Francesco Novello

The Ag-NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nucleoli

The relationship between the Ag-NOR (silverstained Nucleolar Organizer Region) proteins and the functional-structural organization of the nucleolar ribosomal chromatin was studied in regenerating and cortisol-stimulated rat hepatocytes. Statistical analysis of Ag-NOR proteins, carried out with an automated image analyzer, indicated that in regenerating rat hepatocytes the quantity of Ag-NOR proteins mainly increased between the 4th and 12th h of regeneration, reaching a level twice that of resting hepatocytes. Also the synthesis of pre-ribosomal RNA (pre-rRNA) was stimulated after the 4th of regeneration. Cycloheximide administered to rats at a dose of 0.025 mg/100 g body weight (bw) prevented any increase in Ag-NOR proteins but did not hinder the stimulation of pre-rRNA snythesis. In 8 h cortisol-stimulated hepatocytes no significant change in amount of Ag-NOR protein was observed whereas pre-rRNA synthesis was highly increased as in 12 h regenerating hepatocytes. These results indicated that in rat hepatocytes Ag-NOR proteins and stimulation of pre-rRNA synthesis are not related. The relationship between the Ag-NOR proteins and the distribution of the completely extended intranucleolar ribosomal chromatin was also studied in regenerating rat hepatocytes. At 12 h after partial hepatectomy an increased amount of completely extended ribosomal chromatin was observed, contemporaneously with an increased quantity of Ag-NOR proteins. These ribosomal chromatin changes preceded the beginning of DNA synthesis and were prevented by cycloheximide-induced inhibition of protein synthesis.

Spatial redistribution of ribosomal chromatin in the fibrillar centres of human circulating lymphocytes after stimulation of transcription

We have studied the distributional changes of the completely extended ribosomal chromatin present in the fibrillar centres of resting human lymphocytes after phytohemagglutinin (PHA) treatment. In thin sections of resting lymphocytes selectively stained for DNA, the extended non-nucleosomal chromatin was located in a solitary, large agglomerate which corresponds to the solitary, large fibrillar centre observed in uranium-lead-stained sections. At 20 h after PHA stimulation the ribosomal chromatin agglomerate appeared to be fragmented into smaller agglomerates which correspond to numerous fibrillar centres surrounded by a thick rim of dense fibrillar component. The mean area of ribosomal chromatin agglomerates from resting lymphocytes was found to be 0.772 mu 2 + 0.125 SD, whereas in stimulated lymphocytes it was found to be 0.184 mu 2 + 0.052 SD. At 20 h after PHA treatment ribosomal RNA (rRNA) synthesis was 8-fold greater than the control value, whereas DNA synthesis had not started. These results indicate that ribosomal chromatin of resting lymphocyte fibrillar centres contains transcribable sequences, temporally not expressed.

Structure of ribosomal genes of mammalian cells in situ

The structural and functional organization of ribosomal genes was investigated in situ in human circulating lymphocytes and a human tumour cell line, TG cells. Stereo-pair electron micrographs revealed that this ribosomal chromatin is not structured into nucleosomes, but composed of completely extended filaments, 2–3 nm thick. Despite its homogeneous morphological structure only a small portion of ribosomal chromatin present in the dense fibrillar component is transcriptionally active. This was demonstrated in TG cells by exclusive autoradiographic labelling on serial sections of the dense fibrillar component with 3H-uridine and by the distribution of RNase-gold particles in all the ribonucleoprotein (RNP) structures but not in the fibrillar centres. The extended, non-nucleosomal configuration of both transcriptionally inactive and active ribosomal chromatin could be explained by the peculiar protein composition of this chromatin. Staining with the acrolein-silvermethenamine technique for basic proteins indicated that all the completely extended ribosomal chromatin is devoid of histones, even after inactivation of transcription by actinomycin D. Stereo-electron-microscopical visualisation of the Ag-NOR proteins revealed a thread-like structural organization of these proteins with a spatial distribution superimposable on that of the ribosomal chromatin filaments.

