  CAS  Google Scholar 

  49. Greider, C. W. and Blackburn, E., H. (1987) The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell. 51, 887–898.

    Article  PubMed  CAS  Google Scholar 

  50. Cohn, M. and Blackburn, H. E. (1995) Telomerase in yeast. Science 269, 396–400.

    Article  PubMed  CAS  Google Scholar 

  51. Counter, C. M., Meyerson, M., Eaton, E. N. and Weinberg, R. A. (1997) The catalytic subunit of yeast telomerase. Proc. Natl. Acad. Sci. USA 94, 9202–9207.

    Article  PubMed  CAS  Google Scholar 

  52. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning, a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  53. Meyne, J., Baker, R. J., Hobart, H. H., Hsu, T. C., Ryder, O. A., Ward, O. G., et al. (1990) Distribution of non-telomeric sites of the (TTAGGG)n in telomeric sequence in vertebrate chromosomes. Chromosoma 99, 3–10.

    Article  PubMed  CAS  Google Scholar 

  54. Faravelli, M., Azzalin, C. M., Bertoni, L., Chernova O., Mondello, C., and Giulotto, E. (2002) Molecular organization of internal telomeric sequences in Chinese hamster chromosomes. Gene 283, 11–16.

    Article  PubMed  CAS  Google Scholar 

  55. Poon, S. S. S., Martens, U. M., Ward, R. K., Lansdorp, P. M. (1999) Telomere length measurements using digital fluorescence microscopy. Cytometry 36, 267–278.

    Article  PubMed  CAS  Google Scholar 

  56. Rufer, N., Dragowska, W., Thornbury, G., Roosnek, E., Lansdorp, P. M. (1998) Telomere length dynamics in a human lymphocytes subpopulations measured by flow cytometry. Nature Biotech. 16, 743–747.

    Article  CAS  Google Scholar 

  57. Hultdin, M., Gronlund, E., Norrback, K.-F., Ericsson-Lindstroem, E., Just, T. and Roos, G. (1998) telomere analysis by fluorescence in situ hybridization and flow cytometry. Nucleic Acids Res. 26, 3651–3656.

    Article  PubMed  CAS  Google Scholar 

  58. Baerlocher, G. M., Mak, J., Tien, T., and Lansdorp, P. M. (2002) Telomere length measurements by fluorescence in situ hybridization and flow cytometry: Tips and pitfalls. Cytometry 47, 89–99.

    Article  PubMed  CAS  Google Scholar 

  59. Szostak, J. W. and Blackburn, E. H. (1982) Cloning yeast telomeres on linear plasmid vectors. Cell 29, 245–255.

    Article  PubMed  CAS  Google Scholar 

  60. Guerrini, A. M., Ascenzioni F., Tribioli C., and Donini P. (1985) Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences. EMBO J. 4, 1569–1573.

    PubMed  CAS  Google Scholar 

  61. Ascenzioni, F. and Lipps, H. J. (1986) A linear shuttle vector for yeast and hypotrichous ciliate Stylonychia. Gene 46, 123–126.

    Article  PubMed  CAS  Google Scholar 

  62. Brown, W. R. (1989) Molecular cloning of human telomeres in yeast. Nature 338, 774–776.

    Article  PubMed  CAS  Google Scholar 

  63. Guerrini, A. M., Ascenzioni, F., Pisani, G., Rappazzo G., Della Valle, G., and Donini, P. (1990) Cloning a fragment from the telomere of the long arm of human chromosome 9 in a YAC vector. Chromosoma 99, 138–142.

    Article  PubMed  CAS  Google Scholar 

  64. Fu, G. and Barker, D. C. (1998) Rapid cloning of telomere-associated sequence using primer-tagged amplification. Biotechniques 24, 386–389.

    PubMed  CAS  Google Scholar 

  65. Tzfati, Y., Fulton, T. B., Roy, J. and Blackburn, E. H. (2000) Template boundary in a yeast telomerase specified by RNA structure. Science 288, 863–867.

    Article  PubMed  CAS  Google Scholar 

  66. Wen, J. M., Sun, L. B., Zhang, M., and Zheng, M. H. (1998) A non-isotopic method for the detection of telomerase activity in tumor tissue: TRAP-silver staining assay. J. Clinic. Pathol. 51, 110–112.

    Article  CAS  Google Scholar 

  67. Dalla Torre, C. A., Maciel, R. M. B., Pinheiro, N. A., Andrade, J. A. D., de Toledo, S. R. C., et al.. (2002) TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines. Bras. J. Med. Biol. Res. 35, 65–68.

    Google Scholar 

  68. Falchetti, M. L., Levi, A., Molinari, P., Verna, R., and D’Ambrosio, E. (1998) Inceased sensitivity and reproducibility of TRAP assay by avoiding direct primers interaction. Nucleic Acids Res. 26, 862–863.

    Article  PubMed  CAS  Google Scholar 

  69. Kim, N. W. and Wu, F. (1997) Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP). Nucleic Acids Res. 25, 2595–2597.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dipartimento di Biologia Cellulare e dello Sviluppo, Universitá “La Sapienza”, Rome, Italy

    Fiorentina Ascenzioni, Pier Assunta Fradiani & Pierluigi Donini

Authors
  1. Fiorentina Ascenzioni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pier Assunta Fradiani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pierluigi Donini
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Istituto di Tecnologie Biomediche Avanzate, Consiglio Nationale Ricerche, Segrate, Italy

    Vittorio Sgaramella  & Sandro Eridani  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc.

About this protocol

Cite this protocol

Ascenzioni, F., Fradiani, P.A., Donini, P. (2004). Telomere Length Analysis and In Vitro Telomerase Assay. In: Sgaramella, V., Eridani, S. (eds) Mammalian Artificial Chromosomes. Methods in Molecular Biology, vol 240. Humana Press. https://doi.org/10.1385/1-59259-434-4:123

Download citation

  • DOI: https://doi.org/10.1385/1-59259-434-4:123

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-096-0

  • Online ISBN: 978-1-59259-434-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation