Chromosome Research

, Volume 1, Issue 1, pp 45–51 | Cite as

Direct detection of repetitive, whole chromosome paint and telomere DNA probes by immunogold electron microscopy

  • J. Steinmüller
  • E. Schleiermacher
  • H. Scherthan
Research Papers


Biotinylated repetitive, whole chromosome paint and telomere DNA probes were investigated at the electron microscope level after non-isotopicin situ hybridization and direct immunogold detection. The protocol described allowed the visualization of a biotinylated chromosome 1 specific satellite DNA probe in the light microscope without silver intensification. This sensitive method was exploited to analyse factors contributing to signal strength in immunogold chromosome painting. Furthermore, it allowed us to investigate the distribution of (TTAGGG)n telomere repeats in human metaphase chromosomes and interphase nuclei. Telomeric and internal (TTAGGG)n repeats were detected at high spatial resolution in human metaphase chromosomes. In the periphery of lymphocyte interphase nuclei, two types of telomere hybridization signals were observed. They differed remarkably in compactness, indicating two types of chromatin conformation present at the interphase telomeresin situ.

Key words

chromosome painting immunogold electron microscopy non-isotopicin situ hybridization telomere probes 


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  1. Blackburn EH (1991) Structure and function of telomeres.Nature 35: 569–573.Google Scholar
  2. Collins C, Kuo WL, Segraves R, Pinkel D, Fuscoe J, Gray JW (1991) Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes.Genomics 11: 997–1006.Google Scholar
  3. Cooke HJ, Hindley J (1979) Cloning of human satellite III DNA: different components are on different chromosomes.Nucl Acids Res 10: 3177–3196.Google Scholar
  4. de Lange T (1992) Human telomeres are attached to the nuclear matrix.EMBO J 11: 717–724.Google Scholar
  5. de Lange T, Shiue L, Myers RMet al. (1990) Structure and variability of human chromosome ends.Mol Cell Biol 10: 518–527.Google Scholar
  6. Dauwerse JG, Wiegant J, Raap AK, Breuning MH, van Ommen GJB (1992) Multiple colors by fluorescencein situ hybridization using ratio-labelled DNA probes create a molecular karyotype.Hum Mol Genet 1: 593–598.Google Scholar
  7. Ferguson-Smith MA (1991) Putting the genetics back into cytogenetics.Am J Hum Genet 48: 179–182.Google Scholar
  8. Fetni R, Drouin R, Lemineux N, Messier P, Richer C (1991) Simultaneous visualization of chromosome bands and hybridization signal using colloidal-gold labeling in electron microscopy.Proc Natl Acad Sci, USA 88: 10916–10920.Google Scholar
  9. Fetni R, Lemineux N, Malfoy B, Dutrillaux B, Messier P, Richer CL (1992) Detection of small, single-copy genes on protein-G-banded chromosomes by electron microscopy.Cytogenet Cell Genet 60: 187–189.Google Scholar
  10. Gosden J, Hanratty D, Starling J, Fantes J, Mitchell A, Porteous D (1991) Oligonucleotide-primedin situ DNA synthesis (PRINS): a method for chromosome mapping, banding and investigation of sequence organization.Cytogenet Cell Genet 57: 100–104.Google Scholar
  11. Hastie ND, Allshire RC (1989) Human telomeres: fusion and interstitial sites.Trends Genet 5: 326–331.Google Scholar
  12. Henderson E, Blackburn EH (1989) An overhanging 3′ terminus is a conserved feature of telomeres.Mol Cell Biol 9: 345–348.Google Scholar
  13. Henderson E, Hardin CC, Walk SK, Tinoco I Jr, Blackburn EH (1987) Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs.Cell 51: 899–908.Google Scholar
  14. Holgate CS, Jackson P, Cowen PN, Bird CC (1983) Immunosilver-staining: a new method of immunostaining with enhanced sensitivity.J Histochem Cytochem 31: 938–944.Google Scholar
  15. Hutchinson NJ, Langer-Safer PR, Ward DC, Hamkalo B (1982)In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold.J Cell Biol 95: 609–618.Google Scholar
  16. Kievits T, Devilee P, Wiegant Jet al. (1990) Direct non radioactivein situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes.Cytometry 11: 105–109.Google Scholar
  17. Kipling D, Cooke HJ (1992) Beginning or end? Telomere structure, genetics and biology.Hum Mol Genet 1: 3–6.Google Scholar
  18. Klobutcher LA, Swanton MT, Donini P, Prescott DM (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.Google Scholar
