Alison F. Beven

Clusters of multiple different small nucleolar RNA genes in plants are expressed as and processed from polycistronic pre-snoRNAs

Small nucleolar RNAs (snoRNAs) are involved in many aspects of rRNA processing and maturation. In animals and yeast, a large number of snoRNAs are encoded within introns of protein‐coding genes. These introns contain only single snoRNA genes and their processing involves exonucleolytic release of the snoRNA from debranched intron lariats. In contrast, some U14 genes in plants are found in small clusters and are expressed polycistronically. An examination of U14 flanking sequences in maize has identified four additional snoRNA genes which are closely linked to the U14 genes. The presence of seven and five snoRNA genes respectively on 2.05 and 0.97 kb maize genomic fragments further emphasizes the novel organization of plant snoRNA genes as clusters of multiple different genes encoding both box C/D and box H/ACA snoRNAs. The plant snoRNA gene clusters are transcribed as a polycistronic pre‐snoRNA transcript from an upstream promoter. The lack of exon sequences between the genes suggests that processing of polycistronic pre‐snoRNAs involves endonucleolytic activity. Consistent with this, U14 snoRNAs can be processed from both non‐intronic and intronic transcripts in tobacco protoplasts such that processing is splicing independent.

Association of homologous chromosomes during floral development

Reduction in chromosome number and genetic recombination during meiosis require the prior association of homologous chromosomes, and this has been assumed to be a central event in meiosis. Various studies have suggested, however, that while the reduction division of meiosis is a universally conserved process, the pre-meiotic association of homologues differs among organisms. In the fruit fly Drosophila melanogaster, some somatic tissues also show association of homologues [1,2]. In the budding yeast Saccharomyces cerevisiae, there is some evidence for homologue association during the interphase before meiotic division [3,4], and it has been argued that such associations lead directly to meiotic homologue pairing during prophase I [5]. The available evidence for mammals suggests that homologous chromosomes do not associate in germ cells prior to meiotic prophase [6]. To study the occurrence of homologue pairing in wheat, we have used vibratome tissue sections of wheat florets to determine the location of homologous chromosomes, centromeres and telomeres in different cell types of developing anthers. Fluorescence in situ hybridization followed by confocal microscopy demonstrated that homologous chromosomes associate pre-meiotically in meiocytes (germ-line cells). Surprisingly, association of homologues was observed simultaneously in all the surrounding somatic tapetum cells. Homologues failed to associate at equivalent stages in a homologue recognition mutant. These results demonstrate that the factors responsible for the recognition and association of homologues in wheat act before the onset of meiotic prophase. The observation of homologue association in somatic tapetum cells demonstrates that this process and meiotic division are separable.

Association of Phosphatidylinositol 3-Kinase with Nuclear Transcription Sites in Higher Plants

The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic. The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established. In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells. Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus. Dual probing of root sections with JIM135 and anti–bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites. These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants.

The nucleolar architecture of polymerase I transcription and processing.

The nucleolus, the site of transcription and processing of the major ribosomal genes, generally reveals three distinct ultrastructural components in conventional thin‐section electron micrographs (fibrillar centres, dense fibrillar component and granular component). We show here that different parts of the transcription and transcript processing pathway can be mapped to the different nucleolar components in pea root cells. This study shows the full three‐dimensional arrangement of the different domains by in situ hybridization and confocal microscopy, and their correspondence with the major ultrastructural components of the nucleolus is revealed by parallel serial section electron microscopy. The active rDNA is widely dispersed in discrete foci, the larger of which, at least, correspond to well‐defined fibrillar centres. A probe to the external transcribed spacer (ETS) sequence of the pre‐rRNA transcripts labels clearly demarcated regions surrounding the foci of rDNA, and which we show correspond to the dense fibrillar component. Finally, a probe to the entire 45S transcript shows a higher concentration in regions corresponding to the granular component, surrounding the dense fibrillar component labelled by the ETS probe. The changes in structure that occur with heat shock show that nucleolar organization is dynamic and dependent upon transcriptional activity. These results show that the various RNA processing events are spatially highly organized and suggest a vectorial or radial model of transcription and transcript processing, where nascent and newly completed transcripts occupy zones surrounding the genes, which are in turn surrounded by regions containing the older more mature transcripts.

The architecture of interphase chromosomes and gene positioning are altered by changes in DNA methylation and histone acetylation

Wheat nuclei have a remarkably well defined interphase organisation, and we have made use of this to determine the relationship between interphase chromosome organisation, the positioning of specific transgenes and induced changes in DNA methylation and histone acetylation, using in situ hybridisation and confocal 3D imaging. After germinating seeds either in the presence of 5-Azacytidine (5-AC), which leads to DNA hypomethylation, or trichostatin A (TSA), which results in histone hyperacetylation, the architecture of the interphase chromosome arms changes significantly even though the overall Rabl configuration is maintained. This suggests that specific chromosome segments are remodelled by these treatments but that there is a strong link of both centromeres and telomeres to the nuclear envelope. In lines carrying multiple transgene integrations at widely separated sites, we show that the multiple transgenes, which are usually colocalised during interphase, are dispersed after 5-AC or TSA treatment and that there is an increase in transgene activity. This suggests that the colocalisation/dispersion of the transgenes may be a function of specific interphase chromosome organisation and that these lines containing multiple transgene copies may all be partially transcriptionally repressed.

Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes.

