Oppenheim A, Siani M, Sandalon Z, Mengeritsky G
Department of Hematology, Hebrew University Hadassah Medical School, Jerusalem, Israel.
J Mol Biol. 1994 May 13;238(4):501-13. doi: 10.1006/jmbi.1994.1310.
The regulatory region of SV40 is composed of multiple elements, including the origin of replication (ori), the encapsidation signal (ses) and the enhancer. Here, the structure of the chromatin and nucleoprotein complexes in a region encompassing ses and part of the enhancer was investigated in detail by in situ probing with DNase I. We have used a model experimental system based on plasmids which carry parts of the SV40 regulatory region. The results demonstrate that a specific nucleoprotein structure at the region is formed early after transfection. The overall structure is maintained throughout the viral life cycle. The observed DNase digestion pattern is consistent with the presence of a mixed population of viral minichromosomes with various, but not random, nucleosomal arrangements in that region. Specific modulations, which are associated with the various stages of the viral life cycle, are superimposed on the general structure. The most dramatic changes occur at nucleotides 34 and 113, located at both ends of ses and flanking the GC-box region. Some of the changes depend on the presence of viral gene product(s), probably a late (capsid) protein. The results further suggest that the condensed minichromosome within the viral particle assumes a highly specific configuration in this region. The nucleoprotein structure is sensitive to modifications of the primary nucleotide sequence and to flanking DNA elements. There is good correlation between distortions in the nucleoprotein structure and the inability of mutant plasmids to be packaged, substantiating the requirement for proper chromatin condensation in viral packaging.
SV40的调控区域由多个元件组成，包括复制起点（ori）、包装信号（ses）和增强子。在此，通过用DNase I进行原位探测，详细研究了包含ses和部分增强子的区域中的染色质和核蛋白复合物的结构。我们使用了基于携带SV40调控区域部分片段的质粒的模型实验系统。结果表明，转染后早期在该区域形成了一种特定的核蛋白结构。整个病毒生命周期中整体结构得以维持。观察到的DNase消化模式与该区域存在具有各种但非随机核小体排列的病毒微型染色体混合群体一致。与病毒生命周期各个阶段相关的特定调节作用叠加在总体结构上。最显著的变化发生在位于ses两端且位于GC盒区域侧翼的核苷酸34和113处。其中一些变化取决于病毒基因产物的存在，可能是一种晚期（衣壳）蛋白。结果进一步表明，病毒颗粒内浓缩的微型染色体在该区域呈现出高度特异性的构型。核蛋白结构对一级核苷酸序列的修饰和侧翼DNA元件敏感。核蛋白结构的扭曲与突变体质粒无法被包装之间存在良好的相关性，证实了病毒包装中适当染色质浓缩的必要性。
