Rydberg B, Holley W R, Mian I S, Chatterjee A
Lawrence Berkeley National Laboratory, University of California, One Cyclotron Road, MS29-100, Berkeley, CA, 94720, USA.
J Mol Biol. 1998 Nov 20;284(1):71-84. doi: 10.1006/jmbi.1998.2150.
A new method was used to probe the conformation of chromatin in living mammalian cells. The method employs ionizing radiation and is based on the concept that such radiation induces correlated breaks in DNA strands that are in spatial proximity. Human dermal fibroblasts in G0 phase of the cell cycle and Chinese hamster ovary cells in mitosis were irradiated by X-rays or accelerated ions. Following lysis of the cells, DNA fragments induced by correlated breaks were end-labeled and separated according to size on denaturing polyacrylamide gels. A characteristic peak was obtained for a fragment size of 78 bases, which is the size that corresponds to one turn of DNA around the nucleosome. Additional peaks between 175 and 450 bases reflect the relative position of nearest-neighbor nucleosomes. Theoretical calculations that simulate the indirect and direct effect of radiation on DNA demonstrate that the fragment size distributions are closely related to the chromatin structure model used. Comparison of the experimental data with theoretical results support a zig-zag model of the chromatin fiber rather than a simple helical model. Thus, radiation-induced damage analysis can provide information on chromatin structure in the living cell.
一种新方法被用于探测活的哺乳动物细胞中的染色质构象。该方法采用电离辐射，其基于这样的概念：这种辐射会在空间上接近的DNA链中诱导相关断裂。处于细胞周期G0期的人皮肤成纤维细胞和处于有丝分裂期的中国仓鼠卵巢细胞被X射线或加速离子照射。细胞裂解后，由相关断裂诱导产生的DNA片段进行末端标记，并在变性聚丙烯酰胺凝胶上根据大小进行分离。对于78个碱基的片段大小获得了一个特征峰，该大小对应于DNA围绕核小体一圈的长度。175至450个碱基之间的其他峰反映了相邻核小体的相对位置。模拟辐射对DNA的间接和直接效应的理论计算表明，片段大小分布与所使用的染色质结构模型密切相关。实验数据与理论结果的比较支持染色质纤维的锯齿形模型而非简单的螺旋模型。因此，辐射诱导损伤分析可以提供关于活细胞中染色质结构的信息。
