O. V. Zatsepina

The RNA polymerase I-specific transcription initiation factor UBF is associated with transcriptionally active and inactive ribosomal genes

We have characterized an anti-NOR (nucleolar organizer region) serum (P419) from a patient with rheumatoid arthritis and show that it contains antibodies directed against the RNA polymerase I-specific transcription initiation factor UBF. This serum reacts with UBF from a variety of vertebrate cells as revealed both by immunoblotting and by indirect immunofluorescence. We have used the P419 serum to study the intracellular localization of this transcription factor at the light and electron microscopic level. In interphase cells, UBF exhibits a pronounced punctate pattern and is found to be associated with necklace-like structures, which appear to reflect the transcriptionally active state of the nucleolus. Inhibition of rRNA synthetic activity caused either by nutritional starvation or by actinomycin D treatment resulted in a marked decrease in the number and in a significant increase in the size of UBF-positive granules. Under all experimental conditions applied, UBF was exclusively found within the nucleolus and was not released into the nucleoplasm or cytoplasm. During mitosis, UBF was found to be concentrated at the chromosomal NOR indicating that a significant quantity, if not all, of this factor remains bound to the ribosomal transcription units. From this we conclude that UBF is associated both with transcriptionally active and inactive rRNA genes and, therefore, changes in the intracellular localization of UBF are very likely not involved in rDNA transcription regulation.

Constitutive and strong association of PAF53 with RNA polymerase I

Abstract.u2002Mouse RNA polymerase I (Pol I) is composed of 14 polypeptides, 3 of which are thought to be loosely associated with, and may be dislodged from, Pol I. To find out whether these polymerase-associated factors (PAF53, 51, and 49) serve a role in growth-dependent control of rDNA transcription, we generated polyclonal antibodies against three subunits of murine Pol I, RPA116, RPA40 and PAF53, and used different experimental approaches, e.g. immunoblot analysis, immunoprecipitation and immunofluorescence studies, to compare the stoichiometry of individual subunits both in different Pol I preparations and in extracts from cells grown under different conditions. This comparative analysis reveals that the molar ratio of the second largest subunit RPA116 to PAF53 is the same, irrespective of whether crude extracts or highly purified Pol I fractions are analyzed. Significantly, the relative level of PAF53 was comparable in exponentially growing or growth-arrested cells, indicating that growth-dependent fluctuations in Pol I activity are not accompanied by alterations in the amount of PAF53. In addition, we show by high resolution immunofluorescence analysis that, under conditions of repressed rDNA transcription, including serum starvation, actinomycin treatment und during mitosis, PAF53 remains attached to the transcriptional machinery. The finding that the Mr 53,000 protein remains in the multiprotein complex under all experimental conditions tested indicates that PAF53 is not a loosely associated regulatory factor but a bona fide subunit of Pol I.

EXPERIMENTAL INDUCTION OF PRENUCLEOLAR BODIES (PNBS) IN INTERPHASE CELLS :INTERPHASE PNBS SHOW SIMILAR CHARACTERISTICS AS THOSE TYPICALLY OBSERVED AT TELOPHASE OF MITOSIS IN UNTREATED CELLS

Recently, it was shown that a short exposure of living mammalian cells to low ionic strength buffers (hypotonic shock) caused partial or almost complete unraveling of interphase nucleoli. However, when the cells were released from the hypotonic shock and transferred to normal isotonic medium, functionally active and structurally integral nucleoli were reassembled at their initial positions within interphase nuclei. Here, we show further that this process is accompanied by the appearance of numerous discrete extranucleolar bodies, which have striking similarities to the prenucleolar bodies (PNBs) observed in untreated cells at telophase of mitosis. (1) Like PNBs at mitosis, hypotonically induced interphase PNBs are composed of RNA-positive granules and fibrils, contain the major nucleolar protein B23 and silver-binding proteins, but lack DNA and RNA polymerase I transcription factor UBF. (2) As for mitotic PNBs, disappearance of the interphase PNB counterparts coincides with the increase in size of reconstructed nucleoli. (3) Addition of actinomycin D does not prevent assembly of interphase PNBs, but does arrest their coalescence with the chromosomal nucleolus-organizing regions and blocks the complete reformation of nucleoli. It is concluded that the assembly of PNBs generally observed at telophase of mitosis can be induced experimentally in nuclei of interphase mammalian cells in vivo. At interphase, this process is probably initiated by changes in the intracellular ionic environment.

A higher concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies

Transient transfection of HeLa cells with a plasmid encoding the full-length human fibrillarin fused to a green fluorescent protein (GFP) resulted in two major patterns of intensity of the nucleolar labeling for the chimeric protein: weak and strong. Both patterns were maintained in fibrillarin-GFP expressing cells after fixation with formaldehyde. When the fixed fibrillarin-GFP expressing cells were used for immunolabeling with antibodies to fibrillarin, only the nucleoli with a weak GFP-signal became strongly labeled, whereas those with the heavy signals were only lightly stained, if at all. A similar pattern was observed if the cells were immunolabeled with antibodies to GFP. These observations suggest that an increase in antigen accumulation within the nucleolus, which could take place under various physiological or experimental conditions, could prevent the antigen from being recognized by specific antibodies. These results have implications regarding contradictory data on localization of various nucleolar antigens obtained by conventional immunocytochemistry.

