Z-DNA, a new in situ marker for transcription.

Z-DNA forms transiently behind the active RNA polymerases, because of the mechanical torsional stress produced during transcription. In this paper, we explore the possibility that the distribution of Z-DNA stretches signals the sites related to nuclear transcription. To localize transcription, the in situ assay for active RNA polymerases, that allows the elongation of the already initiated transcripts but no initiation of new ones (run-on experiments), was carried out in isolated nuclei of Allium cepa L. root meristems. Both nucleolar and non-nucleolar sites appeared labelled. Nucleoli were most active in transcription than the multiple non-nucleolar foci altogether. In situ immunodetection of Z-DNA provided images that were comparable to those obtained after the run-on assay, with one exception: while Z-DNA and transcription sites were scattered throughout the whole nucleus, Z-DNA also accumulated in the nuclear periphery, where no transcription foci were detected in run-on assays. The peripheral Z-conformation signals might correspond to dsRNA segments present in the pre-mRNA in the process of their export to cytoplasm. The Z-containing structures nearly disappeared when non-nucleolar RNA polymerase II-dependent transcription had been previously abolished by the adenosine analogue DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole). This inhibition selectively decreased the amount of all nucleoplasmic Z structures. On the other hand, the inhibition of the nucleolar RNA polymerase I by cordycepin (3'-deoxyadenosine) prevented the presence of Z-DNA in nucleoli. We propose to use the in situ immunodetection of Z-DNA as a marker of the transcription level in both nucleolus and non-peripheral nucleoplasmic regions of nuclei. Co-detection of Z-DNA and of intermediate filament (IF) proteins, the major components of the nuclear matrix, was also carried out. The IFA antibody recognizes a conserved epitope essential for dimerization of the multiple IF proteins. They co-localized with most nucleolar Z-DNA, but not with the nucleoplasmic ones. In the nuclear periphery, the Z-positive signals were adjacent to the IF proteins constituting the lamina, though both signals did not often co-localize.

Dormancy and proliferation in Saccharum officinarumxS. spontaneum hybrids which differ in the number of the introgressed S. spontaneum chromosomes.

Proliferating cells remain transiently blocked at different cycle compartments until specific stressors are removed or until the cells become adapted to their presence. This paper investigates the efficiency of cycle blocks in three sugarcane hybrids with the full noble cane (Saccharum officinarum) genome (2n=8x=80) but differing in the number of introgressed S. spontaneum (2n=8x=64) chromosomes. The My5514, B42231 and C236-51 cultivars possess 20, 30 and 40 additional S. spontaneum chromosomes, respectively. Flow cytometry showed that over 90% of cells were accumulated with a 2C DNA content in their dormant primordia. The presence of S. spontaneum chromosomes decreased the low stringency of the 4C block. The greater the number of these chromosomes, the lower was the number of quiescent cells with a 4C DNA content (P<0.05). Shortly after stimulation of the primordia (85% relative humidity and 30 degrees C), i.e. in the 2 mm long roots, a negative correlation was found between the number of introgressed S. spontaneum chromosomes and the frequency of cells undergoing replication and mitosis. On the other hand, when roots were already proliferating under steady-state conditions (15 mm long roots) the more S. spontaneum chromosomes the cells possessed, the longer the relative time it took for all chromosomes to replicate and segregate, and the longer the relative time they spent in G(2), with the 4C DNA content. The presence of S. spontaneum chromosomes seems to be recognized by these proliferating cells as a stressor which preferentially activates checkpoint pathways operating at the second half of the cycle, but not at its onset.

High resolution detection of rRNA and rDNA in plant nucleoli with different activities by in situ hybridization.

In the present work we perform in situ hybridization with probes to different stretches of rDNA and electron microscopy of nucleoli with different activities, to gain insight into the ultrastructural organization of transcription and processing in the plant nucleolus. The main ultrastructural nucleolar components: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC), are arranged in different ways depending on nucleolar activity. Heterogeneous FCs containing RNP fibrils and nucleolar perichromatin granules are frequently seen in nucleoli in the process of activation. DNA-RNA in situ hybridization with biotinylated probes spanning different sequences of the rDNA unit followed by immunogold detection of biotin, demonstrated the localization of the ribosomal transcripts in DFC, mainly in the zones around the FCs, in GC, and in the periphery of pale FC. The internal region of the heterogeneous FCs is labeled only in cells in the process of activation of transcription after dormancy. The distribution of the U3 probe indicates that the processing of the rRNA takes place in the DFC and inside the heterogeneous FCs, in which transcription occurs. DNA-DNA hybridization demonstrates the presence of rDNA in the compact and extended chromatin located in the interior and at the periphery of FCs and in nucleolar associated chromatin. Our results support the view that the plant nucleolus has a highly dynamic morphological and functional organization composed of a bipartite domain formed by FCs surrounded by DFC, which is associated with rRNA transcription and processing, and the GC representing a store of preribosomal particles.

Nuclear matrix isolated from plant cells.

