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

• Antragstellerin: Dr. Elizabeth Schroeder-Reiter
• Fachliche Zuordnung: Genetik und Genomik der Pflanzen
• Förderung: Förderung von 2007 bis 2014
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 50971437

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.

• DFG-Verfahren: Sachbeihilfen
• Beteiligte Person: Professor Dr. Gerhard Wanner