Rousset S, Nocentini S, Santella R M, Gasparro F P, Moustacchi E
Institut Curie, Biologie, URA 1292 CNRS, Paris, France.
J Photochem Photobiol B. 1993 Apr;18(1):27-34. doi: 10.1016/1011-1344(93)80037-a.
A direct method of immuno-electron microscopy has been employed to simultaneously determine 8-methoxypsoralen (8-MOP) photoinduced monoadducts (MA) and interstrand cross-links (CL), their relative localization along the DNA molecule, and their removal. It has been applied to DNA from cultures of Fanconi anemia (FA) fibroblasts (complementation groups A and D), and of normal human fibroblasts, following treatment by 8-MOP and 365 nm radiation. The immuno-reaction with monoclonal antibody 8G1 was performed on DNA extracted from the cells just after photoreaction, or after a 24 h repair period, and then denatured. Furan-side MA and also a significant proportion of pyrone-side MA were very efficiently immuno-detected. Only 1-2% CL were IgG-labeled. This is why CL were directly visualized and quantified on denatured DNA from the same cellular samples as used for immuno-detection. Results demonstrate that FA group A cells are not only impaired in the incision of CL, but also of MA. The response of FA group D cells is intermediate between normal and FA group A cells.
已采用一种免疫电子显微镜直接方法来同时测定8-甲氧基补骨脂素（8-MOP）光诱导的单加合物（MA）和链间交联（CL），它们沿DNA分子的相对定位及其去除情况。该方法已应用于经8-MOP和365nm辐射处理后的范可尼贫血（FA）成纤维细胞（互补组A和D）以及正常人成纤维细胞的DNA。在用单克隆抗体8G1进行免疫反应时，对光反应刚结束后或经过24小时修复期后从细胞中提取的DNA进行反应，然后使其变性。呋喃侧的MA以及相当一部分吡喃酮侧的MA都能被非常有效地免疫检测到。只有1-2%的CL被IgG标记。这就是为什么在与用于免疫检测的相同细胞样本的变性DNA上直接对CL进行可视化和定量分析。结果表明，FA A组细胞不仅在CL的切口方面存在缺陷，在MA的切口方面也存在缺陷。FA D组细胞的反应介于正常细胞和FA A组细胞之间。
