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- Theodor-Boveri-Institut für Biowissenschaften (48) (remove)
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.
A monoclonal murine antibody (No-I 14) is described which reacts specifically with a polypeptide of molecular weight (M,) 180000 present in low-speed nuclear pellets from oocytes and somatic cells of Xenopus laevis and X. borealis and in isolated amplified nucleoli. Two-dimensional gel electrophoresis has revealed the acidic nature of this polypeptide (isoelectric at pH of ca 4.2 in the presence of 9.5 M urea). A relatively large proportion of the protein is extracted at elevated ionic strength( i.e., at 0.4-0.5 M alkali salt) in a form sedimenting at approx. 7-8S , compatible with a monomeric state. It is also extracted by digestion with RNase but not with DNase. In immunofluorescence microscopy, antibody No-114 stains intensely nucleoli of oocytes and all somatic cells examined , including the residual nucleolar structure of Xenopus erythrocytes which are transcriptionally inactive. During mitosis the antigen does not remain associated with the nucleolar organizer regions (NOR) of chromosomes but is released and dispersed over the cytoplasm until telophase when it re-associates with the reforming interphase nucleoli. At higher resolution the immunofluorescent region is often resolved into a number of distinct subnucleolar components of varied size and shape. Immunoelectron microscopy using colloidal gold-coupled secondary antibodies reveals that the M, 180000 protein is confined to the dense fibrillar component of the nucleolus. This conclusion is also supported by its localization in the fibrillar part of segregated nucleoli of cells treated with actinomycin D. We conclude that nucleoli contain a prominent protein of M, 180000 which contributes to the general structure of the dense fibrillar component of the interphase nucleolus , independent of its specific transcriptional activity.
High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody
(1987)
A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.
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.
The disintegration of the nuclear envelope has been examined in nuclei and nuclear envelopes isolated from amphibian oocytes and rat liver tissue, using different electron microscope techniques (ultrathin sections and negatively or positively stained spread preparations). Various treatments were studied, including disruption by surface tension forces, very low salt concentrations, and non ionic detergents such as Triton X-lOO and Nonidet P-40. The high local stability of the cylinders of nonmembranous pore complex material is emphasized. As progressive disintegration occurred in the membrane regions, a network of fibrils became apparent which interconnects the pore complexes and is distinguished from the pore complexassociated intranuclear fibrils. This network might correspond to an indistinct lamella, about 15 - 20 nm thick, located at the level of the inner nuclear membrane, which is recognized in thin sections to bridge the interpore distances. With all disintegration treatments a somewhat higher susceptibility of the outer nuclear membrane is notable, but a selective removal does not take place. Final stages of disintegration are generally characterized by the absence of identifiable, membrane- like structures. Analysis of detergent-treated nuclei and nuclear membrane fractions shows almost complete absence of lipid components but retention of significant amount of glycoproteins with a typical endomembrane-type carbohydrate pattern. Various alternative interpretations of these observations are discussed. From the present observations and those of Aaronson and Blobel (1,2), we favor the notion that threadlike intrinsic membrane components are stabilized by their attachment to the pore complexes, and perhaps also to peripheral nuclear structures, and constitute a detergent-resistant, interpore skeleton meshwork.
Nuclei of amphibian oocytes contain large amounts of actin, mostly in unpolymerized or short-polymer form. When antibodies to actin or actin-binding proteins (fragmin and the actin modulator from mammalian smooth muscle) are injected into nuclei of living oocytes of Pleurodeles waltlii, transcription of the lampbrush chromosomes, but not of the rRNA genes, is inhibited. When transcription is repressed by drugs or RNA is digested by microinjection of RNAase into oocyte nuclei, an extensive meshwork of actin filament bundles is seen in association with the isolated lampbrush chromosomes. These observations indicate a close relationship between the state of nuclear actin and transcriptional activity and suggest that nuclear actin may be involved in transcriptional events concerning protein-coding genes.
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.
Semi -iso la ted annul a te lamellae were prepared from single newt oocy tes (Triturus alpestris) by a modified Call a n-T omlin technique. Such preparations were examined with the electron mi croscope, and the negative sta ining a ppearance of th e a nnulate lamellae is described . The annul a te lamellae can be de tected either adhering to the nuclear envelope or being detached from it. Sometimes they a re obse rved to be connected with slender tubular-like structures interpreted as pa rts of the endoplasmic reti culum. The results obta ined from negativ e sta ining a re combined with those from sections. Especially, the structural data on th e a nnula te lamellae and the nuclear envelope of the very same cell were compa red . Evidence is presented th a t in the oocytes studied the two kinds of porous cisternae, n amely a nnul a te lamellae and nuclear envelope, a re markedly distinguished in that the annul a te lamellae ex hibit a much higher pore frequency (generally about twice tha t found for the corresponding nuclear envelope) and have al so a rela tive pore area occupying as much as 32 % to 55 % of th e cistern al surface (compa red with 13 % to 22 % in the nuclear envelopes). T he pore di ame ter a nd all other ultras tructural details of the pore complexes, however, a re equi valent in both kinds of porous cisternae. Like the annuli of the nuclear pore complexes of various a nimal and pl ant cells, the a nnuli of the a nnula te lamellae pores reveal al so an eightfold symmetry of their subunits in negatively stained as well as in ectioned ma teria l. Furthermore, th e a nnul a te lamellae a re shown to be a site of activity of the Mg-Na-Kstimul a ted ATPase.