COVALENTLY CLOSED CIRCULAR DUPLEX DNA OF EPSTEIN-BARR VIRUS IN A HUMAN LYMPHOID-CELL LINE
COVALENTLY CLOSED CIRCULAR DUPLEX DNA OF EPSTEIN-BARR VIRUS IN A HUMAN LYMPHOID-CELL LINE
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DOI：
10.1016/0022-2836(76)90331-4
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发表时间：
1976-01-01
影响因子：
5.6
通讯作者：
JEHN, U
中科院分区：
文献类型：
--
作者：
LINDAHL, T;ADAMS, A;JEHN, U
通过CsCl密度梯度离心和中性甘油梯度离心，从伯基特淋巴瘤衍生的人淋巴细胞系Raji中纯化非整合形式的EB病毒DNA。这种细胞内形式的病毒DNA在中性和碱性溶液中以病毒基因组大小的共价闭合环状DNA分子的典型速率沉积。用低剂量的X射线处理导致分子不连续地转化为具有开放环状DNA的沉降性质的形式（含有一个或多个单链断裂的环状双链体分子）。通过电子显微镜直接观察大的环状DNA分子进一步证实了部分细胞内病毒DNA的共价闭合环状双链体结构。在EB病毒阴性人淋巴细胞系的相应DNA组分中未检测到此类环状分子。在溴化乙锭/CsCl密度梯度离心实验中，纯化的非整合病毒DNA表现为扭曲的、共价闭合的DNA环，其具有与多瘤病毒DNA相同的初始超螺旋密度。后一种额外的纯化技术允许从非生产细胞中分离纯度> 90%的细胞内EB病毒DNA。通过轮廓长度测量确定的来自Raji细胞的环状病毒DNA的分子量在实验误差内与来自病毒颗粒的线性DNA的分子量相同。
A non-integrated form of Epstein-Barr virus DNA was purified from the Burkitt lymphoma-derived human lymphoid cell line Raji by CsCl density gradient centrifugation and neutral glycerol gradient centrifugation. This intracellular form of the virus DNA sediments at a rate typical of a covalently closed circular DNA molecule of the size of the virus genome in both neutral and alkaline solution. Treatment with low doses of X-rays leads to a discontinuous conversion of the molecules to a form with the sedimentation properties of open circular DNA (a circular duplex molecule containing one or more single-strand breaks). The direct observation of large circular DNA molecules by electron microscopy further confirms the covalently closed circular duplex structure of part of the intracellular viral DNA. Such circular molecules were not detected in corresponding DNA fractions from Epstein-Barr virus-negative human lymphoid cell lines. In ethidium bromide/CsCl density gradient centrifugation experiments, the purified non-integrated virus DNA behaves as twisted, covalently closed DNA circles with the same initial superhelix density as polyoma virus DNA. The latter additional purification technique permits the isolation of intracellular Epstein-Barr virus DNA in > 90% pure form from non-producer cells. The molecular weight of the circular virus DNA from Raji cells, determined by contour length measurements, is the same within experimental error as that of the linear DNA from virus particles.