Repair of ionizing radiation damage in primate alpha DNA transfected into rat cells.
Repair of ionizing radiation damage in primate alpha DNA transfected into rat cells.
复制标题
转染大鼠细胞的灵长类 α DNA 中电离辐射损伤的修复。
DOI：
10.1080/09553009214551791
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发表时间：
1992
影响因子：
2.6
通讯作者：
Mendez,F
中科院分区：
文献类型：
--
作者：
Bases,R;Mendez,F
研究了转染大鼠 NRK 细胞并恢复后受辐射的灵长类 αDNA 修复的时间过程。选择大鼠细胞进行转染，因为它们没有 αDNA。质粒 pBUC4α10 在其 5 kbp DNA 中含有 10 个串联的 172 bp αDNA 亚基，经过辐照并通过电穿孔导入大鼠细胞。然后从不含外来大鼠 DNA 的 NRK 细胞核中回收转染的 αDNA，从而可以在时程实验中研究转染的 αDNA 的命运。转染后2·5小时内，αDNA不断进入细胞核进行处理。转染后 2·5 小时即可检测到 NRK 核中 αDNA 中的受损碱基库。转染后 5-150 分钟的 αDNA 测序凝胶分析中，辐射诱导的 αDNA 片段的电泳迁移率介于单位核苷酸长度之间，这一点很突出。这些中等迁移率碎片最初在 T½ 为 6-25 分钟时消失；具有 3'-磷酰基末端的 DNA 片段在 T½ 为 10-20 分钟时消失。推测中等迁移率的αDNA是DNA修复的中间体。尚未在转染细胞中处理的残留DNA碱基损伤随后可以在体外通过使用热和哌啶或大肠杆菌的核酸内切酶III进行β-消除转化为链断裂而被暴露。转染前 4 小时对受体 NRK 细胞进行 5 Gy 照射，可延长发现中等迁移率物质的时间，这与在 300 Gy 之前用小剂量辐射预处理的猴 CV-1 细胞 DNA 中观察到的中等迁移率物种频率增加一致（Baseset al. 1990）。
The time-course of repair of irradiated primate αDNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no αDNA. Plasmid pBUC4α10, containing 10 tandem 172 bp αDNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected αDNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected αDNA in time-course experiments. αDNA continuously entered nuclei for processing in the first 2·5 h after transfection. The pool of damaged bases in αDNA in NRK nuclei was detectable 2·5 h after transfection.Radiation-induced αDNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of αDNA for 5–150 min after transfection. These intermediate mobility fragments initially disappeared with T½of 6–25 min; DNA fragments with 3'-phosphoryl termini disappeared with T½of 10–20 min. The αDNAs of intermediate mobility are presumed to be intermediates in DNA repair. Residual DNA base damage which had not been processed in the transfected cells could later be unmaskedin vitroby conversion to strand breaks by β-elimination using heat and piperidine or endonuclease III ofE. coli.Irradiation of the recipient NRK cells with 5 Gy 4 hours before transfection prolonged the time during which intermediate mobility species could be found, consistent with the increased frequency of intermediate mobility species observed in DNA of monkey CV-1 cells pretreated with small doses of radiation before 300 Gy (Baseset al. 1990).