Reversible differential decondensation of unfixed Chinese hamster chromosomes induced by change in calcium ion concentration of the medium

Differential decondensation of isolated unfixed Chinese hamster metaphase chromosomes was obtained by decreasing the calcium ion concentration in the surrounding medium. A banded appearance of the swollen chromosomes could be observed either directly by phase contrast microscopy or after glutaraldehyde fixation and staining. There was a gradual transition from homogeneously dense to banded and finally to extensively decondensed chromosomes. The patterns induced at different stages were similar to those observed on fixed chromosomes after standard banding procedures (i.e., G-, C-, Cd−, Ag-NOR-staining). Chromosome decondensation could be reversed by the addition of calcium ions to the medium. Ca++-dependent reversible differential chromosome decondensation was not observed if the chromosomes were previously treated with 0.35 M NaCl. Chromosome regions which had incorporated BrdU into their DNA were more resistant to a decrease in calcium ion concentration than BrdU non-substituted regions.

Isolation of the Chromocenter Fraction from Mouse Liver Nuclei

A new method for isolation of the constitutive heterochromatin (chromocenters) from interphase nuclei of mouse liver has been developed. This method allows separation of chromocenters of different size. Chromocenter fractions are essentially free of nucleoli and other contaminants. In contrast to nuclei and nucleoli, the chromocenter fraction is characterized by simpler protein composition, this fraction having a reduced number of proteins (especially high molecular weight proteins). Chromocenters contain all histone fractions; however, the relative proportion of histone H1 is lower and histone H3 is higher than in the total nuclear chromatin. The amount of non-histone proteins of 51, 63, 73, and 180 kD is higher in the chromocenter fraction than in nuclei and nucleoli. The use of immunocytochemistry and immunoblotting methods revealed the presence of the specific kinetochore component, CENP A protein. This suggests tight association of some molecular kinetochore components with chromocenters in the interphase.

The topology of early- and late-replicating chromatin in differentially decondensed chromosomes

In this study we used a novel technique to reveal both longitudinal and transverse differentiation within mammalian mitotic chromosomes. Structural changes in chromosomes that we term ‘differential decondensation’ were produced in cells that were first incubated in hypotonic medium (15% Hanks’ solution), then adapted to normotonic conditions and thereafter exposed to a second short hypotonic shock. Such a double hypotonic treatment (DHT) is not critical for cell viability, but considerably elongates the G2 phase of the cell cycle. Giemsa staining of differentially decondensed chromosomes corresponds to standard G-banding, but does not need the standard post-fixation treatment. Using ‘dynamic’ BrdU banding, we show that such ‘differential’ staining is a result of differential resistance of the R- and G-bands to DHT. Thus, early-replicating foci, markers of R-bands, are localized in the peripheral chromatin halo, whereas late-replicating foci, corresponding to G-bands, remain associated with the axial regions of chromatids. Remarkably, despite these major changes in the structure of the chromosomal bands, the replication foci still preserve their discrete structure.

Structural-functional model of the mitotic chromosome

In the present review the structural role of noncoding DNA, mechanisms of differential staining of mitotic chromosomes, and structural organization of different levels of DNA compactization are discussed. A structural-functional model of the mitotic chromosome is proposed based on the principle of discreteness of structural levels of DNA compactization.

Isolation of the constitutive heterochromatin from mouse liver nuclei.

A method for isolation of constitutive heterochromatin (chromocenters) from nuclei of mouse liver cells is described. This method is based on the higher resistance of chromocenters to low ionic strength treatment as compared with that of nucleoli and euchromatin. The method allows separation of chromocenters that are essentially free of nucleoli and other nuclear contaminants. In contrast to nuclei and nucleoli, isolated chromocenters are characterized by a simpler protein composition and contain a smaller number of proteins (especially of high molecular weight proteins). They possess telomeric DNA and telomerase activity that suggests a tight association of chromocenters with the telomerase complex in mouse hepatocyte nuclei.

Cytological indicators of overall suppression of protein synthesis revealed by staining with a new monoclonal antibody

In this work we have described the nucleolus response on inhibition of protein synthesis visualized by labeling with a new monoclonal antibody. An A3 antigen is observed in the form of numerous foci within a nucleolus and as a few foci over chromosomes in mitotic cells. Regions of A3 antigen localization are susceptible to pepsin treatment but are insensitive to RNase A treatment. This fact indicates that A3 antigen is a protein. On ultrastructural level A3 antigen is localized predominately at the periphery of fibrillar centers. Taken together, A3 antigen properties make it possible to suggest that it is a component of RNA polymerase I transcription machinery. A3 antigen has an intriguing property, namely, the ability to migrate from the nucleolus to the nucleoplasm upon the inhibition of protein synthesis with anisomycin, puromycin, and cycloheximide. The results show that localization of the A3 antigen revealed by the new monoclonal antibody may serve as a cytological indicator of the overall protein synthesis in vitro.