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Derived from candidate sequences of a barley EST database two proteins with homology to the coiled coil region of the human kinetochore protein (KP) CENP-E were generated and classified as centromere protein E-like 1 and 2 (Cpell and Cpe12). Specific antibodies produced against recombinant Cpe11 and Cpe12 proteins labeled the centromere on mitotic chromosomes of barley and field bean and recognized specifically proteins from nuclear/chromosomal protein extracts on immunoblots. No function was predicted for homologues of Cpe11 within the databases for Arabidopsis and rice genomes. However, the centromeric location of Cpe11 and Cpe12 suggests they may have a function within the kinetochore. Plant homologues to barley Cpe12 are N-type kinesins, suggesting that Cpe12 is functionally homologous to human CENP-E.

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.

In an earlier fluorescent in-situ hybridization (FISH) study on petunia (ten Hoopen et al. 1996), we found a considerable discrepancy between the genetic map and the physical map with respect to T-DNA insertions on metaphase chromosomes. For some transgenes we found a preference to integrate near the telomeres. Here, we studied the spatial position of transgenes in interphase nuclei by FISH and 3D-confocal microscopy to elucidate a possible structural preference for the nuclear localization of transgenes. Three transgenes located near telomeres on three different metaphase chromosomes showed a much more internal distribution in interphase root meristem than the telomeres, whereas a proximal transgene appeared to be distributed in a random fashion. The results point to local differences in chromatin compacting along a chromosome. These differences might explain a preference for T-DNA insertion in distal regions of the chromosome.