Shelton E R, Wassarman P M, DePamphilis M L
J Biol Chem. 1980 Jan 25;255(2):771-82.
Mature Simian virus 40 (SV40) chromosomes were isolated from infected CV-1 monkey cells by a hypotonic extraction method that not only allowed replicating viral chromosomes to faithfully continue DNA replication in vitro, but also was found to assemble nascent DNA into nucleosomes with a structure and arrangement typical of native viral chromosomes. Detailed analysis of the DNA and nucleoprotein products from micrococcal nuclease and DNase I digestion of mature viral chromosomes assembled in intact cells showed that the structure of SV40 and CV-1 cellular nucleosomes was the same. Furthermore, the histone composition of viral chromosome was indistinguishable from that of its host. In contrast to the identity in nucleosome structure, nucleosome spacing on isolated SV40 chromosomes was not as regular as on cellular chromatin. When 1% of the DNA was solubilized by micrococcal nuclease, as many as 20 cellular DNA bands were clearly resolved by gel electrophoresis, but only 6 to 7 viral DNA bands were observed and they were broader and less well resolved. In addition, micrococcal nuclease digested SV40 chromosomes at a faster initial rate and to a greater extent than CV-1 chromatin present in the same tube. Either BglI or EcoRI restriction endonuclease cut a single site in 30% of the SV40 chromosomes which suggested that viral nucleosomes were not located in a unique phase with respect to DNA sequence, but appeared to be randomly spaced around the genome. Viral chromosome structure was basically unaltered in hypotonically extracted chromosomes exposed to 200 or 600 mM NaCl and in isotonically extracted chromosomes prepared in the presence of Triton X-100 and EDTA. These results confirm and extend our previous data on the arrangement of SV40 nucleosomes inside isolated nuclei and demonstrate that the structure of viral chromosomes was not altered by the isolation procedures employed. The data are consistent with a model in which an average of 22 nucleosomes, randomly distributed around the SV40 genome, are separated by non-nucleosomal spacer regions which account for about 20% of the total DNA.
通过低渗提取法从感染的CV - 1猴细胞中分离出成熟的猴病毒40（SV40）染色体，该方法不仅能使复制中的病毒染色体在体外忠实地继续进行DNA复制，还发现能将新生DNA组装成具有天然病毒染色体典型结构和排列的核小体。对完整细胞中组装的成熟病毒染色体经微球菌核酸酶和DNase I消化后的DNA和核蛋白产物进行详细分析表明，SV40和CV - 1细胞的核小体结构相同。此外，病毒染色体的组蛋白组成与其宿主的组蛋白组成难以区分。与核小体结构相同形成对比的是，分离出的SV40染色体上的核小体间距不如细胞染色质上的规则。当1%的DNA被微球菌核酸酶溶解时，凝胶电泳能清晰分辨出多达20条细胞DNA条带，但仅观察到6至7条病毒DNA条带，且它们更宽且分辨率更低。此外，微球菌核酸酶消化SV40染色体的初始速率比同一试管中的CV - 1染色质更快，消化程度也更高。BglI或EcoRI限制性内切酶在30%的SV40染色体上切割单个位点，这表明病毒核小体相对于DNA序列并非位于独特的相位，而是似乎在基因组周围随机分布。在暴露于200或600 mM NaCl的低渗提取染色体以及在Triton X - 100和EDTA存在下制备的等渗提取染色体中，病毒染色体结构基本未改变。这些结果证实并扩展了我们之前关于分离细胞核内SV40核小体排列的数据，并表明所采用的分离程序未改变病毒染色体的结构。数据与一个模型一致，即平均22个核小体随机分布在SV40基因组周围，由占总DNA约20%的非核小体间隔区隔开。
