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.
Key words:Microscopy Immunofluorescence DNA fiber Chromatin spreads Whole cell immunofluorescence Fission yeast
- 21.Shaw, P. (2006) Comparison of widefield/deconvolution and confocal microscopy for three-dimensional imaging, in Handbook of Biological Confocal Microscopy 3rd ed. (Pawley, J., ), pp. 452–67, Plenum, NY.Google Scholar
- 29.Yokochi, T., and Gilbert, D.M. (2007) Replication labeling with halogenated thymidine analogs. Curr Protoc Cell Biol Chapter 22, Unit 22 10.Google Scholar