Visualizing yeast chromosomes and nuclear architecture.
We describe here optimized protocols for tagging genomic DNA sequences with bacterial operator sites to enable visualization of specific loci in living budding yeast cells. Quantitative methods for the analysis of locus position relative to the nuclear center or nuclear pores, the analysis of chromatin dynamics and the relative position of tagged loci to other nuclear landmarks are described. Methods for accurate immunolocalization of nuclear proteins without loss of three-dimensional structure, in combination with fluorescence in situ hybridization, are also presented. These methods allow a robust analysis of subnuclear organization of both proteins and DNA in intact yeast cells.
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
Peter
P
Meister
Lutz R
LR
Gehlen
Elisa
E
Varela
Véronique
V
Kalck
Susan M
SM
Gasser
D002467:Cell Nucleus / Q000378:metabolism*; D015825:Chromosomes, Fungal / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence; D018613:Microscopy, Confocal; D004718:Saccharomycetales / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism