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Techniques & Methods

Methodology development has always been a priority in our lab. We are happy to help you perform these techniques and master quantitative cell biology!

Here we provide access to some key protocols and methods papers listed by topics.

The dynamics of chromosome loci in the interphase nucleus

Chromatin dynamics are usually analysed by tracking a GFP-tagged locus over time with either spinning disk or point-scan confocal miscroscopy. The analysis requires several steps. Some references relevant to the technique are:

  • Sage, D., Hediger, F., Gasser, S.M. and Unser, M. (2003) "Automatic Tracking of Chromosomal Loci by Time-Lapse Fluorescent Microscopy" in IEEE International Symp. on Image and Signal Processing and Analysis (ISPA 2003).
  • Neumann, F.R., Hediger, F., Taddei, A. and Gasser, S.M. (2005) "Tracking Individual Chromosomes with Integrated lacO sites and GFP-lacI repressor: Analysing Position and Dynamics of Chromosomal loci in S. cerevisiae" in Cell Biology A Laboratory Handbook, Vol 2 (Celis, J. ed.), 359-367
  • 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 Transactions on Image Processing, 14, 1372-1383.
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  • Hediger, F., Taddei, A., Neumann, F.R. and Gasser, S.M. (2003) "Methods for visualizing chromatin dynamics in living yeast." In Methods in Enzymology, Chromatin Remodeling and modification complexes Vol 375, (Wu, C. and Allis, C.D., eds.), Academic Press, 345-365.
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The localisation of chromatin loci in the yeast Sacharomyces cerevisiae nucleus

The localisation of chromatin loci has to be determined with a large sample number due to the dynamics of chromatin as described above. The sub-nuclear position of a tagged-locus is usually determined in relation to a tagged nuclear envelope or nuclear pore protein in living cells using a 3D stack of images. One can also measure the distance between two differently tagged loci with similar methods (as in Schober et al., 2008). Finally for looking at genomic loci without GFP technology, we have optimized DNA FISH and IF. See the following papers and protocols for methodology:

  • Neumann et al., (2012). "Materials and Methods for measuring movement in yeast".
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  • Neumann, F.R., Hediger, F., Taddei, A. and Gasser, S.M. (2005) "Tracking Individual Chromosomes with Integrated lacO sites and GFP-lacI repressor: Analysing Position and Dynamics of Chromosomal loci in S. cerevisiae" in Cell Biology A Laboratory Handbook, Vol 2 (Celis, J. ed.), 359-367
    PDF not available.
  • Schober, H., Kalck, V., Vega-Palas, M.A., VanHouwe, 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 Research, 18, 261 -271
    (see supplemental material).
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    Supplementary Material
  • Meister, P., Gehlen, L.R. Varela, E., Kalck, V. and Gasser S.M. (2010) "Visualizing Yeast Chromosomes and Nuclear Architecture", in Methods in Enzymology Vol 470, (J. Abelson and M. Simon, eds.) Academic Press, 535-567.
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For tagging yeast genomic loci easily use this method:

  • Rohner, S., Gasser*, S.M. and Meister, P. (2008) *corresponding author "Modules for cloning-free chromatin tagging in Saccharomyces cerevisiae", Yeast, 25, 235-239
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For coupled FISH/IF in yeast we recommend the FISH/IF protocol

Visualizing the position of genes in living, differentiating cells in C. elegans

In the following reference, we describe techniques we have developed for live imaging of developmentally-regulated tagged gene arrays in living worms. We also describe a fluorescence in situ hybridization (FISH) method to visualize endogenous loci to analyze the position of chromatin elements during C. elegans development.

  • Meister, P. Towbin, B.D., Pike, B.L., Ponti, A., and Gasser, S.M. (2010) "Developmentally regulated promoters drive tissue-specific intranuclear gene positioning in C. elegans." Genes & Dev., 24: 766-782.
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For measuring the subnuclear position of tagged-chromatin in both S. cerevisiae and C. elegans we use the ImageJ imaging analysis software, see http://rsbweb.nih.gov/ij/ with the Plugin 'Point Picker' http://bigwww.epfl.ch/thevenaz/pointpicker/.

Reconstitution of silent chromatin

The Sir2-3-4 complex has been isolated from baculoviral overexpression and shown to be a 1:1:1 heterotrimer. This method was published in the following references:

  • Martino, F., Kueng, S., Robinson,, P., Tsai-Pflugfelder, M., van Leeuwen,, F., Ziegler, M., Cubizolles,, F., Cockell,, M.M., Rhodes,, D. and Gasser, S.M. (2009) "Reconstitution of yeast silent chromatin: multiple contact sites and O-AADPR binding load SIR complexes onto nucleosomes in vitro" Molecular Cell, 33, 323-334.
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    Supplementary Material
  • Cubizolles, F., Martino, F., Perrod, S. and Gasser, S.M. (2006) "A homotrimer-heterotrimer switch in Sir2 structure differentiates rDNA and telomeric silencing", Molecular Cell, 21, 825-836.
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Chromatin Immunoprecipitation (ChIP)

We have developed and optimized ChIP in yeast to determine the role of remodelers at breaks and the stability of polymerases at stalled replication forks. This is described in the protocol written up by ex-postdocs Haico Van Attikum and Jennifer Cobb, which can be downloaded here. Our ChIP protocol has been used for different proteins with small variations as described in the papers listed below:

  • Cobb, J.A., Bjergbaek, L., Shimada, K., Frei, C. and Gasser, S.M.(2003) "DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1." EMBO J. 22, 4325-4336
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  • van Attikum, H., Fritsch, O., Hohn, B. and Gasser, S.M. (2004) "INO80 recruitment by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair." Cell, 119, 777-788.
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    Supplementary Material
  • Shimada, K., Oma, Y., Schleker, T., Kugou, K., Ohta,, K., Harata, M., and Gasser, S. M. (2008) "Ino80 chromatin remodeling complex promotes recovery of stalled replication forks", Current Biol. 8, 566-575.
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  • 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.
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    Supplementary Material
Nuclear extracts for transcription and replication in vitro

This protocol was optimized in the mid 1990's to allow us to study replication in vitro. The extracts for transcription and replication are very similar, except they cells are harvested at different stages of the cell cycle. Yeast nuclear isolation is laborious but highly fruitful once it is mastered!

  • Braguglia, D., Heun P., Pasero, P., Duncker, B. and Gasser, S.M. (1998) "Semi-conservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template." J. Mol. Biology, 281, 631-649
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  • Pasero, P., Braguglia, D. and Gasser, S.M. (1997) "ORC-dependent and Origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei." Genes & Dev., 11, 1504-1518
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Analysing yeast DNA damage and checkpoint response