Tracking of Single and Multiple Genomic Loci in Living Yeast Cells

  • Imen Lassadi
  • Kerstin BystrickyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 745)


Nuclear organization is involved in numerous aspects of cellular function. In yeast, analysis of the nuclear position and dynamics of the silent and active mating-type loci has allowed to gain insight into the mechanisms involved in directing mating-type switching. The fluorescent repressor operator systems (FROS) have proven to be a powerful technique to tag DNA sequences to investigate chromosome position and dynamics in living cells. FROS rely on the transgenic expression of a bacterial repressor fused to a fluorescent protein which can bind to its respective operator DNA sequence integrated as multicopy tandem arrays at a specific genomic site. Different FROS exist which facilitate the tagging of up to three different loci simultaneously. This chapter describes detailed protocols for FROS usage and analysis in the yeast Saccharomyces cerevisiae.

Key words

Saccharomyces cerevisiae chromosome dynamics fluorescent proteins live-cell microscopy DNA nuclear organization LacO TetO 


  1. 1.
    Belmont, A.S. (2001) Visualizing chromosome dynamics with GFP. Trends Cell Biol 11, 250–257.PubMedCrossRefGoogle Scholar
  2. 2.
    Belmont, A.S., and Straight, A.F. (1998) In vivo visualization of chromosomes using lac operator-repressor binding. Trends Cell Biol 8, 121–124.PubMedCrossRefGoogle Scholar
  3. 3.
    Rose, M.D., Winston, F., and Hieter, P. (1990) Methods in yeast genetics: A laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  4. 4.
    Michaelis, C., Ciosk, R., and Nasmyth, K. (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91, 35–45.PubMedCrossRefGoogle Scholar
  5. 5.
    Straight, A.F., Belmont, A.S., Robinett, C.C., and Murray, A.W. (1996) GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion. Curr Biol 6, 1599–1608.PubMedCrossRefGoogle Scholar
  6. 6.
    Bupp, J.M., Martin, A.E., Stensrud, E.S., and Jaspersen, S.L. (2007) Telomere anchoring at the nuclear periphery requires the budding yeast Sad1-UNC-84 domain protein Mps3. J Cell Biol 179, 845–854.PubMedCrossRefGoogle Scholar
  7. 7.
    Bystricky, K., Laroche, T., van Houwe, G., Blaszczyk, M., and Gasser, S.M. (2005) Chromosome looping in yeast: telomere pairing and coordinated movement reflect anchoring efficiency and territorial organization. J Cell Biol 168, 375–387.PubMedCrossRefGoogle Scholar
  8. 8.
    Ebrahimi, H., and Donaldson, A.D. (2008) Release of yeast telomeres from the nuclear periphery is triggered by replication and maintained by suppression of Ku-mediated anchoring. Genes Dev 22, 3363–3374.PubMedCrossRefGoogle Scholar
  9. 9.
    Miele, A., Bystricky, K., and Dekker, J. (2009) Yeast silent mating type loci form heterochromatic clusters through silencer protein-dependent long-range interactions. PLoS Genet 5, e1000478.PubMedCrossRefGoogle Scholar
  10. 10.
    Schober, H., Kalck, V., Vega-Palas, M.A., Van Houwe, G., Sage, D., Unser, M., Gartenberg, M.R., and Gasser, S.M. (2008) Controlled exchange of chromosomal arms reveals principles driving telomere interactions in yeast. Genome Res 18, 261–271.PubMedCrossRefGoogle Scholar
  11. 11.
    Taddei, A., Hediger, F., Neumann, F.R., Bauer, C., and Gasser, S.M. (2004) Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins. EMBO J 23, 1301–1312.PubMedCrossRefGoogle Scholar
  12. 12.
    Berger, A.B., Cabal, G.G., Fabre, E., Duong, T., Buc, H., Nehrbass, U., Olivo-Marin, J.C., Gadal, O., and Zimmer, C. (2008) High-resolution statistical mapping reveals gene territories in live yeast. Nat Methods 5, 1031–1037.PubMedCrossRefGoogle Scholar
  13. 13.
    Cabal, G.G., Genovesio, A., Rodriguez-Navarro, S., Zimmer, C., Gadal, O., Lesne, A., Buc, H., Feuerbach-Fournier, F., Olivo-Marin, J.C., Hurt, E.C., and Nehrbass, U. (2006) SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope. Nature 441, 770–773.PubMedCrossRefGoogle Scholar
  14. 14.
    Gard, S., Light, W., Xiong, B., Bose, T., McNairn, A.J., Harris, B., Fleharty, B., Seidel, C., Brickner, J.H., and Gerton, J.L. (2009) Cohesinopathy mutations disrupt the subnuclear organization of chromatin. J Cell Biol 187, 455–462.PubMedCrossRefGoogle Scholar
  15. 15.
    Taddei, A., Van Houwe, G., Hediger, F., Kalck, V., Cubizolles, F., Schober, H., and Gasser, S.M. (2006) Nuclear pore association confers optimal expression levels for an inducible yeast gene. Nature 441, 774–778.PubMedCrossRefGoogle Scholar
  16. 16.
    Gartenberg, M.R., Neumann, F.R., Laroche, T., Blaszczyk, M., and Gasser, S.M. (2004) Sir-mediated repression can occur independently of chromosomal and subnuclear contexts. Cell 119, 955–967.PubMedCrossRefGoogle Scholar
  17. 17.
    Heun, P., Laroche, T., Shimada, K., Furrer, P., and Gasser, S.M. (2001) Chromosome dynamics in the yeast interphase nucleus. Science 294, 2181–2186.PubMedCrossRefGoogle Scholar
