NORs and their transcription competence during the cell cycle

Abstract

In human cells ribosomal genes are organized as clusters, NORs, situated on the short arms of acrocentric chromosomes. It was found that essential components of the RNA polymerase I transcription machinery, including UBF, can be detected on some NORs, termed "competent" NORs, during mitosis. The competent NORs are believed to be transcriptionally active during interphase. However, since individual NORs were not observed in the cell nucleus, their interphase status remains unclear. To address this problem, we detected the competent NORs by two commonly used methods, UBF immunofluorescence and silver staining, and combined them with FISH for visualization of rDNA and/or specific chromosomes. We found that the numbers of competent NORs on specific chromosomes were largely conserved in the subsequent cell cycles, with certain NOR-bearing homologues displaying a very stable pattern of competence. Importantly, those and only those NORs that were loaded with UBF incorporated bromo-uridine in metaphase after stimulation with roscovitine and in telophase, suggesting that competent and only competent NORs contain ribosomal genes transcriptionally active during interphase. Applying premature chromosome condensation with calyculin A, we visualized individual NORs in interphase cells, and found the same pattern of competence as observed in the mitotic chromosomes.

Fig. 1

Karyotype of the HeLa cells assessed by multicolour fluorescence in situ hybridization (M-FISH). There are trisomies of acrocentric chromosomes 13, 14, 15, 21, 22. One marker chromosome M13 appears as isochromosome 15; p-arms of the chromosome 13 are included into the marker M14 together with q-arms of the chromosome 19. One of the chromosomes 22 is defined as marker M16 since it includes some material from the chromosome 8.

Fig. 2

Localization of NORs and competent NORs on the spread mitotic chromosomes of HeLa cells A, B: simultaneous rDNA (A, red in B) and chromosome 15 detection (green in B). Thirteen rDNA signals are clearly recognized in A. Three acrocentrics and one metacentric chromosome are labelled with a chromosome 15-specific probe (B). C, D: silver staining (C, red in B) combined with the probe for chromosome 15 (green in D). Large silver signals (arrows) are seen on all four painted chromosomes. Compare with the low intensity rDNA signals in A and B. E, F, G: detection of rDNA (E; red in G), silver staining (F) and chromosome 22 (green in G). Two very large rDNA signals (arrows in E, G) correspond to relatively small silver signals (arrows in F, G). The third rDNA signal belonging to a smaller painted chromosome (arrowheads in E, G) is less intensive and corresponds to a silver-negative NOR (arrowhead in F, G). Bar: 20 mm.

Fig. 3

Localization of NORs and competent NORs on the spread mitotic chromosomes of LEP cells. A, B: Simultaneous detection of rDNA (A, red in B), chromosome 13 (B, large acrocentrics painted in green) and chromosome 22 (small acrocentrics in green); rDNA signals (arrows) are seen on all four painted chromosomes. Ten NORs can be easily recognized in A. C, D: silver staining (red) combined with the probes for chromosomes 15 (D, large acrocentrics painted in green) and 21 (small acrocentrics in green). Silver signals (arrows) are seen on all the painted chromosomes. Seven silver signals are visible in C.

Fig. 4

Distribution of the NORs (green) and competent NORs (red) among the different chromosomes in HeLa and LEP cells. One of the chromosomes 15 of the HeLa cells is taken in parenthesis to show the variability of the chromosome number. Asterisks indicate two chromosomes of LEP cells that may be competent or non-competent with comparable frequencies. For simplicity the competent NORs are always shown on both chromatids. The different size of the red and green dots illustrates the different intensities of the rDNA and UBF/Ag signals.

Fig. 5

Co-localization of transcriptionally active and competent NORs in telophase (A-C) and metaphase after roscovitine treatment (D-F). A, D: transcription signal (BrU); B, E: UBF. C, F co-localization of transcription (green) and UBF (red) signals.

Figure 6

A-F. UBF-positive NORs (A-E, red in B-F) in the interphase HeLa cells after calyculin A treatment A, B: G1 phase; C, D: S phase; E, F: G2 phase. DAPI counterstaining is in blue. Nine competent NORs appear as single in G1, mostly double in G2. Condensation of the chromosomes is complete only in G2 phase. G, H: Simultaneous detection of UBF (G, red in H) and chromosome 22 (green in H) in G2 cells. Two chromosomes 22 carry UBF signals (arrows); the third chromosome is UBF-negative (compare with the Fig. 3F, G).