HOMOGENEOUS RECONSTITUTED OLIGONUCLEOSOMES, EVIDENCE FOR SALT-DEPENDENT FOLDING IN THE ABSENCE OF HISTONE-H1
HOMOGENEOUS RECONSTITUTED OLIGONUCLEOSOMES, EVIDENCE FOR SALT-DEPENDENT FOLDING IN THE ABSENCE OF HISTONE-H1
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DOI：
10.1021/bi00449a026
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发表时间：
1989-11-14
期刊：
影响因子：
2.9
通讯作者：
VANHOLDE, KE
中科院分区：
文献类型：
--
作者：
HANSEN, JC;AUSIO, J;VANHOLDE, KE
使用盐透析的方法，我们已经将组蛋白八聚体重建到由12个串联重复组成的DNA模板上，每个串联重复包含海胆5S rRNA基因的片段[Simpson，R.T.，Thomas，F.，和Brubaker，J. M.（1985）Cell 42，799 - 808]。在这些模板中，每个海胆重复序列包含用于优选核小体定位的序列。在分析超离心中的沉降速度和沉降平衡研究表明，在组蛋白/DNA摩尔比为1.0 - 1.1时，可以定期获得完全加载的寡核小体（12个核小体/模板）的非常均匀的制备物。用微球菌核酸酶消化寡核小体，然后对纯化的核小体结合的DNA序列进行限制性作图，产生复杂但一致的核小体定位模式。大约50%的核小体似乎在每个重复序列的位置1 - 146处定相，而剩余的核小体占据沿着模板的许多其他较小的离散位置，这些位置沿着相差10 bp的倍数。从0 - 0.2 M NaCl中的oligonucleosomes的沉降速度研究，我们观察到一个可逆的平均沉降系数增加了近30%，伴随着发展的异质性沉积。这些结果与理论预测相结合，表明在缺乏富含赖氨酸的组蛋白的情况下，染色质的线性延伸存在于溶液中，在延伸的（低盐）构象和一个或多个折叠的（高盐）结构之间存在盐依赖性平衡。此外，通过100 mM NaCl，观察到组蛋白八聚体从这些线性寡核小体的盐依赖性解离。
Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., and Brubaker, J. M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remained of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.