Microscopy techniques to examine DNA replication in fission yeast.
Temporal and spatial visualization of replication proteins and associated structures within the narrow confines of a yeast nucleus is technically challenging. Choosing the appropriate method depends upon the parameters of the experiment, the nature of the molecules to be observed, and the hypothesis to be tested. In this chapter, we review three broad types of visualization: whole cell fluorescence or immunofluorescence, which is useful for questions of timing and chromatin association; nuclear spreads, which provide greater resolution within the chromatin for colocalization and region-specific effects; and chromatin fibers, which allow observation of labeled proteins and newly synthesized DNA on a linear chromosome. We discuss applications of these protocols and some considerations for choosing methods and fluorophores.
Green
Marc D
MD
Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA, USA.
Sabatinos
Sarah A
SA
Forsburg
Susan L
SL
D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D015825:Chromosomes, Fungal / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis*; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence / Q000379:methods*; D009172:Mycology / Q000379:methods; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D002843:Chromatin; D004271:DNA, Fungal; D005456:Fluorescent Dyes; D018809:Proliferating Cell Nuclear Antigen; D029702:Schizosaccharomyces pombe Proteins; C085710:mcm2 protein, S pombe; D001973:Bromodeoxyuridine