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

利用共济失调性毛细血管扩张症对人体放射敏感基因进行分离与分析

• 批准号: 04808032
• 负责人: EJIMA Yosuke
• 金额: $ 1.47万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04808032/
• 关键词: AT; Radiosensitivity; Human chromosome 11; Radiation hybrid; IRS-PCR; Genetic disease; 放射線ハイブリド; コスミド; 染色体移入

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.

这项研究的目的是确定人类遗传性疾病共济失调毛细血管扩张症（AT）的基因，其特征是对电离辐射的敏感性增强。一个AT衍生的微细胞杂种，2859/4-1，在人类染色体11 q23处有一个缺失，伴随着辐射抗性的丧失。通过微细胞融合法获得了携带该11 q23缺失染色体的小鼠细胞株，并使用IRS（interspersed repeatable sequence）-PCR分析了缺失区域。在AT位点附近发现了两个PCR标记.使用具有人X/11染色体A9（3552）-2的小鼠细胞株来产生携带微小的人11 q23片段的辐射杂交体。将11 q23片段导入AT细胞后，发现AT基因包含在一个杂种RH 12/1中，而不包含在另外两个杂种MH 12/1和MH 12/3.3中。进一步分析了这三个杂种在11 q23处多态性DNA标记的存在。在三个杂交种之间没有发现标记谱的差异，表明候选区域低于已知DNA标记的分辨率。另一个有趣的是，RH 12/1没有D11 S384，这应该是最接近AT的标记，这表明一个新的可能性，AT基因可能位于端粒D11S384.4。从RH 12/1构建粘粒文库。在256个人源性克隆中，21个克隆起源于11号染色体。在21例正常人中未发现AT基因。利用IRS-PCR技术，分离到一个新的质粒pBM 8.9，并定位在RH 12/1特异性区域，这意味着pBM 8.9可能是一个强有力的候选标记，以指定的cosmoparavirus AT位点。

项目成果

M. V. Kato: "Parental Origin of germ-line and somatic mutations in the retinoblastoma gene" Human Genetics. 94. 31-38 (1994)

M. V. Kato：“视网膜母细胞瘤基因种系和体细胞突变的父母起源”人类遗传学。

Y.EJIMA: "Ataxia-Telangiectasia" Springer-Verlag,Berlin(R.A.Gatti and R.B.Painter,Eds.), 11 (1993)

Y.EJIMA：“共济失调-毛细血管扩张症”施普林格出版社，柏林（R.A.Gatti 和 R.B.Painter，编辑），11 (1993)

Y. Ejima: "Determination of the chromosomal site for the human radiosensitive ataxia telangiectasia gene by chromosome transfer" Mutation Research. 250. 337-343 (1991)

Y. Ejima：“通过染色体转移测定人类放射敏感性共济失调毛细血管扩张基因的染色体位点”突变研究。

Y.Ejima: "Ataxia-telangiectasia" Springer-Verlag (R. A. Gatti and R. B. Painter Eds.), 283 (1993)

Y.Ejima：“共济失调-毛细血管扩张症”Springer-Verlag（R. A. Gatti 和 R. B. Painter 编辑），283 (1993)

K. Ishizaki: "Increased UV-induced SCEs but normal repair of DNA damage in p53-deficient mouse cells" Int. J. Cancer. 58. 254-257 (1994)

K. Ishizaki：“p53 缺陷小鼠细胞中紫外线诱导的 SCE 增加，但 DNA 损伤修复正常” Int。