Bednar J, Horowitz R A, Dubochet J, Woodcock C L
Département d'Analyse Ultrastructurale, Université de Lausanne, Switzerland.
J Cell Biol. 1995 Dec;131(6 Pt 1):1365-76. doi: 10.1083/jcb.131.6.1365.
Cryoelectron microscopy has been used to examine the three-dimensional (3-D) conformation of small oligonucleosomes from chicken erythrocyte nuclei after vitrification in solutions of differing ionic strength. From tilt pairs of micrographs, the 3-D location and orientation of the nucleosomal disks, and the paths of segments of exposed linker can be obtained. In "low-salt" conditions (5 mM NaCl, 1 mM EDTA, pH 7.5), the average trinucleosome assumes the shape of an equilateral triangle, with nucleosomes at the vertices, and a length of exposed linker DNA between consecutive nucleosomes equivalent to approximately 46 bp. The two linker DNA segments converge at the central nucleosome. Removal of histones H1 and H5 results in a much more variable trinucleosome morphology, and the two linker DNA segments usually join the central nucleosome at different locations. Trinucleosomes vitrified in 20 mM NaCl, 1 mM EDTA, (the salt concentration producing the maximal increase in sedimentation), reveal that compaction occurs by a reduction in the included angle made by the linker DNA segments at the central nucleosome, and does not involve a reduction in the distance between consecutive nucleosomes. Frequently, there is also a change in morphology at the linker entry-exit site. At 40 mM NaCl, there is no further change in trinucleosome morphology, but polynucleosomes are appreciably more compact. Nevertheless, the 3-D zig-zag conformation observed in polynucleosomes at low salt is retained at 40 mM NaCl, and individual nucleosome disks remain separated from each other. There is no evidence for the formation of solenoidal arrangements within polynucleosomes. Comparison of the solution conformation of individual oligonucleosomes with data from physical measurements on bulk chromatin samples suggests that the latter should be reinterpreted. The new data support the concept of an irregular zig-zag chromatin conformation in solution over a range of ionic strengths, in agreement with other in situ (McDowall, A.W., J.M. Smith, and J. Dubochet. 1986, EMBO (Eur. Mol. Biol. Organ.) J.5: 1395-1402; Horowitz, R.A., D.A. Agard, J.W. Sedat, and C.L. Woodcock, 1994. J. Cell Biol. 125:1-10), and in vitro conclusions (van Holde, K., and J. Zlatanova. 1995. J. Biol. Chem. 270:8373-8376). Cryoelectron microscopy also provides a way to determine the 3-D conformation of naturally occurring chromatins in which precise nucleosome positioning plays a role in transcriptional regulation.
冷冻电子显微镜已被用于研究鸡红细胞核中的小寡核小体在不同离子强度溶液中玻璃化后的三维（3-D）构象。从倾斜的显微照片对中，可以获得核小体盘的三维位置和方向，以及暴露的连接子片段的路径。在“低盐”条件下（5 mM NaCl，1 mM EDTA，pH 7.5），平均三核小体呈等边三角形形状，核小体位于顶点，连续核小体之间暴露的连接子DNA长度约相当于46 bp。两条连接子DNA片段在中央核小体处汇聚。去除组蛋白H1和H5会导致三核小体形态更加多变，并且两条连接子DNA片段通常在不同位置连接中央核小体。在2 mM NaCl，1 mM EDTA（产生沉降最大增加的盐浓度）中玻璃化的三核小体表明，压缩是通过减少中央核小体处连接子DNA片段所成的夹角来实现的，并且不涉及连续核小体之间距离的减小。通常，连接子进出位点的形态也会发生变化。在4 mM NaCl时，三核小体形态没有进一步变化，但多核小体明显更紧凑。然而，在低盐条件下多核小体中观察到的三维锯齿状构象在4 mM NaCl时得以保留，并且单个核小体盘彼此保持分离。没有证据表明多核小体中形成了螺线管排列。将单个寡核小体的溶液构象与大量染色质样品的物理测量数据进行比较表明，后者应重新解释。新数据支持在一系列离子强度下溶液中不规则锯齿状染色质构象的概念，这与其他原位研究（McDowall, A.W., J.M. Smith, and J. Dubochet. 1986, EMBO (Eur. Mol. Biol. Organ.) J.5: 1395 - 1402; Horowitz, R.A., D.A. Agard, J.W. Sedat, and C.L. Woodcock, 1994. J. Cell Biol. 125:1 - 10）以及体外研究结论（van Holde, K., and J. Zlatanova. 1995. J. Biol. Chem. 270:8373 - 8376）一致。冷冻电子显微镜还提供了一种确定天然存在的染色质三维构象的方法，其中精确的核小体定位在转录调控中起作用。
