Immunohistochemical demonstration of nuclear S1 proteins in various cells

Summary S1 proteins are present in the nuclear structures sensitive to DNases and RNase. To examine localization of these proteins, an antibody was raised in a rabbit. Indirect immunofluorescence staining revealed that S1 proteins located in the extranucleolar nuclear regions of quiescent myocardial and cerebellar cells as well as actively duplicating mouse 3T3 fibroblasts. They located in euchromatin regions of thymus lymphocytes, with a characteristic aster-like immunofluorescence pattern, and on the border of condensed chromatin areas by deposition of immunogold particles in ultrathin sections of thymus. Thus, S1 proteins may be in a nuclear function assigned to the border of heterochromatin areas, and other than synthesis of DNA or of ribosomal RNA. Possible involvement of S1 proteins in the extranucleolar RNA synthesis is discussed.     

Localization of phosphoprotein C23 in nucleoli by immunological methods

Antiserum to a major phosphorylated nucleolar protein. C23 (MW 103000, pI 5.2) from Novikoff hepatoma was produced in rabbits. By immunodiffusion analysis, the antiserum produced precipitin bands and with various crude extracts of nucleoli, but not with extranucleolar or cytosol fractions. The specificity of the antibody was assessed using acid-urea polyacrylamide gel electropherograms of acid-soluble nucleolar proteins in which the separated proteins were transferred to nitrocellulose sheets. The purified antibody reacted predominantly with protein C23 as visualized by the immunoperoxidase procedure. By the indirect immunofluorescence technique, protein C23 was localized predominantly to nucleoli of Novikoff hepatoma or normal rat liver cells. In Novikoff hepatoma cells, traces of fluorescence were seen near the inner layer of the nuclear envelope. Additional narrow regions of fluorescence extended from the nucleoli into the extranucleolar areas of some Novikoff cells. The nucleolar areas of fluorescence were smaller but brighter in the normal liver than in Novikoff hepatoma, consistent with the small size of rat liver nucleoli. These data indicate that the major location of protein C23 is the nucleolus.     

THE ACTION OF α-AMANITIN ON RNA SYNTHESIS IN CHINESE HAMSTER OVARY CELLS : Ultrastructural and Biochemical Studies

α-Amanitin acts in vitro as a selective inhibitor of the nucleoplasmic form B RNA polymerases. Treatment of Chinese hamster ovary (CHO) cells with this drug leads principally to a severe fragmentation of the nucleoli. While the ultrastructural lesions induced by α-amanitin in CHO cells and in rat or mouse liver are quite similar, the results diverge concerning the effect on RNA synthesis. It has been shown that in rat or mouse liver α-amanitin blocks both extranucleolar and nucleolar RNA synthesis. Our autoradiographic and biochemical evidence indicates that in CHO cells high molecular weight extranucleolar RNA synthesis (HnRNA) is blocked by the α-amanitin treatment, whereas nucleolar RNA (preribosomal RNA) synthesis remains unaffected even several hours after the inhibition of extranucleolar RNA synthesis. Furthermore, the processing of this RNA as well as its transport to the cytoplasm seem only slightly affected by the treatment. Finally, under these conditions, the synthesis of the low molecular RNA species (4–5S) still occurs, though less actively. The results are interpreted as evidence for a selective impairment of HnRNA synthesis by α-amanitin in CHO cells.     

Inhibition of nucleolar and chromatin RNA synthesis in Hela cells by excess thymidine

Summary Inhibition of RNA synthesis in Hela cells, evoked by high doses of thymidine, was followed. The author used labelling with ³H-uridine and quantitative autoradiography in two groups of experiments: first, of 4 mM thymidine was being followed for the period of 1, 6, 12 and 24 hours; second, the effect of 0.33, 1, 3, 9 and 27 mM of thymidine for the period of 21 hours. A significant decrease of both the nucleolar and extranucleolar chromatin RNA synthesis was detected as a due to the exposure to 4 mM thymidine for the period of 1–6 hours or to the exposure to 1 mM thymidine for the period of 21 hours. A further deepening of the inhibition of the extranucleolar RNA synthesis appears by protracting the period of action or by increasing the concentration of thymidine in the medium.The author is of the opinion that the inhibition of the nucleolar RNA synthesis is very likely a result of the blockade of the DNA reduplication and of the cell synchronization. The inhibition of the extranucleolar RNA synthesis is on the other hand caused directly, without the intermediate of DNA.     

