Controlling Phase separation and Higher Order Structure of Chromosomal DNA Molecules

控制染色体 DNA 分子的相分离和高阶结构

• 批准号: 14598002
• 负责人: OANA Hidehiro
• 金额: $ 2.3万
• 依托单位: The University of Tokyo (2003)Kyoto University (2002)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14598002/
• 关键词: DNA; chanize of higher order structure; phase separation in single molecule; optical tweezers; fluorescence microscopy; spermine

The biochemical characteristics of lambda DNA chains in folded/unfolded states upon cleavage by the restriction enzyme ApaLI were investigated in the presence of spermine. These characteristics of DNA chains depending on their higher-order structure were studied at the single-molecule level using fluorescence microscopy. With a low concentration of spermine, lambda DNA takes a random coiled conformation and allows digestion by the enzyme, while under a high concentration of spermine, lambda DNA takes a compact folded structure and inhibits such attack. Together with comparative experiments on short oligomeric DNA, our results suggest that the transition in the higher-order structure causes on/off type switching of sensitivity to the enzyme. In addition, we proposed a noninvasive methodology for manipulating single Mb-size whole-genome DNA molecules. Cells were subjected to osmotic shock and the genorne DNA released from the burst cells was transferred to a region of higher salt concentration using optical tweezers. The trapped genome DNA exhibits a conformational transition from a condensed state to an elongated state, accompanied by the change in its environment. The applicability of optical tweezers to the on-site manipulation of giant genome DNA is suggested, i.e., lab-on-a-plate.

在精胺存在下，研究了限制性内切酶ApaLI切割后折叠/未折叠状态的λ DNA链的生化特性。利用荧光显微镜在单分子水平上研究了DNA链的这些特征，这些特征取决于它们的高阶结构。在低浓度的精胺下，λ DNA采取随机卷曲构象并允许酶消化，而在高浓度的精胺下，λ DNA采取紧凑的折叠结构并抑制这种攻击。再加上短的寡聚DNA的比较实验，我们的研究结果表明，在高阶结构的过渡导致对酶的敏感性的开/关型切换。此外，我们提出了一种非侵入性的方法来操纵单个Mb大小的全基因组DNA分子。使细胞经受渗透压休克，并使用光学镊子将从爆发细胞释放的基因组DNA转移到较高盐浓度的区域。被捕获的基因组DNA表现出从凝聚态到伸长态的构象转变，伴随着其环境的变化。提出了光镊在大基因组DNA现场操作中的应用，即，平板实验室

项目成果

Oana, Hidehiro et al.: "Folding transition of large DNA completely inhibits the action of a restriction endonuclease as revealed by single-chain observation"FEBS Letters. 532. 143-146 (2002)

Oana、Hidehiro 等人：“单链观察表明，大 DNA 的折叠转变完全抑制限制性内切酶的作用”FEBS Letters。

Oana, Hidehiro et al.: "Folding transition of large DNA completely inhibits the action of a restriction endonuclease as revealed by single-chain observation"FEBS Letters. 530. 143-146 (2002)

Oana、Hidehiro 等人：“单链观察表明，大 DNA 的折叠转变完全抑制限制性内切酶的作用”FEBS Letters。

小穴英廣ら: "DNAの折り畳み移転と遺伝子活性"バイオサイエンスとインダストリー. 61. 163-168 (2003)

Hidehiro Koana 等人：“DNA 折叠转移和基因活性”《生物科学与工业》61. 163-168 (2003)。

Oana, Hidehiro et al.: "Folding transition of DNA and gene activity"Biscience & Industry. 61. 163-168 (2003)

Oana、Hidehiro 等人：“DNA 的折叠转变和基因活性”Biscience

Y.Yoshikawa et al.: "Ascorbic acid induces a marked conformational change in long duplex DNA"Eur.J.Biochem.. 270. 3101-3106 (2003)

Y.Yoshikawa 等人：“抗坏血酸诱导长双链 DNA 中显着的构象变化”Eur.J.Biochem.. 270. 3101-3106 (2003)