Islation and analysis of human radiosensitive gene by the use of ataxia telangiectasia

这项研究的目的是确定人类遗传病共济失调毛细血管扩张症(AT)的基因，该疾病的特征是对电离辐射的敏感性增强。一个AT来源的微细胞杂交种，2859/4-1，在人类染色体11q23处有缺失，伴随着辐射抗性的丧失。利用微细胞融合技术获得一株携带11q23缺失染色体的小鼠细胞株，并对缺失区域进行IRS(Interspered Repeatitive Sequence)-PCR分析。结果1.鉴定出两个与AT基因座相近的PCR衍生DNA标记。具有人类X/11染色体的小鼠细胞株A9(3552)-2被用来产生携带人类11q23微小片段的辐射杂交体。将11q23片段导入AT细胞，发现AT基因存在于一个杂交种RH12/1中，而不存在于另外两个杂交种MH12/1和MH12/3.3中。进一步分析了这三个杂交种在11q23上是否存在多态DNA标记。三个杂交种的标记图谱没有发现差异，表明候选区域低于已知DNA标记的分辨率。另一个有趣的是，RH12/1没有D11S384，这表明AT基因可能位于D11S384.4的端粒。从RH12/1构建了粘粒文库，在256个人源性粘粒中，有21个克隆来自11号染色体，在这21个粘粒中没有发现AT基因。利用IRS-PCR技术，在RH12/1特异性区域分离和定位了一个新的质粒pBM8.9，表明pBM8.9可能是一个很强的候选标记，可以用来识别AT位点附近的宇宙。

The aim of the research is to identify the gene for the human genetic disease ataxia telangiectasia (AT) that is characterized by an enhanced sensitivity to ionizing radiation.1. An AT-derived microcell hybrid, 2859/4-1, had a deletion at human chromosome 11q23 with a concomitant loss of radioresistance. A mouse cell strain carrying this 11q23-deleted chromosome was generated by microcell fusion, and IRS (interspersed repetitive sequence) -PCR was used to analyze the deleted region. Two PCR-derived DNA markers close to AT locus were identified.2. A mouse cell strain with a human X/11 chromosome, A9 (3552) -2, was used to generate radiation hybrids that carry minute human 11q23 fragments. Introduction of the 11q23 fragments back into AT cells revealed that AT gene is included in one hybrid, RH12/1, but not in the other two hybrids, MH12/1 and MH12/3.3. The three hybrids were analyzed further for the presence of polymorphic DNA markers at 11q23. There found no difference in the marker profile among the three hybrids, indicating that the candidate region is below the resolution of known DNA markers. Of another interest was that RH12/1 did not have D11S384 that should be the closest marker to AT.This suggest a novel possibility that AT gene may be located telomeric to D11S384.4. A cosmid library was constructed from RH12/1. Among the 256 human-derived cosmids, 21 clones were originated from chromosome 11. AT gene was not found in the 21 cosmids. Using the IRS-PCR,a novel plasmid, pBM8.9, was isolated and mapped in the RH12/1-specific region, implying that pBM8.9 could be a strong candidate marker to specify the cosmids aroung AT locus.