ELECTRON-MICROSCOPY OF DNA CROSSLINKED WITH TRIMETHYLPSORALEN - TEST OF SECONDARY STRUCTURE OF EUKARYOTIC INVERTED REPEAT SEQUENCES
ELECTRON-MICROSCOPY OF DNA CROSSLINKED WITH TRIMETHYLPSORALEN - TEST OF SECONDARY STRUCTURE OF EUKARYOTIC INVERTED REPEAT SEQUENCES
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DOI：
10.1073/pnas.73.8.2644
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发表时间：
1976-01-01
影响因子：
11.1
通讯作者：
PARDUE, ML
中科院分区：
文献类型：
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作者：
CECH, TR;PARDUE, ML
反向重复（回文）序列在真核生物基因组中广泛存在，可能存在两种交替的构型，即线性形式和十字形。为了研究这些形式的相对频率，将完整的小鼠组织培养细胞[SVT2]的DNA与4,5',8 - 三甲基补骨脂素（me3 - 补骨脂素）共价交联，以防止在DNA分离过程中DNA二级结构的重排。在乙二醛存在下使DNA变性后，通过电子显微镜（EM）确定me3 - 补骨脂素交联的分布。由于me3 - 补骨脂素交联的频率高且分布相对均匀，得出的结论是几乎所有的反向重复序列都被交联。通过电子显微镜未检测到大量的十字形结构。为了确定me3 - 补骨脂素本身是否可能破坏十字形结构，首先通过热处理在分离的四膜虫rDNA中产生十字形结构，然后在体外进行交联。发现交联是稳定而不是破坏这些十字形结构。所检测的小鼠组织培养细胞的反向重复序列在细胞内主要是线性形式而非十字形结构。
Inverted repeat (palindrome) sequences, which are widespread in eukaryotic genomes, probably exist in 2 alternate configurations, a linear form and a cruciform. To investigate the relative frequency of these forms, the DNA of intact mouse tissue culture cells [SVT2] was covalently crosslinked with 4,5'',8-trimethylpsoralen (me3-psoralen) in order to prevent rearrangement of the DNA secondary structure during DNA isolation. The distribution of me3-psoralen crosslinks was determined by EM after denaturation of the DNA in the presence of glyoxal. Because of the high frequency and the relatively uniform distribution of the me3-psoralen crosslinks, it was concluded that almost all of the inverted repeat sequences were crosslinked. No significant number of cruciforms was detected by EM. To determine whether the me3-psoralen might itself be disrupting cruciform structures, cruciforms were first produced in isolated Tetrahymena rDNA by heat treatment and then crosslinked in vitro. The crosslinking was found to stabilize rather than disrupt these cruciforms. The inverted repeat sequences of the mouse tissue culture cells tested are predominantly in linear forms rather than in cruciform structures inside the cell.