Nucleolar organizer expression in Allium cepa L. chromosomes

Abstract

Roots from Allium cepa L. (cv. Francesa) bulbs in which a maximum of two nucleoli per nucleus developed were selected for this study. Five rDNA clusters were detected by fluorescent in situ hybridization on chromosomal squashes (2n = 16) with a rhodamine-labelled wheat rDNA repeat. The rDNA clusters were located on four chromosomes: the largest cluster occurred on the small arm of a single homologue of the smallest pair 8. Its homologue showed two different small rDNA clusters, one near each telomere. The two homologues of the satellited chromosomes 6 also showed different rDNA contents, which were intermediate to those found in pair 8. The same five well-differentiated hybridization signals were observed in interphase cells that were inactive in transcription because they were in dormant roots, or in proliferating ones in which the synthesis of the large rRNA precursor was prevented. After multipolarizing agent was applied in anaphase followed by inhibition of cytokinesis, multinucleate autotetraploid cells were formed, which often contained more than four nucleoli. Thus, at least two of the three nucleolar organizer regions that consistently failed to develop a nucleolus in normal mononucleate cells were capable of developing nucleoli when segregated into different nuclei in multinucleate cells.

Behaviour of ribosomal genes and nucleolar domains during activation in sugarcane (Saccharum officinarum L.) root primordia: from the unsoaked quiescent state to the steady state of proliferation

“…After washing three times in distilled water for 10 min, they were kept overnight in a 2% aqueous solution of AgNO 3 at 70ºC, in the dark. After a further washing the roots were placed again in the formaldehyde-hydroquinone mixture for 1 h. Afterwards, roots were squashed and mounted on gelatin-covered slides as previously described (Panzera et al, 1996).…”

“…After washing in water to remove fixative, root tips were digested with an enzyme solution containing 2 ml of 2% cellulase (Onozuka R10, Yakult Honsha Co., Tokyo) and 20% liquid pectinase (from Aspergillus niger, Sigma Chemical Co., St. Louis, Mo), for 50 min at 37ºC. The fixed root tips were squashed onto clean microscope slides in a drop of 45% acetic acid as previously described (Panzera et al, 1996). The samples were washed again in the above buffer, before freezing the slides to remove the cover, and were finally air-dried.…”

“…Preparations and probe denaturation, in situ hybridisation, post-hybridisation washing and detection were all performed as previously described (Panzera et al, 1996). Microscopy analyses were made in an epifluorescence Axiophot Zeiss system.…”

“…The structure of the nucleolus is organised around the tandemly repeats of rDNA genes, which form the chromosomal nucleolar organising region (NORs) during mitosis. In spite of the specific differences observed in the number and chromosomal location of rDNA copies and NORs, and also in their expression, specially in plants in which the nucleolar dominance is relatively common (Reder, 1985;Panzera et al, 1996), the active nucleolus in eukaryotes follows a general pattern of organisation with three universal domains. The first is the fibrillar centre (FC) which contains ribosomal genes.…”

“…In the last decade the development of fluorescence in situ hybridization (FISH) and high resolution in situ hybridisation (HRISH) led to a great advance in the investigation of ribosomal gene organisation and expression in different plant species (Motte et al, 1991;Leicht et al, 1992;Highett et al, 1993, Panzera et al, 1996Thompson et al, 1997;Bassy et al, 2000).…”