6th ESACP Congress, Heidelberg, April 7-11, 1999

A044
ELECTRON-MICROSCOPIC TOMOGRAPHY OF SILVER-STAINED INTERPHASE AND METAPHASE NUCLEOLAR ORGANIZER REGIONS
Cheutin T, O'Donohue MF, Kaplan H, Lucas L, Beorchia A, Thiry M, Ploton D

Unité Médian, EA 2063, IFR 53, Reims, France

Amplified rDNA genes (NORs) represent a good model to study the supramolecular volumic organization of active genes (interphase) or inactive genes (mitosis). Due to the presence of argyrophilic proteins (Ag-NOR proteins) in the vicinity of rDNA genes, it is possible to evidence cytochemically rDNA genes and to study their volumic organization at a high resolution by using electron-microscopic tomography. Our study was performed by using a Scanning and Transmission Electron Microscope (STEM) working at 300 kV which has a better resolution and a higher contrast compared to a conventionnal TEM. Whole mounted, silver-stained cells or 2- to 4-µm thick sections of plastic-embedded silver-stained cells were tilted from -60° to +60° with a pitch of 2° with a eucentric goniometer stage. Images were directly numerized, then aligned and these data were used for tomographic reconstruction (ART method). The reconstructed volume was then used for 3D visualization and for tomographic study with a final resolution of 20 nm. This approach was used to study interphasic NORs within entire cells and to examine metaphasic NORs within thick sections. This work allowed us to obtain for the first time a three-dimensional view of nucleolar transcriptionally active sites and of their inactive counterpart within metaphase NORs at a 20 nm resolution. Such an approach appears extremely useful to provide a detailed analysis of the 3D organization of interphase and metaphase chromatin in situ.