The chemical nature of photoreactivable lesions in DNA.
The chemical nature of photoreactivable lesions in DNA.
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DNA 中光反应性损伤的化学性质。
DOI：
10.1073/pnas.53.6.1430
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发表时间：
1965
影响因子：
11.1
通讯作者：
F. Bollum
中科院分区：
文献类型：
--
作者：
J. Setlow;M. Boling;F. Bollum
取出 1ml 并分析流感嗜血杆菌细胞转化对链霉素抗性的能力。5 将混合物在黑暗中 370°C 孵育 25 分钟，转化活性量没有产生可观察到的变化。在一些实验中，在添加转化DNA之前，用光活化酶和光处理合成的多核苷酸。将 10 zg/ml 的 0.2 ml 辐照聚合物样品与 10 ul 酵母储备酶在白色瓷点板中混合。黑光照射后，用 1.15 ml 磷酸盐缓冲液稀释 0.1 ml 混合物，加入 0.15 ml 转化 DNA，并将 0.2 ml 样品置于点板中，如前所述进行光活化。在 370°C 的黑光下长达 3 小时，酵母酶没有表现出失活的迹象。胸腺嘧啶二聚体测定：使用氚标记的胸苷三磷酸制备聚 dA:dT (H3)，通过闪烁计数测定其比活性约为 3000 cpm/ug。经辐照的光再活化样品在 1750°C 下用甲酸水解，并如前所述在 Whatman #1 纸上进行二维色谱分析。6 将水解的小牛胸腺 DNA 添加到每个样品中，以帮助在纸上定位化合物。切下纸条，用水洗脱，放入 15 毫升二恶烷-萘液体中7，并在闪烁光谱仪中计数。
ml were withdrawn and assayed for ability to transform H. influenzae cells to streptomycin resis- tance.5 Incubation of the mixture at 370C in the dark for 25 min produced no observable change in the amount of transforming activity. In some experiments synthetic polynucleotides were treated with photoreactivating enzyme plus light before addition of the transforming DNA. Samples of 0.2 ml of irradiated polymer at 10 zg/ml were mixed with 10 ,ul stock yeast enzyme in white porcelain spot plates. After blacklight illumination, 0.1 ml of the mix was diluted with 1.15 ml phosphate buffer, 0.15 ml transforming DNA was added, and 0.2-ml samples were placed in spot plates for photoreactivation as before. The yeast enzyme showed no evidence of inactivation for as long as 3 hr under the blacklight at 370C. Thymine dimer assay: Tritium-labeled thymidine triphosphate was used to make poly dA:dT (H3) with a specific activity of about 3000 cpm/ug, determined by scintillation counting. Irradiated, photoreactivated samples were hydrolyzed with formic acid at 1750C and chromato-graphed in two dimensions on Whatman #1 paper as previously described.6 Hydrolyzed calf thymus DNA was added to each sample for aid in locating the compounds on the paper. Paper strips were cut, eluted in water, placed in 15-ml dioxane-naphthalene fluid,7 and counted in a scintillation spectrometer.