The effects of zinc chloride on the RNP structures in HEp-2 cells: accumulation of perichromatin granules

The effects of zinc on the ribonucleoprotein (RNP) constituents of HEp-2 cells have been analyzed. Pulse-chase autoradiographic experiments show a preferential inhibition of nucleolar RNA synthesis and a block in the transport of nucleolar and extranucleolar RNA in zinc-treated cells. Concomitantly with the disturbance in RNA metabolism and in protein synthesis, nucleolar condensation, accumulation of perichromatin granules and fibrils, condensation of interchromatin fibrils, and appearance of dense granular bodies occur. Accumulation of perichromatin fibrils and condensation of interchromatin fibrils appear to be related to the block in the transport of heterogeneous nuclear RNA. Depletion of certain proteins required for the assembly of RNP particles could share in the abnormal behavior of RNA and lead to the accumulation of perichromatin granules and the appearance of dense granular bodies.     

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.     

Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro

RNA synthetic activity of human 2-16-cell embryos developing in vitro was studied by [3H]uridine light-microscope autoradiography. Parallelly cut thin sections were examined in the electron microscope. The first extranucleolar RNA synthesis was detected in 4-cell embryos, but nucleoli were never labelled until the 3rd cleavage (6-8-cell embryos). In 6-cell embryos the nucleolar labelling was mostly confined to a narrow peripheral zone. In later cleavage stages most of the blastomeres showed intensive labelling of nucleoli and extranucleolar chromatin. However, rather low levels of extranucleolar RNA synthesis and the absence of nucleolar activity were often seen even in blastomeres of fully compacted morulae. The activation of nucleolar RNA synthesis entailed a noticeable increase in the number of ribosomes (estimated by electron microscope morphometry) that followed a marked drop during the period between the 2-cell and 8-cell stages. The results indicate that the concentration of ribosomes in the preovulatory oocyte is a major factor of its developmental potential.     

Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5alpha-dihydrotestosterone-protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5alpha-dihydrotestosterone-receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5alpha-dihydrotestosterone-receptor complexes than is the ;nucleoplasmic' enzyme. Stimulation occurs less readily in the presence of Mn(2+) and at high ionic strength than in the presence of Mg(2+) and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5alpha-dihydrotestosterone-receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.     

Inhibiting effect of the new cytotoxic antibiotic daunomycin on nucleic acids and mitotic activity of HeLa cells

The effect has been studied of Actinomycin D, Daunomycin (Da.), and Da. N acetyl derivative on mitotic activity and on the nucleic acid synthesis of in vitro HeLa cell cultures. The experiments were carried out by means of the radioautographic technique using stripping films. The relative uptake of thymidine-H³ and uridine-H³ was determined by means of the reduced silver grain count present in the nuclei of controls and treated cells. The mitotic activity and thymidine incorporation were noticeably reduced by Daunomycin and Actinomycin, whereas both processes appeared less affected by Da. N acetyl derivative. As regards nuclear RNA synthesis, all three antibiotics at low doses chiefly inhibit nucleolar RNA synthesis. On the other hand, whilst Actinomycin at higher doses causes an almost total inhibition of the synthesis of the whole nuclear RNA, in Daunomycin- and Da. N acetyl derivative-treated cells extranucleolar RNA synthesis is less susceptible to inhibition.     

Delayed flow-through cytoplasm of newly synthesized Balbiani ring 75S RNA.

