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Strukture and function of plant and mammalian chromosomes: High resolution scanning electron microscopic structural analysis and localization of DNA and proteins with heavy metal staining and immunogold labeling

植物和哺乳动物染色体的结构和功能：利用重金属染色和免疫金标记对 DNA 和蛋白质进行高分辨率扫描电子显微镜结构分析和定位

基本信息

批准号：
50971437
负责人：
Dr. Elizabeth Schroeder-Reiter
金额：
--
依托单位：
Department Biologie II (aufgelöst)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2007
资助国家：
德国
起止时间：
2006-12-31 至 2013-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/50971437?language=en
关键词：
Strukture function plant mammalian chromosomes

项目摘要

High resolution scanning electron microscopy (SEM) offers the possibility for investigation of chromosome topography from low magnification surveys down to the ultrastructural level of 10 nm elementary fibril and, under best conditions, to the level of DNA (2 nm). A new application for investigation of chromosome architecture, focused ion beam milling in combination with low-voltage field emission scanning electron microscopy (FIB/FESEM), allows direct visualization of chromosome interior for the first time in addition to 3D reconstruction of structures from serial FIB sections. With these and additional analytical techniques, such as metal impregnation and immuno(nano)gold labeling, it is possible to compare structural and functional elements of chromatin, offering a valuable approach to combined ultrastructural and immunocytological questions. Global structures common to eukaryotic chromosomes will continue to be characterized by investigating structural chromosome variants (chromosomes from different organisms – plant and animal, chromosomes of different sizes, centromere variants, nucleolus organizing regions/NORs). In particular, histone variants and DNA-sequences will be investigated in the centromere and NOR regions in order to elucidate how functionally defined chromatin and epigenetic elements are manifested in chromosomal substructural details. Structural analysis of Drosophila melanogaster polytene chromosomes in comparison with those from mutants for chromosome structure are planned, as well as optimizing techniques for isolation of Arabidopsis thaliana and human chromosomes. Current techniques for chromosome isolation, metal impregnation, immunogold detection and in situ hybridization will be further optimized to improve labeling efficiency under conditions that best preserve chromosome substructures and that are conducive to highest possible resolution.

高分辨率扫描电子显微镜（SEM）提供了从低放大率调查到10 nm基本原纤维的超微结构水平，并在最佳条件下，到DNA水平（2 nm）的染色体地形调查的可能性。一个新的应用程序的染色体结构的调查，聚焦离子束铣削结合低压场发射扫描电子显微镜（FIB/FESEM），允许直接可视化的染色体内部的第一次除了从连续FIB部分的结构的三维重建。通过这些和其他分析技术，如金属浸渍和免疫（纳米）金标记，可以比较染色质的结构和功能元素，为结合超微结构和免疫细胞学问题提供有价值的方法。真核生物染色体共有的全局结构将继续通过研究结构染色体变体（来自不同生物体的染色体-植物和动物，不同大小的染色体，着丝粒变体，核仁组织区/NOR）来表征。特别是，组蛋白变体和DNA序列将在着丝粒和NOR区域进行研究，以阐明功能定义的染色质和表观遗传元件如何在染色体亚结构细节中表现出来。果蝇多线染色体的结构分析与染色体结构的突变体相比，以及优化技术分离拟南芥和人类染色体的计划。目前的染色体分离，金属浸渍，免疫金检测和原位杂交技术将进一步优化，以提高标记效率的条件下，最好地保存染色体亚结构，并有利于尽可能高的分辨率。