Perichromatin fibrils and chromatin ultrastructural pattern

Abstract The relationship between the synthesis of acidic nuclear proteins, phosphoproteins, RNA and chromatin ultrastructural pattern was studied in regenerating rat hepatocytes after partial hepatectomy. α-Amanitin induced, as early as 30 min after injection, a reduction of RNA synthesis to about 50% of the control level; the degree of inhibition had remained the same at 2 h after poisoning. No change was detected either in acidic nuclear protein synthesis or in phosphorylation for the whole time examined. The DNA-containing structures, demonstrated by the Gautier staining procedure, were in a dispersed pattern either in untreated regenerating hepatocytes or 30 min after α-amanitin administration to rats; but they did appear in a condensed form 1 h and more especially 2 h after toxin injection. In untreated regenerating hepatocytes, the regressive EDTA staining method for RNP revealed a large quantity of perichromatin fibrils which remained unchanged 30 min after α-amanitin treatment and were diminished at 1 h and strongly reduced 2 h thereafter. Cycloheximide treatment promptly reduced the synthesis of nuclear acidic proteins while leaving unchanged the synthesis of RNA; the quantity of perichromatin fibrils and the loosened appearance of DNA-containing structures were the same as in the control rat nuclei. Our results showed that the ultrastructural pattern of chromatin was not directly related either to the synthesis of RNA or to acidic nuclear proteins or to the phosphorylation of phosphoproteins; on the contrary, a strict relationship with the quantity of perichromatin fibrils was demonstrated. The possible interaction of perichromatin fibrils with other chromatin components was discussed as a possible regulatory mechanism of chromatin pattern.

Relationship between the extended, non-nucleosomal intranucleolar chromatin in situ and ribosomal RNA synthesis

In the present study we have investigated the relationship between the structure of intranucleolar chromatin in situ and the synthesis of rRNA. Using thin sections selectively stained for DNA we observed that intranucleolar chromatin of cortisol-stimulated rat hepatocytes consisted of clumps and fibres, both showing a nucleosomal configuration, and by loose agglomerates of extended DNA filaments, with a thickness of 2-3 nm, which never formed nucleosomal structures. After inhibition of rRNA synthesis by actinomycin D, the agglomerates of extended DNA filaments without nucleosomal configuration were noticed to be still present even at 3 h after drug treatment. In human resting lymphocytes, with a very low rate of rRNA synthesis, a large roundish, loose agglomerate of DNA filaments without nucleosomal configuration was found in the central zone of the nucleolar body. After a 16-fold increase in rRNA synthesis induced by a 48-h stimulation with phytohemagglutinin (PHA) the loose agglomerates appeared to be more numerous, but markedly reduced in size. We concluded that the extended non-nucleosomal configuration is a permanent feature of intranucleolar loose chromatin agglomerates and not a consequence of transcriptional activity.

Relationship between the fine structural organization of chromatin and nucleic acid synthesis in regenerating rat hepatocytes.

The changes in the fine structure of chromatin induced in regenerating rat hepatocytes by hydroxyurea and α-amanitin have been studied in thin sections stained for DNA with the Fuelgen-like method of Cogliati and Gautier (1973, C.R. Acad. Sci. Ser. D 276, 3041–3044). At 24 hr after partial hepatectomy, concurrently with a high synthesis of DNA and RNA, the chromatin appeared mainly in the dispersed form. Both the extranucleolar and the nucleolar chromatin were particularly enriched in the thin fibers, with a thickness of about 12 nm, as previously described ( Derenzini, 1979 , J. Ultrastruct. Res. 69, 239–248). The treatment with hydroxyurea inhibited almost completely the synthesis of DNA, 30 and 60 min after injection, while it left unchanged the synthesis of RNA. No change in the gross chromatin ultrastructure and in the presence of the 12-nm fibers in the dispersed chromatin was noticed. α-Amanitin inhibited the synthesis of heterogeneous and ribosomal RNA to the same extent 1 and 2 hr after poisoning whereas it did not alter the synthesis of DNA. No modification in the gross ultrastructural pattern of chromatin was noticed at 1 hr after toxin injection whereas the 12-nm fibers in the nucleolar and extranucleolar dispersed chromatin were clearly reduced. Thicker 20-nm fibers became concurrently visible; these fibers are composed of knobby structures peculiar to the condensed chromatin. The gross chromatin ultrastructure was greatly modified at 2 hr after α-amanitin treatment when a marked clumping of the 20-nm fibers gave rise to the formation of highly condensed chromatin masses.

