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ナノ可視化による染色体構造に関する研究

CHANNARONG, SARTSANGA 神戸大学

2022.03.25

概要

令和4年2月18日10:40-12:00にZoomによる公開ゼミにて、CHANNARONG SARTSANGA氏の本論文審査報告会を実施し、同日12:00-12:30に本論文審査委員5人による審査を行った。

本論文の「Studies of chromosome structure by nano-visualization(ナノ可視化による染色体構造に関する研究)」は、14,0年前の発見以来、未知の構造体である染色体について、最新のナノ可視化を可能にする顕微鏡分析法を用いて、植物染色体の内部および外部構造を解明するという最先端の研究である。顕微鏡分析法ならびに画像解析の開発を行って、微細な染色体構造解明に迫る試みである。研究の新規性としては、ナノレベルでの高解像が期待できるヘリウムイオン顕微鏡(HIM)、走査型電子顕微鏡(SEM)、高圧電子顕微鏡(HVTEM)、および超高圧電子顕微鏡(UHVTEM)という可視光ではない高度な顕微鏡を使用して、植物染色体の外部および内部のクロマチン構造を解析することに世界で初めて成功したことである。

本論文の主要構成は次の5点である。1)生物試料としてこれまで研究が蓄積されてきたオオムギ染色体試料について、高収量で高品質の染色体調整法の開発に成功したこと、2)ヘリウムイオンを線源とする顕微鏡であるヘリウムイオン顕微鏡(HIM)による染色体外部のクロマチン構造の解明を行ったこと、3)走査型電子顕微鏡(SEM)によるカルシウムイオンの染色体凝縮についての効果を解明したこと、4)クロマチンスキャッフォールドタンパク質(TopoIIα)の染色体局在性の解明を行ったこと、5)内部浸透度が高く2Aの解像度が期待できる2種類の高圧電子顕微鏡(HVTEM)ならびに超高圧電子顕微鏡(UHVTEM)を用いた、テロメア、仁形成領域、動原体等の染色体機能部位のクロマチン構造に着目したTopollαの三次元構造の解明を行ったことである。

本論文の第1章は緒論、第2章では、脱水処理を避けた改良型染色体調整法として、高度に同調化(58%)させることに成功し、31Chromosome/μLという高収率で染色体を単離する技術を開発した。イオン液体コーティングを施した自然状態に極めて近いオオムギ染色体のHIM像の取得と画像解析の結果、染色体表面のクロマチン繊維のサイズは11.6士3.511111であり、Piskadlo et al.(2016)の提唱するクロマチンネットワークモデルと一致したことを証明した。また、染色体機能部位である染色体末端と動原体付近で凝縮度が異なることを明らかにした。第3章では、2価カルシウムイオンの染色体凝縮への効果について検証し、カルシウムイオン除去で染色体が弛緩すること、染色体の垂直方向ではI.75倍に伸張すること、並行方向についても弛緩する構造変化を明らかにした。第4章では、オオムギのスキャッフォールドタンパク質(Topolla)抗体を作製し、免疫染色法を用いてTopollαの局在を明らかにした。そしてTopollαは染色体腕に沿って分布すること、仁形成領域および動原体領域に高度に集中していること、それが機能と関連していると議論した。また、Topollαを選択的に染色するナノゴールド免疫染色法を植物染色体に初めて適応し、HVTEMでTopoilαを検出するための有用な手段となることを証明した。第5章では、HVTEMでTopollαの二次元染色体局在解析を行い、UHVTEMで三次元構造解析を試みている。

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