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

点击翻译按钮获取中文摘要

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.