Methods for visualizing chromatin dynamics in living yeast.

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

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