| Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody
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Author:
Date:
2017-05-20
[Abstract] Eukaryotic nuclear DNA wraps around histone proteins to form a nucleosome, a basic unit of chromatin. Posttranslational modification of histones plays an important role in gene regulation and chromosome duplication. Some modifications are quite stable to be an epigenetic memory, and others exhibit rapid turnover or fluctuate during the cell cycle. Histone H4 Lys20 monomethylation (H4K20me1) has been shown to be involved in chromosome condensation, segregation, replication and repair. H4K20 methylation is controlled through a few methyltransferases, PR-Set7/Set8, SUV420H1, and SUV420H2, and a demethylase, PHF8. In cycling cells, the level of H4K20me1 increases during G2 and M phases and decreases during G1 phase. To monitor the local concentration and global fluctuation of histone ...
[摘要] 真核核DNA包裹组蛋白，形成核小体，是染色质的基本单位。组蛋白的翻译后修饰在基因调控和染色体重复中起重要作用。一些修饰是相当稳定的，作为表观遗传记忆，其他修饰在细胞周期中表现出快速更替或波动。组蛋白H4 Lys20单甲基化（H4K20me1）已显示参与染色体凝聚，分离，复制和修复。通过几种甲基转移酶PR-Set7 / Set8，SUV420H1和SUV420H2以及脱甲基酶PHF8控制H4K20甲基化。在循环细胞中，H4K20me1的水平在G2期和M期增加，G1期下降。为了监测活细胞中组蛋白修饰的局部浓度和全局波动，我们开发了一种基因编码的探针，称为薄荷素（修饰特异性细胞内抗体; Sato等人，2013和2016）。通过测量核细胞与细胞质的强度比，可以监测单个细胞中H4K20me1的相对水平。该详细方案允许甲基转移酶对基于HatoK等人的质粒H4K20me1-mintbody活性细胞中H4K20me1水平的影响进行半定量分析（2016）。
背景 ...
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| Analysis of Developing Pollen Grains within Intact Arabidopsis thaliana Anthers by Olympus Two-Photon Laser Scanning Microscopy
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Author:
Date:
2015-12-05
[Abstract] The method consists of imaging developing pollen grains as they form within intact, immature Arabidopsis thaliana anthers. Using two-photon excitation in the infrared wavelength range, the intrinsic fluorescence (autofluorescence) of developing pollen grains and surrounding sporophytic tissues of the anther wall, including the tapetum, middle layer, endothecium and epidermis, can be visualized in the three-dimensional space of an intact anther. In contrast to conventional confocal microscopy, the application of red-shifted light by two-photon microscopy improves depth penetration into specimens, while the scattering of light and subsequent phototoxicity is minimized, making this a superior method for imaging the developing pollen grains and tapetal cells enclosed within anthers. ...
[摘要] 该方法包括当它们在完整的未成熟拟南芥花药内形成时使发育的花粉粒成像。使用在红外波长范围内的双光子激发，发育花粉颗粒和花药壁的周围孢子体组织（包括绒毡层，中间层，内皮和表皮）的固有荧光（自发荧光）可以在三维空间的完整的花药。与传统的共聚焦显微镜相反，通过双光子显微镜应用红移光改善深度穿透到标本，同时光的散射和随后的光毒性最小化，使这是成像发展中的花粉粒和绒毡层细胞的优良方法封闭在花药内。所描述的技术被优化用于检测花粉壁的自体荧光组分，包括花粉球蛋白和花粉外壳，并且提供关于野生型和花粉壁突变植物的花药中的自发荧光代谢物的空间和发育数据（Quilichini等al。，2014）。活的，完整的花药的双光子成像的使用具有潜在的未来研究，目的在于了解花粉发育期间配子体和孢子体组织之间的空间关系以及代谢物或荧光标记的蛋白质在发育的花药内的分布。
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| Novel Method for Site-specific Induction of Oxidative DNA Damage to Study Recruitment of Repair Proteins to Heterochromatin and Euchromatin
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Author:
Date:
2014-06-05
[Abstract] ROS-induced DNA damage is repaired in living cells within a temporal and spatial context, and chromatin structure is critical to a consideration of DNA repair processes in situ. It’s well known that chromatin remodeling factors participate in many DNA damage repair pathways, indicating the importance of chromatin remodeling in facilitating DNA damage repair. To date, there has been no method to induce site-specific oxidative DNA damage in living cells. Therefore, it is not known whether the DNA repair mechanisms differ within active or condensed chromatin. We recently established a novel method, DTG (Damage Targeted at one Genome-site), to study DNA damage response of reactive oxygen species (ROS)-induced DNA damage in living cell at one genome loci with active or inactive ...
[摘要] ROS诱导的DNA损伤在时间和空间背景下在活细胞中修复，并且染色质结构对于原位DNA修复过程的考虑是关键的。众所周知，染色质重塑因子参与许多DNA损伤修复途径，表明染色质重塑促进DNA损伤修复的重要性。到目前为止，还没有方法诱导活细胞中的位点特异性氧化性DNA损伤。因此，不知道DNA修复机制在活性或凝集的染色质中是否不同。我们最近建立了一种新的方法，DTG（损害靶向一个基因组位点），研究活性氧（ROS）诱导的DNA损伤活动细胞中的DNA损伤反应在一个基因组活性或无活性转录。为此，我们在U2OS细胞中在X染色体上整合了四环素响应元件（TRE）盒（〜90kb）（Lan等人，2010），然后融合KillerRed（KR）刺激的ROS诱导物，其可以特异性产生ROS诱导的DNA损伤，tet-阻遏物（tetR-KR，OFF）或转录激活物（TA-KR，ON）（Lan等人， ，2014）（图1）。 TetR-KR或TA-KR分别结合TRE盒并在异源或真核细胞状态下诱导ROS损伤。如何染色质状态调节DNA损伤反应过程可以通过使用这种强大的方法来检查。
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