Tsukamoto T, Hashiguchi N, Janicki S M, Tumbar T, Belmont A S, Spector D L
Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Nat Cell Biol. 2000 Dec;2(12):871-8. doi: 10.1038/35046510.
Chromatin structure is thought to play a critical role in gene expression. Using the lac operator/repressor system and two colour variants of green fluorescent protein (GFP), we developed a system to visualize a gene and its protein product directly in living cells, allowing us to examine the spatial organization and timing of gene expression in vivo. Dynamic morphological changes in chromatin structure, from a condensed to an open structure, were observed upon gene activation, and targeting of the gene product, cyan fluorescent protein (CFP) reporter to peroxisomes was visualized directly in living cells. We found that the integrated gene locus was surrounded by a promyelocytic leukaemia (PML) nuclear body. The association was transcription independent but was dependent upon the direct in vivo binding of specific proteins (EYFP/lac repressor, tetracycline receptor/VP16 transactivator) to the locus. The ability to visualize gene expression directly in living cells provides a powerful system with which to study the dynamics of nuclear events such as transcription, RNA processing and DNA repair.
染色质结构被认为在基因表达中起关键作用。利用乳糖操纵子/阻遏物系统和绿色荧光蛋白（GFP）的两种颜色变体，我们开发了一种系统，可在活细胞中直接可视化一个基因及其蛋白质产物，从而使我们能够在体内研究基因表达的空间组织和时间安排。在基因激活时，观察到染色质结构从浓缩结构到开放结构的动态形态变化，并且在活细胞中直接可视化了基因产物青色荧光蛋白（CFP）报告基因定位于过氧化物酶体的过程。我们发现整合的基因位点被早幼粒细胞白血病（PML）核体所包围。这种关联不依赖于转录，但依赖于特定蛋白质（增强黄色荧光蛋白/乳糖阻遏物、四环素受体/病毒蛋白16反式激活因子）在体内与该位点的直接结合。在活细胞中直接可视化基因表达的能力提供了一个强大的系统，可用于研究诸如转录、RNA加工和DNA修复等核事件的动态过程。