Residual nuclear matrices can be successfully obtained from isolated nuclei of different monocot and dicot plant species using either high ionic or low ionic extraction protocols. The protein composition of isolated nuclear matrices depends on the details of isolation protocols. They are stable and present in all cases, a tripartite organization with a lamina, nucleolar matrix, and internal matrix network, and also maintain some of the basic architectural features of intact nuclei. In situ preparations demonstrate the continuity between the nuclear matrix and the plant cytoskeleton. Two-dimensional separation of isolated plant nuclear matrix proteins reveals a heterogeneous polypeptide composition corresponding rather to a complex multicomponent matrix than to a simple nucleoskeletal structure. Immunological identification of some plant nuclear matrix components such as A and B type lamins, topoisomerase II, and some components of the transcription and splicing machineries, internal intermediate filament proteins, and also specific nucleolar proteins like fibrillarin and nucleolin, which associate to specific matrix domains, establish a model of organization for the plant nuclear matrix similar to that of other eukaryotes. Components of the transcription, processing, and DNA-anchoring complexes are associated with a very stable nucleoskeleton. The plant matrix-attached regions share structural and functional characteristics with those of insects, vertebrates, and yeast, and some of them are active in animal cells. In conclusion, the available data support the view that the plant nuclear matrix is basically similar in animal and plant systems, and has been evolutionarily conserved in eukaryotes.

Identification and localization of a nucleolin homologue in onion nucleoli.

A protein homologous to nucleolin, a major nucleolar protein with multifunctional features involved in pre-rRNA synthesis and early processing, has been identified and localized in situ in onion root meristematic cells by different techniques, which have included the use of an antibody raised against hamster nucleolin. The protein was identified on Western blots of nucleolar proteins as a 64-kDa band, by means of the anti-nucleolin antibody, bismuth staining, and the silver staining-nucleolar organizer (Ag-NOR) method. The experiments also suggested that nucleolin could be a target of these two cytochemical stainings. Although the 64-kDa band corresponds to a major nucleolar protein, it is a minor one among total nuclear proteins. The same techniques were used in situ at the ultrastructural level, and the immunogold detection of the nucleolin homologue was quantitatively evaluated. The protein accumulates in the transition area from nucleolar fibrillar centers to the dense fibrillar component, which is considered to be the structural result of ribosomal gene transcription. Out of this transition area, the dense fibrillar component may be divided into two regions, proximal and distal with respect to fibrillar centers, which show, respectively, the significant and unsignificant presence of nucleolin; we interpret this fact as the expression of the topological arrangement of pre-rRNA processing. Fibrillar centers themselves showed a weak but significant labeling with the anti-nucleolin antibody. However, bismuth staining was absent from the interior of fibrillar centers, indicating that the nucleolin in them is not phosphorylated. Ag-NOR staining uniformly covered fibrillar centers and the dense fibrillar component (at least in its proximal region), but it did not stain condensed chromatin inclusions in heterogeneous fibrillar centers, showing that the binding of nucleolin to chromatin is associated with its decondensation. This work provides additional evidence of the high phylogenetic conservation of molecular motifs which take part in ribosome biogenesis.

Immunolocalization of DNA at nucleolar structural components in onion cells.

Intranucleolar DNA, including ribosomal DNA (rDNA), was localized in situ in proliferating onion cells under the electron microscope using an anti-DNA monoclonal antibody and a postembedding indirect immunogold procedure. In the interphase nucleolus of this species, characterized by a very high amount of rRNA genes, we found DNA concentrated mostly in fibrillar centres (FCs) and in the region of the dense fibrillar component (DFC) immediately surrounding them. Clusters of gold particles were frequently seen covering both of these structural components of the nucleolus at the same time. Moreover, the same technique, applied to transcriptionally arrested quiescent onion cells, showed the nucleolar DFC devoid of DNA. Also, in mitotic cells at telophase, the prenucleolar material, which has the same morphological and cytochemical features as the DFC, does not contain DNA. These data suggest the existence of at least two subcomponents of the DFC in the onion cell nucleolus, one associated with pre-rRNA synthesis, and the other, with further processing of transcripts, already released from the rDNA template. We conclude that the first subcomponent forms part of the "transition between FC and DFC", which is the in situ structural counterpart of pre-rRNA synthesis. This transition is morphologicaly sizeable in onion cells, because of their high number of rRNA genes and the large size of the DFC mass; however, it would be largely detectable in situ in other cell systems, where the whole DFC comprises only a thin layer and the amount of rDNA is considerably reduced.

A short-lived nuclear phosphoprotein encoded by the human ets-2 proto-oncogene is stabilized by activation of protein kinase C.

The human ets-2 gene is a homolog of the v-ets oncogene of the E26 virus and codes for a 56-kilodalton nuclear protein. The ets-2 protein is phosphorylated and has a rapid turnover, with a half-life of 20 min. When human lymphocytic CEM cells were treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the level of the ets-2 protein was quickly elevated 5- to 20-fold. This effect of TPA was mimicked by a synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol, and was blocked by the protein kinase C inhibitor H7, indicating that protein kinase C is involved in the induction. The increase in the ets-2 protein was due to stabilization of the protein, because the protein had a half-life of more than 2 h in the presence of TPA and the ets-2 mRNA level did not increase significantly upon TPA treatment. The protein synthesis inhibitor cycloheximide enhanced the effect of TPA on the ets-2 protein and could itself slow turnover of the protein. Properties of the ets-2 protein, such as nuclear localization, phosphorylation, rapid turnover, and response to protein kinase C, indicate that this protein belongs to a group of oncogene proteins which are generally thought to have regulatory functions in the nucleus (e.g., myc, fos, myb, and p53). Our results suggest that protein kinase C, either directly or indirectly, regulates the level of the ets-2 protein by posttranslational mechanisms.