| Project/Area Number |
13670788
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pediatrics
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| Research Institution | Kanazawa university |
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Principal Investigator |
SAIKAWA Yutaka Kanazawa university hospital, Pediatrics, Assistant Professor, 医学部附属病院, 講師 (60283107)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Human hemeoxygenase-1 / hereditary disease / gene correction / Alu repeat / homologous recombination / site-specific recombinase / ヒトヘムォキシゲナーゼ1遺伝子 |
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| Research Abstract |
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
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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
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