Barr F G, Davis R J, Eichenfield L, Emanuel B S
Division of Human Genetics and Molecular Biology, Children's Hospital of Philadelphia, PA 19104.
Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):942-6. doi: 10.1073/pnas.89.3.942.
We have explored the mechanism of genomic rearrangement in a hamster fibroblast cell culture system in which rearrangements are induced 5' to the endogenous thymidine kinase gene by chemical carcinogen treatment. The wild-type region around one rearrangement breakpoint was cloned and sequenced. With this sequence information, the carcinogen-induced rearrangement was cloned from the corresponding rearranged cell line by the inverse polymerase chain reaction. After the breakpoint fragment was sequenced, the wild-type rearrangement partner (RP15) was isolated by a second inverse polymerase chain reaction of unrearranged DNA. Comparison of the sequence of the rearrangement breakpoint with the wild-type RP15 and 5' thymidine kinase gene regions revealed short repeats directly at the breakpoint, as well as nearby A + T-rich regions in each rearrangement partner. Pulsed-field electrophoresis analysis demonstrated that this rearrangement is an interstitial deletion of 35 kilobases. Southern blot analysis of the RP15 region in unrearranged parental cells showed a demethylated CpG island and a complex of DNase I-hypersensitive sites adjacent to the breakpoint in the region deleted by the rearrangement. Therefore, these studies reveal interesting sequence and chromatin features near the rearrangement breakpoints and suggest a role for nuclear organization in the mechanism of carcinogen-induced genomic rearrangement.
我们在仓鼠成纤维细胞培养系统中探索了基因组重排的机制，在该系统中，通过化学致癌物处理在内源性胸苷激酶基因的5'端诱导重排。克隆并测序了一个重排断点周围的野生型区域。利用该序列信息，通过反向聚合酶链反应从相应的重排细胞系中克隆了致癌物诱导的重排。对断点片段进行测序后，通过对未重排DNA进行第二次反向聚合酶链反应分离出野生型重排伴侣（RP15）。将重排断点的序列与野生型RP15和5'端胸苷激酶基因区域进行比较，发现在断点处直接存在短重复序列，以及每个重排伴侣中附近的富含A+T的区域。脉冲场电泳分析表明，这种重排是一个35千碱基的间质缺失。对未重排亲代细胞中RP15区域的Southern印迹分析显示，在重排缺失区域的断点附近有一个去甲基化的CpG岛和一个DNase I超敏位点复合体。因此，这些研究揭示了重排断点附近有趣的序列和染色质特征，并提示核组织在致癌物诱导的基因组重排机制中发挥作用。