Incorporation by RNA polymerases of BrUTP into both plant root tissue and isolated plant nuclei as a method for localization of the sites of transcription has been used. In this paper pea root tissue was used, and under the conditions employed, nearly all the incorporation occurs in the nucleolus, and thus must be catalysed by RNA polymerase I. Immunofluorescence and confocal microscopy shows that incorporation occurs in a pattern consisting of many small foci distributed widely through the dense fibrillar component of the nucleoli. Immunogold labelling using silver-enhanced Nanogold probe at the electron microscopic level confirms the sites of transcription as small foci approximately 200 nm in diameter. Simultaneous fluorescence in situ hybridization with a probe to the external transcribed spacer (ETS) region of the pre-rRNA shows that the structures revealed by this probe and the BrUTP immunofluorescence labelling are very similar. A probe to the transcribed portion of the rDNA (18S) also shows a good correlation to the sites of BrUTP incorporation within the nucleolus. On the other hand a probe to the non-transcribed intergenic spacer region (NTS) shows very little coincidence with the sites of BrUTP incorporation, and double fluorescence in situ labelling with both 18S and NTS probes confirms this difference in localization. These results suggest that most BrUTP foci correspond to single transcribed genes.

Splicing-independent processing of plant box C/D and box H/ACA small nucleolar RNAs

Small nucleolar RNAs (snoRNAs) are involved in various aspects of ribosome biogenesis and rRNA maturation. Plants have a unique organisation of snoRNA genes where multiple, different genes are tightly clustered at a number of different loci. The maize gene clusters studied here include genes from both of the two major classes of snoRNAs (box C/D and box H/ACA) and are transcribed as a polycistronic pre-snoRNA transcript from an upstream promoter. In contrast to vertebrate and yeast intron-encoded snoRNAs, which are processed from debranched introns by exonuclease activity, the particular organisation of plant snoRNA genes suggests a different mode of expression and processing. Here we show that single and multiple plant snoRNAs can be processed from both non-intronic and intronic transcripts such that processing is splicing-independent and requires endonucleolytic activity. Processing of these different snoRNAs from the same polycistronic transcript suggests that the processing machineries needed by each class are not spatially separated in the nucleolus/nucleus.

The architecture of interphase chromosomes and nucleolar transcription sites in plants.

Fluorescence in situ hybridization (FISH) coupled with confocal microscopy has been used to reveal the interphase chromosome organization in plants. In wheat and several other related species, we have shown that the interphase chromosomes are in a very well-defined organization, with centromeres and telomeres located at opposite sides of the nuclear envelope-a classic Rabl configuration. In transgenic wheat lines, FISH analysis of metaphase chromosomes has shown that multiple transgene copies can be integrated along a single chromosome, with large regions of intervening genomic sequence. These multiple copies are often colocalized in interphase, suggesting either an ectopic association or a highly reproducible interphase chromatin configuration. Bromo-uridine (BrU) incorporation has been used to label transcription sites in the nucleolus. Using pea root tissue, we have combined BrU incorporation with preembedding 1-nm gold detection to image the nucleolar transcription sites by electron microscopy. This has revealed many distinct elongated clusters of silver-gold particles. These clusters are 200-300 nm in length and are thicker at one end than the other. We suggest that each cluster corresponds to a single transcribed gene. Serial sectioning of several entire nucleoli has enabled the reconstruction of all the nucleolar transcription sites, and we have estimated that there are 200-300 transcribed genes per nucleolus.

ATP‐dependent regulation of nuclear Ca2+ levels in plant cells

Localised alterations in cytoplasmic Ca2+ levels are an integral part of the response of eukaryotic cells to a plethora of external stimuli. Due to the large size of nuclear pores, it has generally been assumed that intranuclear Ca2+ levels reflect the prevailing cytoplasmic Ca2+ levels. Using nuclei prepared from carrot (Daucus carota L.) cells, we now show that Ca2+ can be transported across nuclear membranes in an ATP‐dependent manner and that over 95% of Ca2+ is accumulated into a pool releasable by the Ca2+ ionophore A.23187. ATP‐dependent nuclear Ca2+ uptake did not occur in the presence of ADP or ADPγS and was abolished by orthovanadate. Confocal microscopy of nuclei loaded with dextran‐linked Indo‐1 showed that the initial ATP‐induced rise in [Ca2+] occurs in the nuclear periphery. The occurrence of ATP‐dependent Ca2+ uptake in plant nuclei suggests that alterations of intranuclear Ca2+ levels may occur independently of cytoplasmic [Ca2+] changes.

The nucleus: a highly organized but dynamic structure

The nucleus in plants and animals is a highly structured organelle containing several well‐defined subregions or suborganelles. These include the nucleolus, interphase chromosome territories and coiled bodies. We have visualized transcription sites in plants at both light‐ and electron‐microscopy level by the incorporation of BrUTP. In the nucleolus many dispersed foci are revealed within the dense fibrillar component, each of which probably corresponds to a single gene copy. In the nucleoplasm there are also many dispersed foci of transcription, but not enough to correspond to one site per transcribed gene. We have shown that in wheat, and probably many other plant species, interphase chromosome territories are organized in a very regular way, with all the chromosomes in the Rabl configuration, all the centromeres clustered at the nuclear membrane and all the telomeres located at the nuclear membrane on the opposite side of the nucleus. However, despite this regular, polarized structure, there is no sign of polarization of transcription sites, or of any preferred location for them with respect to chromosome territorial boundaries. The nucleus is also highly dynamic. As an example, we have shown by the use of a green fluorescent protein fusion to the spliceosomal protein U2B′′ that coiled bodies move and coalesce within the nucleus, and may act as transport structures within the nucleus and nucleolus.