Wong M L, Hsu M T
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029.
Nucleic Acids Res. 1989 May 11;17(9):3535-50. doi: 10.1093/nar/17.9.3535.
Electron microscopic analysis of bis-psoralen crosslinked adenovirus type 5 virion DNA revealed supercoiled domains in an otherwise linear DNA. The existence of supercoiled arrangement in all the virion DNA was demonstrated by the sensitivity of Ad5 DNA in pentonless virus particles to the supercoiling-dependent endonucleolytic activity of Bal31 and S1 nucleases. These nucleases were found to cleave Ad5 virion DNA at specific sites. The observation of stable cleavage sites in the limit digestion of virion DNA by Bal31 suggests that cleavage sites represent boundaries of core proteins which impede the exonuclease activity of Bal31. These data suggest that specific arrangement of core proteins on Ad5 virion DNA. Based on this analysis we determined positions of core proteins in viral genome using indirect end labeling technique. The size of supercoiled domains of virion DNA was estimated by electron microscopy and also by boundaries of mutually exclusive Bal31 cleavage sites at limit digestion condition. Our data suggest each supercoiled domain is equal to about 12% of Ad5 genome length and about 8 loops can be accommodated in Ad5 virion. However sequences at two extreme ends of the viral genome were found to be outside of supercoiled domains. An interesting correlation between supercoiled domains and gene domains of Ad5 genome was noticed.
对双补骨脂素交联的5型腺病毒病毒粒子DNA进行电子显微镜分析，发现在原本呈线性的DNA中存在超螺旋结构域。通过无五邻体病毒颗粒中的Ad5 DNA对Bal31和S1核酸酶的超螺旋依赖性内切核酸酶活性的敏感性，证实了所有病毒粒子DNA中都存在超螺旋排列。发现这些核酸酶在特定位点切割Ad5病毒粒子DNA。在Bal31对病毒粒子DNA进行极限消化时观察到稳定的切割位点，这表明切割位点代表了阻碍Bal31核酸外切酶活性的核心蛋白的边界。这些数据表明Ad5病毒粒子DNA上核心蛋白的特定排列。基于此分析，我们使用间接末端标记技术确定了病毒基因组中核心蛋白的位置。通过电子显微镜以及在极限消化条件下相互排斥的Bal31切割位点的边界，估计了病毒粒子DNA超螺旋结构域的大小。我们的数据表明，每个超螺旋结构域约等于Ad5基因组长度的12%，Ad5病毒粒子中可容纳约8个环。然而，发现病毒基因组两端的序列位于超螺旋结构域之外。注意到Ad5基因组的超螺旋结构域与基因结构域之间存在有趣的相关性。
