Cao H, Widlund H R, Simonsson T, Kubista M
Department of Biochemistry and Biophysics, Lundberg Institute, Chalmers University of Technology, Medicinaregatan 9C, Göteborg, SE-413 90, Sweden.
J Mol Biol. 1998 Aug 14;281(2):253-60. doi: 10.1006/jmbi.1998.1925.
Nucleosomes, the building blocks of chromatin, are responsible for DNA packaging in eukaryotic cell nuclei. They play a structural role in genome condensation, and influence transcription and replication. Properties of the DNA sequence, such as curvature and flexibility, direct the location of nucleosomes. DNA sequences that position nucleosomes have been identified and rules that govern their properties have been formulated. However, DNA sequences that are refractory to nucleosome formation have been less well characterised and it is possible that they may perturb or alter chromatin structure. Here we identify such sequences by selecting those that refrain from nucleosome formation from a large pool of synthetic DNA fragments with a central region of 146 random base-pairs fitted with adapters for PCR amplification. These were used for in vitro salt-induced reconstitution of nucleosomes under thermodynamic equilibrium conditions. Fragments that did not form nucleosomes were purified, amplified by PCR, and the reconstitution was repeated. After 17 rounds of negative selection, the material was highly enriched in sequences reluctant to form nucleosomes. Cloning and sequencing revealed that 35% of the molecules had long repeats of TGGA, and their affinity for histone octamers was about half that of average DNA.
核小体是染色质的组成单位，负责真核细胞核中的DNA包装。它们在基因组压缩中起结构作用，并影响转录和复制。DNA序列的特性，如曲率和柔韧性，决定了核小体的位置。已鉴定出定位核小体的DNA序列，并制定了控制其特性的规则。然而，难以形成核小体的DNA序列的特征尚未得到很好的描述，它们可能会扰乱或改变染色质结构。在这里，我们通过从大量合成DNA片段中选择那些不形成核小体的序列来鉴定此类序列，这些片段的中央区域有146个随机碱基对，并装有用于PCR扩增的接头。这些片段用于在热力学平衡条件下体外盐诱导的核小体重组。未形成核小体的片段被纯化，通过PCR扩增，然后重复重组过程。经过17轮阴性选择后，该材料中富含不易形成核小体的序列。克隆和测序显示，35%的分子具有TGGA的长重复序列，它们对组蛋白八聚体的亲和力约为平均DNA的一半。
