Morgan W F, Fero M L, Land M C, Winegar R A
Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143.
Mol Cell Biol. 1988 Oct;8(10):4204-11. doi: 10.1128/mcb.8.10.4204-4211.1988.
The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.
细胞遗传学终点指标姐妹染色单体交换（SCE）和染色体畸变被广泛用作诱变致癌物诱导DNA损伤的指标。染色体畸变似乎直接源于DNA双链断裂，但导致SCE形成的损伤尚不清楚。大多数诱导SCE的化合物会在DNA中诱导一系列损伤。为了研究双链断裂在SCE形成中的作用，我们构建了一个质粒，该质粒会产生一种特定的损伤，即交错末端（“粘性”）DNA双链断裂。这个名为pMENs的质粒包含一个选择标记neo，它是一个编码新霉素抗性的细菌基因，以及与小鼠金属硫蛋白基因启动子相连的细菌限制性内切酶EcoRI的编码序列。EcoRI识别DNA中的G↓AATTC序列，并产生具有四个核苷酸突出端的交错末端DNA双链断裂。用pMENS转染的细胞对抗生素G418具有抗性，并含有EcoRI基因的整合拷贝，可通过DNA滤膜杂交检测到。添加重金属CdSO4后，通过抗EcoRI抗体检测发现细胞内产生了EcoRI。添加CdSO4后的细胞遗传学分析表明染色体畸变频率显著增加，但对SCE频率影响很小。尽管存在一些细胞间的异质性，但这些结果证实DNA双链断裂确实会导致染色体畸变，但这些断裂不足以引起SCE的形成。