With a nonaqeous microdissection technique, the cytoplasm of Chironomus salivary gland cells can be separated into concentric zones situated at increasing distances from the nuclear envelope. This dissection technique is used here to investigate the cytoplasmic distribution of 75S RNA of Balbiani ring origin. The Balbiani ring 75S RNA has properties of a messenger RNA coding for secretory proteins. After a pulse of RNA precursor to the living animal, labeled Balbiani ring 75S RNA is found mainly in the cytoplasm located closer to the nuclear envelope, with smaller amounts toward the periphery of the cell. This gradient, initially very steep, lasts for a least 2 days, but less than 6 days. Experiments with 5-fluorouridine indicate that the formation of the gradient does not depend upon simultaneous export of ribosomal subunits. After a pretreatment of the animals with the protein synthesis inhibitor cycloheximide, however, newly synthesized 75S RNA distributes evenly in the cytoplasm-that is, this treatment prevents the formation of the 75S RNA gradient. The gradient in salivary glands of normally cultured animals is therefore likely to be the result of diffusion restriction of the labeled 75S RNA. Thus the 75S RNA located closer to the nuclear envelope is the most recently exported 75S RNA. An explanation of these results is the the 75S RNA associates with the membranes of the endoplasmic reticulum early or immediately after nuclear release. This association should occur in the cytoplasm surrounding the nucleus and may occur either as single particles and/or as parts of polysomes.     

Autoradiographic study of RNA synthesis in adrenal cells of the young rat in primary culture

Primary cultures of young rat adrenal cells were used in a radioautographic study of the effect of ACTH on RNA synthesis. Control and ACTH-stimulated cultures were incubated with 5.6 [3H]-uridine for 30 min and chased for 15 and 30 min. Labelling over nucleolar and extranucleolar areas was always lower in the ACTH-stimulated animals at all experimental times. At 30 min chase-time a relatively high labelling of nuclei in both experimental conditions suggest the slow metabolism of RNA in adrenal. The results confirm the inhibitor effect of adrenocorticotrophin on [3H]-uridine incorporation.     

A human common nuclear matrix protein homologous to eukaryotic translation initiation factor 4A

Amino acid sequencing and mass spectrometry revealed identity of a human nuclear matrix protein, termed hNMP 265, with a predicted protein of gene KIAA0111. Two-dimensional electrophoresis and Northern hybridization showed the protein to ubiquitously occur in various human cell types. Exhibiting DEAD-box motifs characteristic for RNA helicases, hNMP 265 is highly similar to the human initiation factors eIF4A-I and -II. On the other hand, hNMP 265 greatly differs from the initiation factors by a N-terminal sequence rich in charged amino acids. Sequence searches and alignments indicate proteins related to hNMP 265 in other eukaryotes. Chimeras between hNMP 265 and green fluorescence protein or hapten appeared as speckles in extranucleolar regions in the nucleus, but not in the cytoplasm. Experiments with tagged deletion mutants indicated that the N-terminal amino acid sequence is necessary for nuclear localization. A putative role of hNMP 265 in pre-mRNA processing is discussed.     

Effects of alpha-amanitin, cycloheximide, and thioacetamide on low molecular weight nuclear RNA.

Studies were made on the effects of alpha-amanitin, cycloheximide, and thioacetamide on synthesis and content of low molecular weight nuclear RNA. Cycloheximide, an inhibitor of protein synthesis and the synthesis of 45S pre-rRNA and 5S RNA, also inhibited synthesis of nuclear U1 and U3 RNAs. alpha-Amanitin, an inhibited the synthesis of U1 and U2 low molecular weight nuclear RNA. Thioacetamide, which induces nucleolar hypertrophy and increased nucleolar RNA polymerase activity, markedly increased synthesis of 5.8S RNA and U3 RNA. These results show that syntheses of individual low molecular weight nuclear (LMWN) RNAs are controlled by different regulatory mechanisms. In particular, there appears to be a specific relationship between U3 RNA and functional states of the nucleolus.     

Electron microscopic and radioautographic data on the synthesis of RNA and chromatin structure in cells of the rat cerebral cortex

The intensity of RNA synthesis in cells of the rat brain hemispheres (neurons, astrocytes, oligodendrogliocytes, microgliocytes) was studied by electron microscopic radioautography, and the data obtained were compared with dispersed to condensed chromatin area ratio. The correlation was found between the level of RNA synthesis and dispersed chromatin area. High chromatin dispersity in neuron and intensive NA synthesis in the extranucleolar part of the nucleus made it possible to assume the existence of depression of an especially large genome part and the variability of the proteins produced by this cell.