Establishment of simultaneous and quantitative assay for DNAbinding and transcriptional activity of glucocorticoid receptor.

糖皮质激素受体 DNA 结合和转录活性同步定量测定的建立。

• 批准号: 23770169
• 负责人: MIKUNI Shintaro
• 金额: $ 2.91万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23770169/
• 关键词: タンパク質; 核酸の構造・動態・機能; 転写因子; バイオイメージング; 分子間相互作用; 生物物理; 核酸; 蛋白質; 転写活性; 相関解析; 相互作用; 一分子計測; FCS; FCCS; 核内受容体; DNA

Regulation of transcriptional activity could be initiated by associating the transcriptional factors with its specific sequence of genomic DNA. However, the direct relationship between “the affinity for DNA” and “transcriptional activity” of transcriptional factors is not clarified. To clarify this relationship, we tried to establish the simultaneous and quantitative assay for DNA binding and transcriptional activity of glucocorticoid receptor (GR), which is one of transcriptional factors, using fluorescence cross-correlation spectroscopy (FCCS). As a result, the dissociation constant between the purified GR fused with EGFP and the Alexa647-labeled GR specific sequences (glucocorticoid response element, GRE) could be determined as 0.63 μM, and sequence-specificity of GR could be confirmed by FCCS. Moreover, the dissociation constant between the purified RNA-specific binding protein fused withmCherry (red fluorescent protein) and Alexa488-labeled RNA was also determined as 143 nM by FCCS.

转录活性的调节是由具有基因组DNA的特定序列的转录因子开始的。使用荧光互相关光谱（FCCS），与EGFP融合的转录因子的糖皮质激素受体的旋转活性。可以通过FCC确认。

项目成果

Analysis for the relationship of intranuclear diffusional landscape and transcriptional activity of glucocorticoid receptor

糖皮质激素受体核内扩散景观与转录活性关系分析

• 发表时间: 2011
• 作者: Arai Y;Nagai T.;Shintaro Mikuni
• 通讯作者: Shintaro Mikuni

Determination of dissociation constant between glucocorticoid receptor and DNA using FCCS

使用 FCCS 测定糖皮质激素受体与 DNA 之间的解离常数

• 发表时间: 2012
• 作者: 木塚康彦;北爪しのぶ;谷口直之;Mikuni S and Kinjo M
• 通讯作者: Mikuni S and Kinjo M

細胞機能科学研究室

细胞功能科学实验室

Training course for FCS and FCCS Fluorescence Microscopy: from Basics toAdvanced

FCS 和 FCCS 荧光显微镜培训课程：从基础知识到高级

• 发表时间: 2012
• 作者: Yamada M;Kumamoto K;Mikuni S;Arai Y;Kinjo M;Nagai T;Tsukasaki Y;Watanabe TM;Fukui M;Jin M;Toba S;Hirotsune S.;水谷武臣;Kinjo M and Mikuni S
• 通讯作者: Kinjo M and Mikuni S

Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate

使用三色荧光蛋白底物监测单个凋亡细胞中的半胱天冬酶级联

• DOI: 10.1016/j.bbrc.2010.12.047
• 发表时间: 2011
• 期刊: Biochemical and Biophysical Research Communications
• 影响因子: 3.1
• 作者: Fan Sun;Shintaro Mikuni;Masataka Kinjo
• 通讯作者: Masataka Kinjo