{"created":"2023-07-27T06:55:01.226991+00:00","id":50649,"links":{},"metadata":{"_buckets":{"deposit":"8cfe26a2-04f7-407b-889e-51520f94dfc2"},"_deposit":{"created_by":18,"id":"50649","owners":[18],"pid":{"revision_id":0,"type":"depid","value":"50649"},"status":"published"},"_oai":{"id":"oai:kanazawa-u.repo.nii.ac.jp:00050649","sets":["2812:2813:2831"]},"author_link":["25440","74433"],"item_9_biblio_info_8":{"attribute_name":"書誌情報","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2004-04-13","bibliographicIssueDateType":"Issued"},"bibliographicPageStart":"2p.","bibliographicVolumeNumber":"2001-2002","bibliographic_titles":[{"bibliographic_title":"平成14(2002)年度 科学研究費補助金 基盤研究(C) 研究成果報告書概要"},{"bibliographic_title":"2002 Fiscal Year Final Research Report Summary","bibliographic_titleLang":"en"}]}]},"item_9_creator_33":{"attribute_name":"著者別表示","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{}],"nameIdentifiers":[{},{},{}]}]},"item_9_description_21":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"HO-1欠損症に認められたAlu配列を介したゲノム遺伝子欠失の機序解明を目的として以下の研究を進めた。\n1.HO-1ゲノム遺伝子のイントロンおよびAlu配列内に存在するトポイソメラーゼII結合配列の機能解析。\n相同組み換え修復の開始には二本鎖DNAの切断が必要である。HO-1ゲノム遺伝子のエクソン2を挟むAluおよびイントロン配列にはトポイソメラーゼIIコンセンサス結合配列が複数存在する。これらの中から機能的結合部位を同定するために、トポイソメラーゼII阻害剤を用いた二本鎖DNA切断点の検索を行った。HO-1^<+/+>LCLを阻害剤(VP-16,doxorubicin)添加培養液で処理し、内因性のトポイソメラーゼIIを阻害することによって生じたDNA断片をligation-mediated PCR(LM-PCR)にて増幅した。阻害剤処理ゲノムDNA特異的に複数の増幅バンドが得られ、現在塩基配列を解析中である。\n2.Recombination hotspotの機能解析\nエクソン欠失後のAlu配列の再結合部位には36bpの保持された塩基配列とそれにオーバラップするAluコア配列が認められた(計49bp)。塩基配列および構造の特徴から部位特異的組み換え酵素による欠失パターン、特にFlp/FRT systemとの類似性が示唆された。そこで、再結合部位の塩基配列を種々に改変した49bpのDNAプローブを作成し、結合する蛋白の存在を計10種類のヒト癌細胞株およびマウス胎児細胞株由来の核蛋白を用いてゲルシフト法にて検索した。Aluコア配列を含む逆位配列構造を有する改変DNAプローブに特異的に結合する核蛋白が、複数のヒト癌細胞株およびマウス胎児線維芽細胞(NIH3T3)に検出された。さらにNIH3T3を大量培養し、この結合蛋白の部分精製を開始した。第一段階として、DNA結合蛋白の共通の特性を利用し、ヘパリンカラムによる分画を行った。分画中の蛋白の特異的DNA結合性をゲルシフト法で確認し、陽性分画をプール後、-80℃で保存した。第二段階の精製にはストレプトアビジンアフィニティーカラムにビオチン標識プローブを結合させ、蛋白のプローブへの特異的結合性を用いて精製する。得られた精製蛋白はSDS-PAGEまたはnative gelで分離後、PVDF膜に転写する。クマシーブリリアントブルーで蛋白を染色し、分子量を測定する。得られた蛋白バンドを膜とともに切り出し、プロテアーゼ処理後、生成した断片化ペプチドを質量分析計(MALDI-TOF/MS)で測定する。得られた質量値をもとに、データーベース(MS-Fit, http://falcon.ludwig.ucl.ac.uk/)に登録されている蛋白質の同定をおこなう予定である。","subitem_description_type":"Abstract"},{"subitem_description":"To investigate the pathomechanisms of Alu-mediated genomic deletion observed in a case with hemeoxygenase-1 (HO-1) deficiency, following analyses have been performed ;\n1. Functional analyses of topoisomerase II-binding sites (TBSs) located in the introns and Alu repeats of the human HO-1 gene.\nThe 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 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.\n2. Functional analyses of the hotspot sequence within the Alu-associated recombination site of the HO-1 gene in the case of HO-1 deficiency.