Chromosome no. 1 of Crepis capillaris shows defined 3D-shapes in mitotic prophase.

The shape of mitotic prophase chromosomes has been studied in root tip nuclei by confocal microscopy and 3D-image analysis. Crepis capillaris chromosome no. 1 was used as a test object. Chromosome conformation was studied in early, mid- and in late prophase. In mid- and late prophase, individual chromosomes could be distinguished on the basis of their length. Early prophase chromosomes could not be distinguished as individuals. The central axes of prophase chromosomes were traced with an automated computer procedure and then represented as a string of 3D coordinates. This representation facilitated measurement along the chromosome axis of shape parameters such as curvature (amount of bending), torsion (helical winding) and torsion sign (helical handedness). Stretches of early prophase chromosomes showed full helical turns, which could be left- or right-handed. In the later prophase stages curvature and torsion were statistically analysed. Our data on 40 midprophase chromosomes no. 1 show that they are still highly curved, but full helical turns were no longer found. Instead, an overall meandering pattern was observed. In late prophase, one central loop persisted, flanked by two preferential regions of high curvature.

The 5S rRNA gene clusters have a defined orientation toward the nucleolus in Petunia hybrida and Crepis capillaris.

The 3D localization of the 5S ribosomal RNA genes was studied in cells of the cortex zone of roots in the plant species Petunia hybrida inbred line V26 and in Crepis capillaris. The analysis was carried out on interphase nuclei (both species) and on prophase nuclei (C. capillaris). The 5S ribosomal RNA genes were detected by fluorescence in-situ hybridization and 3D images were obtained by confocal scanning laser microscopy. In both plant species, the 5S ribosomal genes were localized at the short arm of chromosome 2, which, in both plants, also possesses a satellite at its end. Statistical and visual analysis of interphase nuclei showed that: (1) there is a preference for an association of the 5S rRNA gene clusters of the two homologous chromosomes, and (2) the 5S rRNA gene clusters in both species had a preserved spatial position within the interphase nucleus and they tended to be polarized with respect to their neighbouring cells (i.e. a relic telophase orientation). Moreover, tracing of the chromosomal segment between the 5S loci and the active NOR revealed that the homologous chromosomes during early/mid prophase were aligned and that they entered the nucleolus side by side, at least for these chromosome segments. We interpret our data to mean that location of 5S rRNA near the nucleolus favours their functioning in ribosome biogenesis.

Characterisation of the nucleolar organising regions during the cell cycle in two varieties of Petunia hybrida as visualised by fluorescence in situ hybridisation and silver staining.

The cell cycle-dependent spatial position, morphology and activity of the four nucleolar organising regions (NORs) of the Petunia hybrida cultivar Mitchell and the inbred line V26 have been analysed. Application of the silver staining technique and fluorescence in situ hybridisation on fixed root-tip material revealed that these interspecific hybrids possess four NORs of which only those of chromosome 2 are active during interphase, which implies that the NOR activity is not of parental origin. However, at the end of mitosis, activity of all NOR regions could be detected, suggesting that the high demand for ribosomes at this stage of the cell cycle requires temporal activity of all NORs. Using actin DNA probes as markers in fluorescence in situ hybridisation experiments enabled the identification of the individual petunia chromosomes.

The spatial localization of 18 S rRNA genes, in relation to the descent of the cells, in the root cortex of Petunia hybrida.

The 3-D localization of transcription inactive 18 S rRNA genes was studied in interphase nuclei of Petunia hybrida root tip cells. To enable a cell type (i.e. cortex)-specific study in which also the orientation and descent of the cells could be taken into account, a method was developed to preserve the spatial organization of the root meristem. The ribosomal genes were detected by fluorescence in situ hybridization using a biotinylated cDNA probe. 3-D images of 81 nuclei, obtained by confocal scanning laser microscopy, were processed with newly developed computer software. 3-D nucleolar and nuclear dimensions, and the localization of the FISH-spots, were recorded interactively. We compared the absolute and relative position of the genes within and between files of cells of the cortex region of several roots, taking into account the genealogical relationship of the cells. Statistical analysis showed that both the relative and absolute positions of the inactive genes were random, also in more closely related cells within a file of cells. A 'relict telophase orientation' of the genes (i.e. the position of the genes in the daughter cells are mirror images of each other) could only be observed in the G0/1 phase of 'true' daughter cells; the orientation was not preserved throughout the next cell cycle.