Protocol

The Nucleolus

Volume 1455 of the series Methods in Molecular Biology pp 29-40

Date:

Correlative Light and Electron Microscopy of Nucleolar Transcription in Saccharomyces cerevisiae

  • Christophe NormandAffiliated withLaboratoire de Biologie Moléculaire du CNRS, University of Toulouse
  • , Maxime BerthaudAffiliated withCRCNA-UMR 892 INSERMCNRSUniversity of Nantes
  • , Olivier GadalAffiliated withLaboratoire de Biologie Moléculaire du CNRS, University of Toulouse Email author 
  • , Isabelle Léger-SilvestreAffiliated withLaboratoire de Biologie Moléculaire du CNRS, University of Toulouse Email author 

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Abstract

Nucleoli form around RNA polymerase I transcribed ribosomal RNA (rRNA) genes. The direct electron microscopy observation of rRNA genes after nucleolar chromatin spreading (Miller’s spreads) constitutes to date the only system to quantitatively assess transcription at a single molecule level. However, the spreading procedure is likely generating artifact and despite being informative, these spread rRNA genes are far from their in vivo situation. The integration of the structural characterization of spread rRNA genes in the three-dimensional (3D) organization of the nucleolus would represent an important scientific achievement. Here, we describe a correlative light and electron microscopy (CLEM) protocol allowing detection of tagged-Pol I by fluorescent microscopy and high-resolution imaging of the nucleolar ultrastructural context. This protocol can be implemented in laboratories equipped with conventional fluorescence and electron microscopes and does not require sophisticated “pipeline” for imaging.

Key words

Correlative light and electron microscopy (CLEM) Nucleolus RNA polymerase I (Pol I) Ribosomal RNA genes (rDNA) Pol I transcription Transmission electron microscopy (TEM) Fluorescence microscopy Yeast Saccharomyces cerevisiae