3-DIMENSIONAL ORGANIZATION OF RIBOSOMAL DNA IN INTERPHASE NUCLEI OF PISUM-SATIVUM BY INSITU HYBRIDIZATION AND OPTICAL TOMOGRAPHY

The three-dimensional (3D) organization of rDNA-containing chromatin was studied in structurally well preserved, interphase nuclei of Pisum sativum root tips by in situ hybridization using a biotinylated cDNA probe to the 18, 5.8 and 25 S rDNA sequences. The probe was detected by immunofluorescence and optical section images recorded either by video imaging or by using a confocal laser scanning microscope. Detailed 3D reconstructions were made of 12 nucleoli by projection of confocal optical sections. The probe labelled four perinucleolar heterochromatin sites, one pair 1.0-2.1 μm in diameter and the other 0.5-1.0 μm diameter. It also labelled intranucleolar structures including 300-500 nm spots emanating from the perinucleolar sites into the body of the nucleolus. The intranucleolar labelled structures emanating from the perinucleolar sites lay in discrete domains. Medium power observations of 22 fields of cells (6-30 cells per field) were made by optical sectioning using a video camera and computer deblurring. The arrangement of the perinucleolar sites was modelled in each cell and the arrangements examined for nonrandomness. The sites tended to be spaced out around the nucleolar periphery approximating a regular tetrahedral arrangement as if to minimize clustering and the large sites appeared to lie in a plane perpendicular to the root axis. Cells with multiple nucleoli did not have any preferred distribution of sites between nucleoli. These observations are discussed in terms of current models of rDNA organization. © 1990 Springer-Verlag.