We have determined the relationship between overall nuclear architecture, chromosome territories, and transcription sites within the nucleus, using three-dimensional confocal microscopy of well preserved tissue sections of wheat roots. Chromosome territories were visualized by GISH using rye genomic probe in wheat/rye translocation and addition lines. The chromosomes appeared as elongated regions and showed a clear centromere–telomere polarization, with the two visualized chromosomes lying approximately parallel to one another across the nucleus. Labeling with probes to telomeres and centromeres confirmed a striking Rabl configuration in all cells, with a clear clustering of the centromeres, and cell files often maintained a common polarity through several division cycles. Transcription sites were detected by BrUTP incorporation in unfixed tissue sections and revealed a pattern of numerous foci uniformly distributed throughout the nucleoplasm, as well as more intensely labeled foci in the nucleoli. It has been suggested that the gene-rich regions in wheat chromosomes are clustered towards the telomeres. However, we found no indication of a difference in concentration of transcription sites between telomere and centromere poles of the nucleus. Neither could we detect any evidence that the transcription sites were preferentially localized with respect to the chromosome territorial boundaries.

the nucleoplasm, must be equally active, but no obvious Norwich NR4 7UH, UK ultrastructure has yet been associated with them, except by fluorescence in situ hybridization (Highett et al., 1993a). Active rDNA transcription complexes have been visualized Summary by a hypotonic spreading technique first devised by Miller Incorporation by RNA polymerases of BrUTP into both and Beatty (1969) for the amplified rDNA found in Xenopus plant root tissue and isolated plant nuclei as a method for oocytes, and since applied by others to many different cell localization of the sites of transcription has been used. In types and organisms. These spread preparations show the this paper pea root tissue was used, and under the condiaxis of the gene associated with many (50-100) attached tions employed, nearly all the incorporation occurs in the RNA polymerase I molecules, together with nascent RNA nucleolus, and thus must be catalysed by RNA polymerase transcripts radiating away from the polymerases. Often the I. Immunofluorescence and confocal microscopy shows nascent RNAs show a terminal knob, assumed to be an that incorporation occurs in a pattern consisting of many RNP complex, and the increasing length of transcripts small foci distributed widely through the dense fibrillar along the genes has given rise to the description of the component of the nucleoli. Immunogold labelling using transcription complexes as 'Christmas trees'. Various comsilver-enhanced Nanogold probe at the electron microponents involved in the cleavage of the external transcribed scopic level confirms the sites of transcription as small spacer (ETS), such as fibrillarin, have been shown to be foci approximately 200nm in diameter. Simultaneous present in the terminal knobs (Mougey et al., 1993). fluorescence in situ hybridization with a probe to the Until recently, however, there has been little progress in external transcribed spacer (ETS) region of the pre-rRNA determining the organization of transcription and proshows that the structures revealed by this probe and the cessing in situ within the nucleolus. Conventional thin BrUTP immunofluorescence labelling are very similar. A section electron microscopy shows the nucleolus as a probe to the transcribed portion of the rDNA (18S) also densely stained structure, within which differently strucshows a good correlation to the sites of BrUTP incorporatured regions can often be discerned: fibrillar centres (FCs) tion within the nucleolus. On the other hand a probe to are small, lightly stained regions, typically approximately the non-transcribed intergenic spacer region (NTS) shows spherical and less than 1 µm in diameter; these are often very little coincidence with the sites of BrUTP incorporasurrounded by densely staining material-the dense fibriltion, and double fluorescence in situ labelling with both lar component (DFC); the rest of the volume of the nucleolus 18S and NTS probes confirms this difference in localization.contains densely packed particles, assumed to be preThese results suggest that most...

A panel of monoclonal antibodies that recognize a class of cell wall proteins, related to the hydroxyproline‐rich glycoproteins, has been assembled and characterized in relation to their restricted patterns of binding amongst the cells comprising the carrot root apex. The occurrence of the epitopes at the surface of cells and intercellular spaces in the region of the apex between the meristematic initials and the region of cell expansion indicates dynamic patterns that reflect aspects of the development of the anatomical pattern. The monoclonal antibody JIM11 reacts with the surface of cells in the central root cap and the region of the meristem. As the cortex/stele boundary becomes established the reactivity is seen in the inner cortical layers and finally in the whole cortex. Later in development the JIM11 epitope is also expressed by two pairs of pericycle cell files adjacent to the phloem region and also by the epidermis. The JIM12 monoclonal antibody is unreactive with cells in the region of the root cap and the meristem but is reactive with intercellular spaces formed at the junction of the oblique and radial walls in the double‐layered sectors of the pericycle opposite the xylem poles. This epitope is also transiently expressed by the two phloem sieve tube element mother cells. Later in development JIM12 recognizes the future metaxylem cells. The antibody JIM20 recognizes all the cells and intercellular spaces recognized by JIM11 and JIM12. Immuno‐chemical analyses indicate cross‐reactivity with carrot taproot extensin and Solanaceous lectins.

SummaryA new Arabidopsis meiotic mutant has been isolated. Homozygous ahp2-1 (Arabidopsis homologue pairing 2) plants were sterile because of failure of both male and female gametophyte development. Fluorescent in situ hybridisation showed that in ahp2-1 male meiocytes, chromosomes did not form bivalents during prophase I and instead seemed to associate indiscriminately. Chromosome fragmentation, chromatin bridges and unbalanced segregation were seen in anaphase I and anaphase II. The ahp2-1 mutation was caused by a T-DNA insertion in an Arabidopsis homologue of meu13 , which has been implicated in homologous chromosome pairing during meiosis in Schizosaccharomyces pombe. Our results suggest that meu13 function is conserved in higher eukaryotes and support the idea that Arabidopsis, yeast and mouse share a pairing pathway that is not present in Drosophila melanogaster and Caenorhabditis elegans.

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.

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.

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.

SummaryThe rDNA transcription units are enormous macromolecular structures located in the nucleolus and containing 50±100 RNA polymerases together with the nascent pre-rRNA attached to the rDNA. It has not previously been possible to visualize nucleolar transcription units directly in intact nucleoli, although highly spread preparations in the electron microscope have been imaged as`Christmas trees' 2±3 mm long. Here we determine the relative conformation of individual transcription units in Pisum sativum plant nucleoli using a novel labelling technique. Nascent transcripts were detected by a highly sensitive silver-enhanced 1 nm gold procedure, followed by 3D electron microscopy of entire nucleoli. Individual transcription units are seen as conical, elongated clusters approximately 300 nm in length and 130 nm in width at the thickest end. We further show that there were approximately 300 active ribosomal genes in the nucleoli examined. The underlying chromatin structure of the transcribing rDNA was directly visualized by applying a novel limited extraction procedure to ®xed specimens in order to wash out the proteins and RNA, thus speci®cally revealing DNA strands after uranyl acetate staining. Using this technique, followed by post-embedding in situ hybridization, we observed that the nucleolar rDNA ®bres are not extended but show a coiled, thread-like appearance. Our results show for the ®rst time that native rDNA transcription units are linear, compacted Christmas trees.