Protocol

The Nucleolus

Volume 1455 of the series Methods in Molecular Biology pp 41-57

Date:

High-Throughput Live-Cell Microscopy Analysis of Association Between Chromosome Domains and the Nucleolus in S. cerevisiae

  • Renjie WangAffiliated withLaboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS
  • , Christophe NormandAffiliated withLaboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS
  • , Olivier GadalAffiliated withLaboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS Email author 

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Abstract

Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell’s nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.

Key words

Nucleolus FROS Nuclear organization Live-cell imaging Fluorescence microscopy Chromosome domain dynamics Yeast Saccharomyces cerevisiae