\nI 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.","subitem_description_type":"Abstract"}]},"item_9_description_22":{"attribute_name":"内容記述","attribute_value_mlt":[{"subitem_description":"研究課題/領域番号:13670788, 研究期間(年度):2001-2002","subitem_description_type":"Other"},{"subitem_description":"出典：研究課題「ヒトヘムオキシゲナーゼ1遺伝子変異に対する遺伝子修復治療への基礎的検討」課題番号13670788\n（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） \n（https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-13670788/136707882002kenkyu_seika_hokoku_gaiyo/）を加工して作成","subitem_description_type":"Other"}]},"item_9_description_5":{"attribute_name":"提供者所属","attribute_value_mlt":[{"subitem_description":"金沢大学医薬保健研究域医学系","subitem_description_type":"Other"}]},"item_9_identifier_registration":{"attribute_name":"ID登録","attribute_value_mlt":[{"subitem_identifier_reg_text":"10.24517/00056958","subitem_identifier_reg_type":"JaLC"}]},"item_9_relation_28":{"attribute_name":"関連URI","attribute_value_mlt":[{"subitem_relation_name":[{"subitem_relation_name_text":"https://kaken.nii.ac.jp/search/?qm=60283107"}],"subitem_relation_type_id":{"subitem_relation_type_id_text":"https://kaken.nii.ac.jp/search/?qm=60283107","subitem_relation_type_select":"URI"}},{"subitem_relation_name":[{"subitem_relation_name_text":"https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670788/"}],"subitem_relation_type_id":{"subitem_relation_type_id_text":"https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670788/","subitem_relation_type_select":"URI"}},{"subitem_relation_name":[{"subitem_relation_name_text":"https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-13670788/136707882002kenkyu_seika_hokoku_gaiyo/"}],"subitem_relation_type_id":{"subitem_relation_type_id_text":"https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-13670788/136707882002kenkyu_seika_hokoku_gaiyo/","subitem_relation_type_select":"URI"}}]},"item_9_version_type_25":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_ab4af688f83e57aa","subitem_version_type":"AM"}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2022-05-19"}],"displaytype":"detail","filename":"HO-PR-SAIKAWA-Y-kaken 2004-2p.pdf","filesize":[{"value":"91.2 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"HO-PR-SAIKAWA-Y-kaken 2004-2p.pdf","url":"https://kanazawa-u.repo.nii.ac.jp/record/50649/files/HO-PR-SAIKAWA-Y-kaken 2004-2p.pdf"},"version_id":"1daca6aa-7176-4470-a64a-846361516030"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"research report","resourceuri":"http://purl.org/coar/resource_type/c_18ws"}]},"item_title":"ヒトヘムオキシゲナーゼ1遺伝子変異に対する遺伝子修復治療への基礎的検討","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"ヒトヘムオキシゲナーゼ1遺伝子変異に対する遺伝子修復治療への基礎的検討"},{"subitem_title":"Analysis of Alu-mediated genomic deletion in a case with hemeooxygenase-1 deficiency","subitem_title_language":"en"}]},"item_type_id":"9","owner":"18","path":["2831"],"pubdate":{"attribute_name":"公開日","attribute_value":"2022-05-19"},"publish_date":"2022-05-19","publish_status":"0","recid":"50649","relation_version_is_last":true,"title":["ヒトヘムオキシゲナーゼ1遺伝子変異に対する遺伝子修復治療への基礎的検討"],"weko_creator_id":"18","weko_shared_id":-1},"updated":"2023-07-27T13:05:26.228616+00:00"}