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

为了研究在血红素加氧酶-1 （HO-1）缺乏症中观察到的铝介导的基因组缺失的病理机制，我们进行了以下分析：位于人类HO-1基因内含子和Alu重复序列的拓扑异构酶ii结合位点（tbs）的功能分析。铝介导的同源重组在一些遗传性疾病和癌症中的作用已被确立为一种病理机制。由于染色体双链断裂（DSBs）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重复序列中发现的潜在tbs可能是同源重组的靶位点。为了验证这一假设，进行了HO-1基因中功能性tbs的测定。用拓扑异构酶II抑制剂（VP-16和阿霉素）治疗从正常对照建立的LCLs。抑制内源性拓扑异构酶II产生的DNA片段导致dsb的产生，并利用连接介导的PCR技术扩增。在抑制剂处理的lcl中特异性地测定了几种PCR产物。目前正在对模具产品进行测序，以确定dsb的位置。HO-1缺乏症患者HO-1基因alu相关重组位点热点序列功能分析我在HO-1基因缺失位点发现了由保守的36bp Alu序列和Alu核心序列（重组热点）组成的独特反转序列（49 bp）。这种结构特征显示了Flp/FRT系统（酵母中位点特异性重组酶系统）的相似性。为了探索人类系统中新的位点特异性重组酶，设计了几个序列变异的合成寡核苷酸（49 bp）并用于凝胶转移试验。在人类癌细胞系和小鼠胚胎细胞系NIH3T3的核提取物中，发现了特异性结合探针ATS2.2的蛋白，该蛋白含有倒置重组热点序列。我打算用肝素柱层析和亲和层析来部分纯化这些蛋白质。部分纯化的蛋白将用SDS-PAGE测定分子量进行表征，并将用MALDI-TOF/MS进行分析。少

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