[9] Mapping of yeast nucleosomes in Vivo

Publisher Summary The localization of nucleosomes in yeast chromatin has so far been analyzed on isolated chromatin or nuclei because intact cells and spheroplasts are impermeable to macromolecules, thus, preventing the penetration of enzymes normally used in vivo or in vitro as analytical tools (deoxyribonuclease I, micrococcal nuclease, exonuclease III, and restriction endonucleases). The use of nystatin as a permeabilizing agent has opened the possibility of the enzymatic treatment of chromatin in living cells and the development of new analytical approaches to chromatin analysis. Permeabilization with nystatin allows mapping in the spheroplasts of nucleosomes located on large chromosomal segments by the traditional indirect end-labeling procedure. The resulting picture is not substantially different from that conventionally obtained with isolated chromatin. In this case, the advantage is the fact that the risks of protein rearrangements and/or modifications are kept to a minimum.