Kramer P R, Sinden R R
Department of Biochemistry and Biophysics, Texas A&M University, Houston 77030-3303, USA.
Biochemistry. 1997 Mar 18;36(11):3151-8. doi: 10.1021/bi962396q.
Unrestrained DNA supercoiling was measured using a Me3-psoralen photobinding assay within a transcriptionally active hygromycin B phosphotransferase (hph) gene integrated into different chromosomal locations in five transformed human fibrosarcoma cell lines. The level of unrestrained supercoiling in the hph gene varied, from high to low levels, in different chromosomal locations in living human cells. In one cell line, the hph gene contained no unrestrained supercoiling. Consequently, supercoiling was not dictated by the DNA sequence of the active hph gene. The addition of alpha-amanitin, which can inhibit transcription, reduced unrestrained supercoiling by 75% at one chromosomal location, by 50% at two other locations, and had little, if any, effect at two other chromosomal locations. Different levels of supercoiling in separate regions of the chromosome require that the chromosome be organized into independent topological domains in vivo. Evidence for independent topological domains in living cells is presented. From analysis of the relaxation of supercoiling as a function of the number of breaks introduced into the chromosome, the in vivo topological domain size for the human ribosomal RNA genes was estimated between 30,000 and 45,000 kb.
使用甲基三嗪补骨脂素光结合测定法，在整合到五种转化的人纤维肉瘤细胞系不同染色体位置的转录活性潮霉素B磷酸转移酶（hph）基因内，测量无约束的DNA超螺旋。在活的人类细胞的不同染色体位置，hph基因中的无约束超螺旋水平有所不同，从高到低。在一个细胞系中，hph基因没有无约束超螺旋。因此，超螺旋不是由活性hph基因的DNA序列决定的。添加可抑制转录的α-鹅膏蕈碱，在一个染色体位置使无约束超螺旋减少75%，在另外两个位置减少50%，而在另外两个染色体位置几乎没有影响。染色体不同区域的超螺旋水平不同，这表明染色体在体内被组织成独立的拓扑结构域。本文提供了活细胞中独立拓扑结构域的证据。通过分析超螺旋松弛与引入染色体的断裂数之间的函数关系，估计人核糖体RNA基因在体内的拓扑结构域大小在30000至45000 kb之间。
