Thacker J, Tambini C E, Simpson P J, Tsui L C, Scherer S W
MRC Radiobiology Unit, Chilton, Didcot, Oxon, UK.
Hum Mol Genet. 1995 Jan;4(1):113-20. doi: 10.1093/hmg/4.1.113.
The identification of genes controlling cellular response to DNA damage is of considerable importance, and cell lines showing hypersensitivity to DNA-damaging agents can be used as vehicles to map and clone these genes. In this study the hamster cell line irs1, showing hypersensitivity to a number of different DNA-damaging agents, was fused to normal human cells to complement the defect. The resultant hybrids were analysed by Alu-PCR, chromosome painting, and with DNA markers to map the complementing gene (named XRCC2) to a specific chromosomal region. These hybrids showed correction of sensitivity to both X-rays and to mitomycin-C, and contained human chromosome 7, often as their only human component. Hybrids showing unstable retention of human chromosomes were sub-cloned to show that loss of chromosome 7 and loss of resistance to mitomycin-C occurred concordantly. Two separate hybrids were found to have a smaller piece of chromosome 7, and specific DNA probes and microsatellite markers defined this as a contiguous region at 7q35-36. Hybrid irradiation-fusion methods were used to further reduce the size of the complementing genomic region and to localize the gene to an approximately 3-5 Mb region at 7q36.1.
鉴定控制细胞对DNA损伤反应的基因具有相当重要的意义，对DNA损伤剂表现出超敏反应的细胞系可作为定位和克隆这些基因的载体。在本研究中，对多种不同DNA损伤剂表现出超敏反应的仓鼠细胞系irs1与正常人细胞融合以弥补缺陷。通过Alu-PCR、染色体涂染以及使用DNA标记对所得杂种进行分析，将互补基因（命名为XRCC2）定位到特定的染色体区域。这些杂种对X射线和丝裂霉素C的敏感性均得到纠正，并且通常仅含有人染色体7作为其唯一的人类成分。对人染色体保留不稳定的杂种进行亚克隆，结果表明染色体7的丢失与对丝裂霉素C抗性的丧失是一致的。发现两个独立的杂种含有一条较小片段的染色体7，特定的DNA探针和微卫星标记将其确定为7q35 - 36的一个连续区域。采用杂交辐射融合方法进一步缩小互补基因组区域的大小，并将该基因定位到7q36.1上一个约3 - 5 Mb的区域。
