Lipchitz L, Axel R
Cell. 1976 Oct;9(2):355-64. doi: 10.1016/0092-8674(76)90125-2.
We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array. Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA. Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.
我们分析了核小体中特定核苷酸序列被DNA限制性内切酶识别和切割的效率。适用于这类分析的一个系统是用限制性内切酶EcoRI切割牛基因组。牛卫星I占基因组的7%，呈串联重复排列，在该序列中每隔1400个碱基对（bp）有一个EcoRI位点。这种限制性片段易于测量，这使得我们能够分析该序列在核小体阵列中时的可及性。初步研究表明，卫星I序列以类似于总基因组DNA的方式组织成核小体结构。然后，我们检测了完整细胞核中卫星序列上EcoRI切割位点对内切核酸酶切割的可及性。我们发现，虽然来自裸露DNA的几乎所有卫星I序列都被切割成离散的1400 bp片段，但在完整细胞核中只有33%的卫星I DNA以该片段形式释放出来。这些数据表明，细胞核中57%的EcoRI位点可被切割，并且切割可发生在至少一半核小体亚基的核心内。对消化产物的分析表明，核小体在卫星I DNA的EcoRI位点周围呈随机分布。最后，卫星序列可从细胞核中与其相关蛋白质一起被切割成1400 bp长的片段这一观察结果，为作为染色质分离特定序列提供了一种方法。通过蔗糖梯度速度离心，我们分离出了70%纯的卫星I染色质组分。对该染色质组分进行核酸酶消化揭示了核小体亚基的存在，并表明可以以这种方式分离特定序列，而不会使其亚基结构发生严重紊乱。