Relationship between ultrastructure and function of hepatocyte chromatin: A study with adrenalectomized rats after cortisol administration

Using either the routine staining procedure or the Feulgen-like Gautiers method the condensed pattern of hepatocyte chromatin in adrenalectomized rats appeared unchanged 1 hr after cortisol administration, whereas at 4 hr and especially at 8 hr it looked more dispersed, in parallel with an increased synthesis of RNA and nuclear nonhistone proteins (NHP). The quantity of [3H]cortisol bound to deoxyribonucleoproteins (DNP) was the same at 1 and 4 hr after hormone treatment and it was unaffected by amanitin, which induced a chromatin condensation. Eight hours after cortisol injection almost all the DNA structures, revealed by the Gautier technique, were in the unraveled form; concurrently, a large quantity of perichromatin fibrils was visualized with the Bernhard method for ribonucleoproteins (RNP). Amanitin poisoning of 8-hr stimulated rats induced, 1 hr after treatment, a light condensation of the DNA structures and a strong condensation after 2 hr; the perichromatin fibrils were lightly reduced 1 hr after toxin injection and had almost disappeared 2 hr thereafter. Amanitin reduced RNA synthesis to about 25% of control value both 1 and 2 hr after poisoning. No modification of NHP synthesis was observed after toxin treatment. Our results demonstrating a strict relationship only between the chromatin pattern and the quantity of perichromatin fibrils, morphological expression of heterogeneous nuclear (hn) RNA which has been synthesized (J. P. Bachellerie, E. Puvion, and J. P. Zalta,Eur. J. Biochem. 58, 327, 1975; S. Fakan, E. Puvion, and G. Spohr,Exp. Cell Res. 99, 155, 1976), suggest that the chromatin ultrastructural changes induced by cortisol are a consequence and not a cause of gene transcriptional activity.

Electron-microscopical evidence that ribosomal chromatin of human circulating lymphocytes is devoid of histones☆

We have studied the distribution of histones in the nucleolus of human circulating lymphocytes in situ, using thin sections, either treated with antibodies against the core histones revealed by colloidal gold, or stained with the acrolein-silver methenamine technique for basic proteins. Gold particles were not found in the fibrillar centre, nor were silver-stained structures visible in this nucleolar component. Since the fibrillar centre contains the bulk of the ribosomal chromatin which is in a completely extended, non-nucleosomal configuration, our results indicate that this chromatin is devoid of histones.

Effect of α-amanitin poisoning on the synthesis of deoxyribonucleic acid and of protein in regenerating rat liver

Abstract The synthesis of DNA and of protein in regenerating liver was studied in rats poisoned with non-lethal doses of α-amanitin, sufficient to inhibit RNA polymerase II. When α-amanitin was given at 20 h after partial hepatectomy, i.e. in coincidence with the peak of DNA synthesis, this was markedly inhibited within 2–3 h of poisoning. When rats were poisoned at 16 h after operation the peak of DNA synthesis was delayed. α-Amanitin affected protein synthesis with the same time course as, but less severely than DNA synthesis.

Inhibition of ribonucleic acid and of protein synthesis in the organs of rats and mice poisoned with α-amanitin

Abstract The synthesis of RNA in vivo, the activity of RNA polymerase of isolated nuclei, and the synthesis of protein in vivo have been studied in the liver and kidney of rats and mice, at various times after administration of α-amanitin. The synthesis of RNA is impaired reversibly in the liver of both species and irreversibly in mouse kidney, whereas it is almost unaffected in rat kidney. Almost parallel changes were observed in the activity of RNA polymerase II of isolated nuclei. Protein synthesis was decreased in mouse liver and kidney, and much less in rat organs. The administration of α-amanitin was followed by disaggregation of polysomes in mouse liver; microsomes isolated from the same organ had a reduced capacity of incorporating phenylalanine into protein in vitro, which could be restored by addition of polyuridylic acid.