  18. 18.
    Bressan, D.A., Vazquez, J., and Haber, J.E. (2004) Mating type-dependent constraints on the mobility of the left arm of yeast chromosome III. J Cell Biol 164, 361–371.PubMedCrossRefGoogle Scholar
  19. 19.
    Bystricky, K., Van Attikum, H., Montiel, M.D., Dion, V., Gehlen, L., and Gasser, S.M. (2009) Regulation of nuclear positioning and dynamics of the silent mating type loci by the yeast Ku70/Ku80 complex. Mol Cell Biol 29, 835–848.PubMedCrossRefGoogle Scholar
  20. 20.
    Houston, P.L., and Broach, J.R. (2006) The dynamics of homologous pairing during mating type interconversion in budding yeast. PLoS Genet 2, e98.PubMedCrossRefGoogle Scholar
  21. 21.
    Simon, P., Houston, P., and Broach, J. (2002) Directional bias during mating type switching in Saccharomyces is independent of chromosomal architecture. EMBO J 21, 2282–2291.PubMedCrossRefGoogle Scholar
  22. 22.
    Nagai, S., Dubrana, K., Tsai-Pflugfelder, M., Davidson, M.B., Roberts, T.M., Brown, G.W., Varela, E., Hediger, F., Gasser, S.M., and Krogan, N.J. (2008) Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science 322, 597–602.PubMedCrossRefGoogle Scholar
  23. 23.
    Bystricky, K., Heun, P., Gehlen, L., Langowski, J., and Gasser, S.M. (2004) Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques. Proc Natl Acad Sci USA 101, 16495–16500.PubMedCrossRefGoogle Scholar
  24. 24.
    Therizols, P., Duong, T., Dujon, B., Zimmer, C., and Fabre, E. (2010) Chromosome arm length and nuclear constraints determine the dynamic relationship of yeast subtelomeres. Proc Natl Acad Sci USA 107, 2025–2030.PubMedCrossRefGoogle Scholar
  25. 25.
    Fekete, R.A., and Chattoraj, D.K. (2005) A cis-acting sequence involved in chromosome segregation in Escherichia coli. Mol Microbiol 55, 175–183.PubMedCrossRefGoogle Scholar
  26. 26.
    Rohner, S., Gasser, S.M., and Meister, P. (2008) Modules for cloning-free chromatin tagging in Saccharomyces cerevisiae. Yeast 25, 235–239.PubMedCrossRefGoogle Scholar
  27. 27.
    Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21, 3329–3330.PubMedCrossRefGoogle Scholar
  28. 28.
    Dufour, A., Shinin, V., Tajbakhsh, S., Guillen-Aghion, N., Olivo-Marin, J.C., and Zimmer, C. (2005) Segmenting and tracking fluorescent cells in dynamic 3-D microscopy with coupled active surfaces. IEEE Trans Image Process 14, 1396–1410.PubMedCrossRefGoogle Scholar
  29. 29.
    Heun, P., Laroche, T., Raghuraman, M.K., and Gasser, S.M. (2001) The positioning and dynamics of origins of replication in the budding yeast nucleus. J Cell Biol 152, 385–400.PubMedCrossRefGoogle Scholar
  30. 30.
    Rosa, A., Maddocks, J.H., Neumann, F.R., Gasser, S.M., and Stasiak, A. (2006) Measuring limits of telomere movement on nuclear envelope. Biophys J 90, L24–6.PubMedCrossRefGoogle Scholar
  31. 31.
    Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., and Stelzer, E.H. (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009.PubMedCrossRefGoogle Scholar
  32. 32.
    Hajjoul, H., Kocanova, S., Lassadi, I., Bystricky, K., and Bancaud, A. (2009) Lab-on-Chip for fast 3D particle tracking in living cells. Lab Chip 9, 3054–3058.PubMedCrossRefGoogle Scholar
  33. 33.
    Hediger, F., Taddei, A., Neumann, F.R., and Gasser, S.M. (2004) Methods for visualizing chromatin dynamics in living yeast. Methods Enzymol 375, 345–365.PubMedCrossRefGoogle Scholar
  34. 34.
    Meister, P., Gehlen, L.R., Varela, E., Kalck, V., and Gasser, S.M. (2010) Visualizing yeast chromosomes and nuclear architecture. Methods enzymology, Guide to yeast genetics, J. Abelson and M. Simon, eds.,Vol. 470 (New York, NY: Academic Press), 535–567.Google Scholar
  35. 35.
    Sage, D., Neumann, F.R., Hediger, F., Gasser, S.M., and Unser, M. (2005) Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamics. IEEE Trans Image Process 14, 1372–1383.PubMedCrossRefGoogle Scholar
  36. 36.
    Vazquez, J., Belmont, A.S., and Sedat, J.W. (2001) Multiple regimes of constrained chromosome motion are regulated in the interphase Drosophila nucleus. Curr Biol 11, 1227–1239.PubMedCrossRefGoogle Scholar
  37. 37.
    Iannuccelli, E., Mompart, F., Gellin, J., Lahbib-Mansais, Y., Yerle, M., and Boudier, T. (2010) NEMO: a tool for analyzing gene and chromosome territory distributions from 3D-FISH experiments. Bioinformatics 26, 696–697.PubMedCrossRefGoogle Scholar
  38. 38.
    Boeke, J.D., LaCroute, F., and Fink, G.R. (1984) A positive selection for mutants lacking orotidine-5-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197, 345–346.PubMedCrossRefGoogle Scholar
  39. 39.
    Heim, R., Cubitt, A.B., and Tsien, R.Y. (1995) Improved green fluorescence. Nature 373, 663–664.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Laboratoire de Biologie Moléculaire EucaryoteUniversité de ToulouseToulouseFrance
  2. 2.Laboratoire de Biologie Moléculaire Eucaryote (LBME)Université de ToulouseToulouseFrance

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