Large Scale Chromatin Structure

大规模染色质结构

• 批准号: 8157349
• 负责人: james g mcnally
• 金额: $ 5.35万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157349
• 关键词: Caliber; Chromatin Structure; Cytoplasm; DNA; DNA Repair; DNA Structure; Detection; Higher Order Chromatin Structure; Mammalian Cell; Resolution; Three-Dimensional Image; imaging modality

Large-scale chromatin structure is the higher-order folding of chromatin on the scale of several hundred kilobases. Little is known about this folding, but the structures must influence transcription, replication and repair of DNA. To learn more about these structures, we previously examined transcriptionally active domains of 400 - 2000 kb by light microscopy. We found that several different domains exhibit similar structures in light microscope images, namely a series of adjacent puncta or "beads" approximately 0.5 microns in diameter. When these same domains are transcriptionally inactive, only one punctum or "bead" is detected. Thus transcription induces a decondensation from single beaded to multi-beaded structures. To observe these structures at higher resolution we are exploring the applicability of x-ray microscopy. This imaging modality enables visualization of whole, unsectioned mammalian cells at 25 nm resolution. Detection of chromatin relies on the natural contrast afforded by the absorption of organic matter relative to water. Our goal is to determine if chromatin structure can be detected by this approach and if so, then to provide a 3D image of nuclear chromatin at 25 nm resolution. Our current results suggest that there is not sufficient contrast to visualize chromatin, at least by transmission x-ray microscopy. However, we have obtained strikingly detailed images of the cytoplasm by the same approach. We can now clearly visualize all membrane structures in the cell by x-ray microscopy down to 30 nm resolution. The advantage of this new approach is that the method achieves this resolution for intact, unstained 3D specimens, thereby providing an unprecedented 3D view of cellular architecture.

大规模染色质结构是几百千倍酶的色质的高阶折叠。关于这种折叠知之甚少，但是结构必须影响DNA的转录，复制和修复。为了了解有关这些结构的更多信息，我们先前通过光学显微镜检查了400-2000 kb的转录活性域。我们发现，几个不同的域在光学显微镜图像中表现出相似的结构，即直径约0.5微米的一系列相邻点或“珠子”。当这些相同的域在转录上无效时，仅检测到一个点击量或“珠”。因此，转录引起了从单个串珠到多珠结构的反应。 为了在较高的分辨率下观察这些结构，我们正在探索X射线显微镜的适用性。这种成像方式使以25 nm分辨率的整体，无调的哺乳动物细胞可视化。染色质的检测取决于有机物相对于水的吸收带来的自然对比。我们的目标是确定该方法是否可以检测到染色质结构，如果是这样，则在25 nm分辨率下提供核染色质的3D图像。我们目前的结果表明，至少通过透射X射线显微镜可以看到染色质，没有足够的对比。但是，我们通过相同的方法获得了细胞质的惊人详细图像。现在，我们可以通过X射线显微镜降至30 nm的分辨率来清楚地清楚地看到细胞中的所有膜结构。这种新方法的优点是，该方法可以实现完整，未染色的3D样品的分辨率，从而提供了前所未有的3D蜂窝架构视图。