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Analysis of Alu-mediated genomic deletion in a case with hemeooxygenase-1 deficiency

血红素加氧酶 1 缺陷病例中 Alu 介导的基因组缺失分析

基本信息

批准号：
13670788
负责人：
SAIKAWA Yutaka
金额：
$ 1.86万
依托单位：
Kanazawa university
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

To investigate the pathomechanisms of Alu-mediated genomic deletion observed in a case with hemeoxygenase-1 (HO-1) deficiency, following analyses have been performed ;1. Functional analyses of topoisomerase II-binding sites (TBSs) located in the introns and Alu repeats of the human HO-1 gene.The role of Alu-mediated homologous recombination has been established as a pathomechanism in some hereditary diseases and cancers. Since chromosomal double-strand breaks (DSBs) play a crucial role in the homologous recombination processes, potential TBSs found in the introns and Alu repeats, surrounding HO-1 exon 2, could be the target sites of homologous recombination. To test this hypothesis, determination of functional TBSs in the HO-1 gene was performed. HO-1^<+/+> LCLs established from the normal controls were treated with the inhibitors (VP-16 and doxorubicin) of topoisomearase II. The DNA fragments produced by the inhibition of endogenous topoisomerase II resulting in the creation of DSBs w … More ere amplified using a ligation-mediated PCR technique. Several PCR products were determined specifically in the inhibitor-treated LCLs. Sequencing of die products to identify the sites of DSBs are undergoing.2. Functional analyses of the hotspot sequence within the Alu-associated recombination site of the HO-1 gene in the case of HO-1 deficiency.I have found the unique inversion sequences (49 bp) that consist of the conserved 36-bp Alu sequences and Alu core sequences (recombination hotspot) at the deletion site of HO-1 gene. This structural feature showed similarity of the Flp/FRT system (site-specific recombinase system in yeast). To explore a novel site-specific recombinase in the human system, the several synthetic oligonucleotides (49 bp) with sequence variations were designed and used for gel shift assays. In the nuclear extracts derived from human cancer cell lines and a mouse embryonic cell line, NIH3T3, the proteins specifically bound to the probe designated as ATS2.2 that contains the inverted recombination hotspot sequence were identified. I have intended to partially purify these proteins using heparin column chromatography followed by affinity chromatography. The partially purified proteins will be characterized with molecular weight determined by SDS-PAGE and will be analyzed with MALDI-TOF/MS. Less

为探讨血红素加氧酶-1（HO-1）缺乏症患者中观察到的β-内酰胺酶介导的基因组缺失的病理机制，进行了以下分析：1.人HO-1基因内含子和Alu重复序列中拓扑异构酶II结合位点（Topoisomerase II binding sites，TBSs）的功能研究表明，HO-1基因的内含子和Alu重复序列中的TBSs介导的同源重组在某些遗传性疾病和癌症中起重要作用。由于染色体双链断裂（DSB）在同源重组过程中起着至关重要的作用，因此在HO-1外显子2周围的内含子和Alu重复中发现的潜在的TBS可能是同源重组的靶位点。为了检验这一假设，进行了HO-1基因中功能性TBS的测定。用拓扑异构酶II的抑制剂（VP-16和多柔比星）处理从正常对照建立的HO-1^<+/+> LCL。抑制内源性拓扑异构酶II导致DSB产生的DNA片段， ...更多信息使用连接介导的PCR技术扩增。几个PCR产物被确定具体在走廊处理的LCL。正在对产品进行测序，以确定DSB的位点。HO-1基因缺失时，HO-1基因与Alu相关的重组位点内热点序列的功能分析在HO-1基因缺失位点发现了由保守的36 bp Alu序列和Alu核心序列组成的独特的倒位序列（49 bp）（重组热点）。这一结构特征显示了Flp/FRT系统（酵母中的位点特异性重组酶系统）的相似性。为了探索一种新的位点特异性重组酶在人类系统中，几个合成的寡核苷酸（49 bp）的序列变异的设计和用于凝胶位移测定。在来源于人癌细胞系和小鼠胚胎细胞系NIH 3 T3的核提取物中，鉴定了与包含反向重组热点序列的命名为ATS 2.2的探针特异性结合的蛋白质。我打算用肝素柱层析法和亲和层析法部分纯化这些蛋白质。部分纯化的蛋白质将通过SDS-PAGE测定分子量进行表征，并将使用MALDI-TOF/MS进行分析。