Measurement of unrestrained negative supercoiling and topological domain size in living human cells
Measurement of unrestrained negative supercoiling and topological domain size in living human cells
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DOI：
10.1021/bi962396q
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发表时间：
1997-03-18
期刊：
影响因子：
2.9
通讯作者：
Sinden, RR
中科院分区：
文献类型：
--
作者：
Kramer, PR;Sinden, RR
在5个转化的人纤维肉瘤细胞系中，在整合到不同染色体位置的具有转录活性的潮霉素B磷酸转移酶（hph）基因内，使用Me（3）-peptien光结合测定法测量了无限制的DNA超螺旋。在人类活细胞的不同染色体位置，hph基因中不受限制的超螺旋水平从高到低不等。在一个细胞系中，hph基因不包含无限制的超螺旋。因此，超螺旋不是由活性hph基因的DNA序列决定的。添加α-鹅膏蕈碱，它可以抑制转录，减少不受限制的超螺旋在一个染色体位置的75%，在其他两个位置的50%，并有很少的，如果有的话，在其他两个染色体位置的影响。染色体不同区域的超螺旋水平不同，需要染色体在体内被组织成独立的拓扑结构域。在活细胞中的独立拓扑结构域的证据。从超螺旋的松弛作为引入染色体的断裂数的函数的分析，人核糖体RNA基因的体内拓扑结构域的大小估计在30 000和45 000 kb之间。
Unrestrained DNA supercoiling was measured using a Me(3)-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.