  19. Landegent JE, Jansen in de Wal N, Dirks RW, Bass F, van der Ploeg M (1987) Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactivein situ hybridization.Hum Genet 73: 354–357.Google Scholar
  20. Lichter P, Cremer T, Borden J, Manuelidis L, Ward DC (1988) Delineation of individual human chromosomes in metaphase and interphase cells byin situ suppression hybridization using recombinant DNA libraries.Hum Genet 80: 224–234.Google Scholar
  21. Lichter P, Boyle AL, Cremer T, Ward DC (1991) Analysis of genes and chromosomes by non-isotopicin situ hybridization.Genet Anal Techn Appl 8: 24–35.Google Scholar
  22. Messier P-E, Jean P, Richer C-L (1986) Easy transfer of selected mitosis from light to electron microscopy.Cytogenet Cell Genet 43: 207–210.Google Scholar
  23. Meyne J, Ratliff RL, Moyzis RK (1989) Conservation of the human telomere sequence (TTAGGG)n.Proc. Natl Acad Sci, USA 86: 7049–7053.Google Scholar
  24. Moyzis RK, Buckingham JM, Cram LSet al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes.Proc Natl Acad Sci, USA 85: 6622–6626.Google Scholar
  25. Narayanswami S, Hamkalo BA (1987) Hybridization to chromatin and whole chromosome mounts. In: Sommerville J, Scheer U (eds).Electron microscopy in molecular biology. A practical approach. Oxford: IRL Press, pp 215–232.Google Scholar
  26. Narayanswami S, Hamkalo BA (1991) DNA sequence mapping using electron microscopy.Genet Anal Tech Appl 8: 14–23.Google Scholar
  27. Narayanswami S, Lundgren K, Hamkalo BA (1989) Deoxyribonucleic acid sequence mapping on metaphase chromosomes by immunoelectron microscopy.Scanning Microsc. 3: Suppl, 65–76.Google Scholar
  28. Narayanswami S, Dvorkin N, Hamkalo BA (1991) Nucleic acid sequence localization by electron microscopicin situ hybridization.Methods Cell Biol 35: 109–132.Google Scholar
  29. Narayanswami S, Doggett NA, Clark LM, Hildebrand CE, Weier HU, Hamkalo BA (1992) Cytological and molecular characterization of centromeres inMus domesticus andMus spretus.Mammalian Genome 2: 186–194.Google Scholar
  30. Pinkel D, Landegent J, Collins Cet al. (1988) Fluorescencein situ hybridization with human chromosome specific libraries: detection of trisomy 21 and translocations of chromosome 4.Proc Natl Acad Sci, USA 85: 9138–9142.Google Scholar
  31. Pluta AF, Kaine BP, Spear BB (1982) The terminal organization of macronuclear DNA inOxytricha fallax.Nucleic Acids Res 10: 8145–8154.Google Scholar
  32. Raap AK, Marijnen JGJ, Vrolijk J, van der Ploeg M (1986) Denaturation, renaturation, and loss of DNA duringin situ hybridization procedures.Cytometry 7: 235–242.Google Scholar
  33. Scherthan H (1990) Localization of the repetitive telomeric sequence (TTAGGG)n in two muntjac species and implications for their karyotypic evolution.Cytogenet Cell Genet 53: 127–136.Google Scholar
  34. Scherthan H, Köhler M, Vogt P, von Malsch K, Schweizer D (1992) Chromosomalin situ hybridization with double-labeled DNA: signal amplification at the probe level.Cytogenet Cell Genet 60: 4–7.Google Scholar
  35. Sen D, Gilbert W (1988) Formation of paralle four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis.Nature 334: 364–366.Google Scholar
  36. Sundquist WI, Klug A (1989) Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops.Nature 342: 825–829.Google Scholar
  37. Viegas-Pequignot E, Berrard S, Brice A, Apiou F, Mallet J (1990) Localization of a 900 bp-long fragment of the human choline acetyltransferase gene to 10q11.2 by nonradioactivein situ hybridization.Genomics 9: 210–212.Google Scholar
  38. Wells RA, Germino GG, Krishna S, Buckle VJ, Reeders ST (1990) Telomere-related sequences at interstitial sites in the human genome.Genomics 8: 699–704.Google Scholar
  39. Wieacker P, Davies KE, Cooke HJet al. (1984) Toward a complete linkage map of the human X chromosome: regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids.Am J Hum Genet 36: 265–270.Google Scholar
  40. Williamson JR, Raghuraman MK, Cech TR (1989) Monovalent cation induced structure of telomeric DNA: the G-quartet model.Cell 59: 871–880.Google Scholar

Copyright information

© Rapid Communications of Oxford Ltd 1993

Authors and Affiliations

  • J. Steinmüller
    • 1
  • E. Schleiermacher
    • 1
  • H. Scherthan
    • 2
  1. 1.the Institut für Anthropologie der Universität MainzMainzGermany
  2. 2.the Abt. Humanbiologie und Genetik der Universität KaiserslauternKaiserslauternGermany

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