Methods for visualizing chromatin dynamics in living yeast.

摘要: Publisher Summary This chapter describes the techniques for visualization of chromatin in living cells (primarily budding yeast), while pointing out pitfalls and artifacts that can arise during live cell imaging. It also presents analytical tools have been developed quantitation data generated by digital These allow defining a new field quantitative analysis: dynamic behavior DNA real time. To visualize laci repressor yeast, its gene is fused frame to sequences encoding nuclear localization signal, S65T derivative natural green fluorescent protein (GFP), which has red-shifted excitation spectrum higher emission intensity. Fusions optimized forms cyan fluorescent (CFP) or yellow (YFP) be used. The detection movement monitored tracking algorithm uses programming extract optimal spatiotemporal trajectory particle. automated image analysis software consists three components: alignment phase, preprocessing phase. Chromatin mobility nearly identical organisms studied detail date. Notably, tagged sites along yeast chromosomes, on X chromosome Drosophila spermatocytes, various insertions at random positions human chromosomes show similar dynamics.