Three-dimensional organization of the ribosomal genes and Ag-NOR proteins during interphase and mitosis in PtK1 cells studied by confocal microscopy

Abstract

The three-dimensional (3-D) organization of rDNA-containing chromatin and the set of protein markers of active ribosomal genes, the Ag-NOR proteins, were investigated by confocal laser scanning microscopy (CLSM). The rDNA genes of marsupial cells (PtK1) were mapped using biotinylated DNA probes for 45S rDNA sequences and the Ag-NOR protein distribution was revealed by specific Ag-NOR staining. We used PtK1 cells because each nucleolus possesses only one nucleolar organizer region (NOR). In metaphase chromosomes, nonisotopic in situ hybridization demonstrated the presence of rDNA in the secondary constriction of the X chromosomes with an axial distribution and also lateral expansions. 3-D reconstruction of the Ag-NOR protein signals revealed the presence of these proteins in the secondary constriction where they formed a crescent-shaped structure around the axial chromatin pedicule. The organization of the secondary constriction in PtK1 chromosomes is discussed. During interphase, nonisotopic in situ hybridization in intact cell monolayers and isolated nuclei showed the rDNA genes distributed as intense fluorescent spots linked by weak signals in the inner regions of the nucleoli. We conclude that the rDNA is not homogeneously distributed in the internal regions of the nucleoli. In the same nucleolar regions, the Ag-NOR proteins were revealed as granules linked by thin filaments. These images indicate similar 3-D distributions for rDNA probes and Ag-NOR proteins. The beaded organization of the transcriptional regions in the nucleoli is discussed.

Involvement of in situ conformation of ribosomal genes and selective distribution of upstream binding factor in rRNA transcription.

“…For light and confocal microscopy, cell monolayers were grown on glass slides as described previously (Robert-Fortel et al, 1993).…”

“…It consists of a 6.6-kb DNA fragment, including the downstream half of the 18S and 90% of the 28S rDNA coding sequence. Amplification and labeling of the probe by biotin were previously reported (Robert-Fortel et al, 1993). Probe detection and signal amplification were performed according to Robert-Fortel et al (1993) using three antisera applied successively, the last one being conjugated with Texas Red fluorochrome.…”

“…To understand the organization of rDNA in relation with gene activity, a number of studies have aimed at examining the 3-D distribution of rDNA in nuclei. For example, the 3-D distribution of rDNA in the interphase has been investigated by fluorescence in situ hybridization using confocal microscopy (Leitch et al, 1992;Shaw, 1989, 1990;Highett et al, 1993;Robert-Fortel et al, 1993;Junera et al, 1995) and has revealed that the rDNA exists in discrete 3-D domains, including bright foci (cluster of genes) and genes with a dispersed configuration (Highett et al, 1993;Junera et al, 1995;RobertFortel et al, 1993). A complementary approach has been to correlate this 3-D organization with the nucleolar domains defined at the level of electron microscopy.…”

“…The 3-D distribution of the rDNA in nucleoli appeared heterogeneous, and it was proposed that this heterogeneity depends on the transcriptional activity of the rDNA Shaw, 1989, 1990;Leitch et al, 1992;Highett et al, 1993;Robert-Fortel et al, 1993;Junera et al, 1995;Shaw et al, 1995). Therefore, the distribution of the rDNA transcription machinery would be very important to confirm this hypothesis and to know whether transcription could also be dependent or modulated by the 3-D distribution of the transcription factors.…”

Chromosomal localization of ribosomal DNA sequences in an apple rootstock using a digoxygenin detection system

“…There are two recent reports on karyotype analysis for Arabidopsis which has chromosomes of comparable size but fewer in number (2n=10) than Malus (2n=34) [12,13]. This group used digoxygenin labelling coupled with a fluorescent detection system to determine the chromosomal localization of rDNA (9kb probel and of a smaller tandem repeat sequence(380bp probe)．Although there is a recent report[8l of successful in situ hybridization to chromosomes of a woody gymnosperm (Picea)，there are no such reports for woody angiosperms． This paper describes the use of digoxygenin-labelled apple rDNA sequences(18S coding sequence plus spacer)as a probe along with an enzyme一1inked colour reaction to hybridize to MM106 chromosomes．The position of the nucleolar organiser regions，the site of ribosomal RNA transcription [14,15] was determined by silver staining to provide independent verification of the rDNA probe results．The number of active ribosomal gene loci can also be confirmed by counting nucleoli (sites of ribosome biogenesis)in interphase nuclei [13,14,[16][17][18]]．…”