Yeast Nucleoplasmic Extracts and an Application to Visualize Chromatin Assembly on Single Molecules of DNA

In eukaryotic cells, the genomic DNA is packaged into chromatin, the basic unit of which is the nucleosome. Studying the mechanism of chromatin formation under physiological conditions is inherently difficult due to the limitations of research approaches. Here we describe how to prepare a biochemical system called yeast nucleoplasmic extracts (YNPE). YNPE is derived from yeast nuclei, and the in vitro system can mimic the physiological conditions of the yeast nucleus in vivo. In YNPE, the dynamic process of chromatin assembly has been observed in real time at the single-molecule level by total internal reflection fluorescence microscopy. YNPE provides a novel tool to investigate many aspects of chromatin assembly under physiological conditions and is competent for single-molecule approaches.