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- Cytologie (13)
- Amphibian oocytes (3)
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Transcriptionally inactive chick erythrocyte nudei were reactivated by Sendai virusinduced fusion of erythrocytes with rat L6j1 myoblasts. We used antibodies to trace the appearance of a specific protein engaged in transcription of a defined dass of genes, those coding for rRNA, during reactivation. Using immunofluorescence microscopy, we found increasing amounts of rat RNA polymerase I to appear, during a certain period of time after fusion, in the reforming nudeoli of the chick nudei. Amounts of rat RNA polymerase I sufficient to be detected by immunofluorescence microscopy had accumulated in the newly developed chick nudeoli 72- 190 h after fusion was initiated. This time interval coincides with the time when chick rRNA synthesis can first be detected. The results raise the possibility that during these stages of the reactivation process chick rRNA genes are transcribed by heterologous RNA polymerase I moleeules of rat origin.
Upon incubation of cultured rat cells with the adenosine analogue 5,6-dichloro-l-β- D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar "necklaces". We have used this experimental system, which does not inhibit transcription of the rRNA genes, to study by immunocytochemistry the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology. Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosomal protein (S 1) of the small ribosomal subunit permit localization of nucleolar preribosomal particles. The results show that, under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase land argyrophilic proteins. Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene-containing granules. We conclude that, although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits. We further propose that each RNA polymerase-positive granular unit represents a single transcription unit and that each continuous array of granules ("string of nucleolar beads") reflects the linear distribution of rRNA genes along a nucleolar organizer region. Based on the total number of polymerase I-positive granules we estimate that a minimum of 60 rRNA genes are active during interphase of DRB-treated rat cells.
Purified mitochondrial DNA (mitDNA) from ovaries ofXenopus lae vis was injected into the nuclei (germinal vesicles) of large viteUogenic oocytes of the same organism and examined by electron microscopy ofthe spread nuclear contents. Normally located nuclei of untreated oocytes as weil as peripherally translocated nuclei of centrifuged oocytes were used. In addition, oocyte nuclei isolated and incubated under liquid paraffin oil were injected with DNA. The integrity oftranscriptional structures of endogenous chromosomal (Iampbrush chromosomes) and extrachromosomal (nucleoli) genes of the injected nuclei was demonstrated. Microinjected mitDN A was identified as circles of chromatin exhibiting polynucleosome-like organization and a me an contour length of 2.6 J.Lm, corresponding to a compaction ratio of the mitDN A of about 2 : I. This DNA packing ratio is similar to that observed after preparation of various kinds of native chromatin in low salt buffers. The chromatin circles formed from injected mitDNA only very rarely exhibited lateral fibrils suggestive of transcriptional activity. These results suggest that purified mitDNA can be transformed to normally structured chromatin when exposed to oocyte nuclear contents but is rarely , if at all , transcribed in this form and in this environment.
Rabbit antibodies to RNA polymerase I from a rat hepatoma have been used to localize the enzyme in a variety of cells at the light and electron microscopic level. In interphase cells the immunofluorescence pattern indicated that polymerase I is contained exclusively within the nucleolus. That this fluorescence, which appeared punctated rather than uniform, represented transcriptional complexes of RNA polymerase I and rRNA genes was suggested by the observation that it was enhanced in regenerating liver and in a hepatoma and was markedly diminished in cells treated with actinomycin D. Electron microscopic immunolocalization using gold-coupled second antibodies showed that transcribed rRNA genes are located in, and probably confined to, the fibrillar centers of the nucleolus. In contrast, the surrounding dense fibrillar component, previously thought to be the site of nascent prerRNA, did not contain detectable amounts of polymerase I. During mitosis, polymerase I molecules were detected by immunofluorescence microscopy at the chromosomal nucleolus organizer region, indicating that a considerable quantity of the enzyme remains bound to the rRNA genes. From this we conclude that rRNA genes loaded with polymerase I molecules are transmitted from one cell generation to the next one and that factors other than the polymerase itself are involved in the modulation of transcription of DNA containing rRNA genes during the cell cycle.
Natural changes in the transcription of rRNA genes were studied in nucleoli from three oogenic stages of the newt Triturus alpestris with electron microscope, autoradiographic, and biochemical techniques. From determinations of the uridine triphosphate pool sizes and [3H]uridine uptake, phosphorylation, and incorporation into 28S and 18S rRNAs in vivo it was estimated that the rate of rRNA synthesis was about 0.01% in previtellogenic oocytes and 13% in mature oocytes when compared to midvitellogenesis. Spread preparations of nucleoli showed significant morphological changes in the transcriptional complexes. The total number of lateral fibrils, i.e., ribonucleoproteins containing the nascent rRNA precursor, were drastically decreased in stages of reduced synthetic activity. This indicates that rRNA synthesis is regulated primarily at the level of transcription. The resulting patterns of fibril coverage of the nucleolar chromatin axes revealed a marked heterogeneity. On the same nucleolar axis occurred matrix units that were completely devoid of lateral fibrils, matrix units that were almost fully covered with lateral fibrils, and various forms of matrix units with a range of lateral fibril densities intermediate between the two extremes. Granular particles that were tentatively identified as RNA polymerase molecules were not restricted to the transcription l complexes. They were observed, although less regularly and separated by greater distances, in untranscribed spacer regions as well as in untranscribed gene intercepts. The results show that the pattern of transcriptional control of rRNA genes differs widely in different genes, even in the same genetic unit.
The arrangement of transcriptional units in the loops of lampbrush chromosomes from oocyte nuclei of urodele amphibia and from primary nuclei of the green alga Acetabularia have been studied in the electron microscope using spread preparations. Loops with different patterns of arrangement of matrix units (i.e. to a first approximation, transcriptional units) can be distinguished: (i) loops consisting of one active transcriptional unit; (ii) loops containing one active transcriptional unit plus additional fibril-free, i.e. apparently untranscribed, intercepts that may include 'spacer' regions; (iii) loops containing two or more transcriptional units arranged in identical or changing polarities, with or without interspersed apparent spacer regions. Morphological details of the transcriptional complexes are described. The observations are not compatible with the concept that one loop reflects one and only one transcriptional unit but, rather, lead to a classification of loop types according to the arrangement of their transcriptional units. We propose that the lampbrush chromosome loop can represent a unit for the coordinate transcription of either one gene or a set of several (different) genes.
The occurrence of stacked annulate tamellae is documented for a plant cell system, namely for pollen mother cells and developing pollen grains of Canna generalis. Their structural subarchiteeture and relationship to endoplasmie reticulum (ER) and nuclear envelope cisternae is described in detail. The results demonstrate structural homology between plant and animal annulate lamellae and are compatible with, though do not prove, the view that annulate lamcllar cisternae may originate as a degenerative form of endoplasmic retieulum.
The structural organization of transcriptionally active DNA that contains cistrons for precursor molecules of ribosomal RNA is described in positively stained spread preparations from nuclei and nucleoli isolated from the green alga, Acetabularia mediterranea Lmx. These nuclei contain large aggregates of nucleolar subunits in which fibril-covered regions, the putative active cistrons for precursors of ribosomal RNA, alternate with fibril-free intercepts, the "spacers". The length distribution of the different intercepts of this DNA is given, and the pattern is compared with those shown in animal cell systems. The data are discussed in relation to problems of transcription and of amplification of ribosomal RNA genes.
Electron microscopic spread preparations of oocyte nucleoli (lampbrush stage) of various amphibians are quantitatively evaluated and the length distributions of repeat-, matrix-, and spacer-units along the rRNA cistron containing axes are given. The correlation of the matrix unit data with the gel electrophoretic pattern of labelled nuclear RNA from the same oocytes is examined. The mean value of the matrix unit corresponds fairly well to a 2.6 million D peak of pre-rRNA but the distribution of both matrix units and labelled pre-rRNAs shows an asymmetrical heterogeneity indicating the existence of some larger primary transcription products of rDNA. Novel structural aspects are described in the spacer regions which suggest that transcription does also take place in DNP regions between the matrix units. A special "prelude piece" coding for approx. 0.5 million D of RNA is frequently visualized in the spacer segments at the beginning of a matrix unit. Possible artifacts resulting from the preparation, the relative congruence between the data obtained using both methods, and the functional meaning of the findings are discussed against the background of current concepts of structural organization and transcription products of nucleolar DNA.
Segregation of the nucleolar components is described in the differentiated nucleus of the generative cell in the growing Clivia and Lilium pollen tubes. This finding of a natural nucleolar segregation is discussed against the background of current views of the correlations of nucleolar morphology and transcriptional activity.
The nucleolus
(1994)