William J. Steele

Synthesis of 28-S RNA in the nucleolus.

Abstract RNA of isolated nucleoli of rat liver was subjected to sucrose density-gradient centrifugation. The major components were 45-S, 35-S and 28-S RNA, although a smaller peak of 6-S RNA and several other minor components were also present. Extranucleolar nuclear RNA contained mainly 28-S, 18-S and 6-S RNA. With radioactive orotic acid as a precursor and with the aid of actinomycin D as a blocking agent of new RNA synthesis, evidence was obtained that the 45-S RNA is biosynthesized in the nucleolus and converted there into 35-S RNA and 28-S RNA. It was found that the nucleolar RNA contains little 18-S RNA. The base composition of whole nucleolar RNA supports the evidence that the nucleolus synthesizes precursors of the 50–60-S subunits of the ribosomes. Evidence for the rapid turnover of the 45-S RNA in nucleoli was provided by studies on the kinetics of the decay of the 45-S peak in actinomycin D-treated nucleoli which showed that the half-life of the 45 S was approx. 8 min.

Isolation of Nucleoli

The studies which have been presented have provided support for the concept that the nucleolus is an important center of ribonucleic acid metabolism in the cell. They suggest that the activity of the nucleolus may be independent of the overall ribonucleic acid synthesis generally believed to occur on the double strands of chromosomal DNA. Apparently, the nucleolus does not function simply as a storehouse for ribonucleic acids synthesized on the chromatin, since a block of most of the RNA synthesis in the nucleus does not affect the rate of synthesis in the nucleolus. The role of the nucleolus in synthesis of RNA for cytoplasmic particles has been commented on previously, particularly in connection with the interesting studies on enucleolation by Perry et al. [30–32]. It appears that there are two parts to the biosynthesis of ribosomes, one of which occurs in the nucleolus as has been pointed out by these authors. The second is that in which the chromatin plays an important part in either directing the release of ribosomal particles or in the completion of these particles so that they may be released into the cytoplasm. Thioacetamide has proved to be an interesting tool for separating the synthetic activities of the nucleolus from those of the remainder of the nucleus. Further studies on the composition of the nucleoli may provide evidence for the individuality of the types of RNA formed by the nucleolus and the chromatin, and perhaps may answer some questions regarding the differences between the synthetic activities in neoplastic cells and other cells.

Ultrastructural aspects of the ribonucleo-protein network in nuclei of walker tumor and rat liver☆

Abstract Electron microscopic studies were made on isolated nuclei of Walker tumor and rat liver extracted with dilute and concentrated salt solutions. The presence of a dense network of fibrils and particles attached to the nucleolus was demonstrated in the Walker tumor. This network corresponds in appearance and location to the nuclear ribonucleoprotein network observed previously by light microscopy [16]. After the extractions with saline solutions, the particles remained in the network but were largely absent from the nucleoli. These particles are similar to the interchromatinic particles seen in unextracted nuclear preparations and in nuclei in situ. Electron microscopic studies after enzymatic digestion of freshly isolated rat liver nuclei with RNase, DNase and pepsin show that the dense particles in the nuclear ribonucleoprotein network are composed largely of ribonucleoproteins.

Studies on the ribonucleic acid components of the nuclear ribonucleoprotein network.

Abstract Parallel studies were carried out on the sedimentation and base composition of RNA of the nuclear residue fraction. Comparative studies were performed on the RNA of isolated nucleoli. To obtain the nuclear residue fraction, isolated nuclei were fractionated by successive extraction with salt solutions into (a) the “nuclear sap”, (b) the deoxyribonucleoprotein fraction and (c) the nuclear residue fraction. The main difference between the sedimentation profiles of RNA of nucleoli and the nuclear residue is the polydispersity of the rapidly sedimenting RNA and the greater amount of 18-S RNA and 23-S RNA in the nuclear residue. A difference profile suggests that a number of types of RNA are present in the nuclear residue with sedimentation coefficients different from the RNA of isolated nucleoli. Studies of the rapidly labeled RNA ([32P]Pi or [14C]orotate) and total RNA of the nuclear residue fraction following administration of actinomycin D indicate that the rapid decrease in the amount of RNA in this fraction was correlated with the inhibition of synthesis of rapidly labeled, rapidly sedimenting G-C-rich RNA. Sequentially, 55-S RNA, 45-S RNA, 35-S RNA and 28-S RNA of isolated nucleoli and the nuclear residue decreased in amount and isotope when they were prelabeled. Turnover studies revealed that the half-life of nucleolar 55-S RNA and 45-S RNA were approx. 8.5 min and 11 min, respectively. The main difference between the sedimentation profiles of RNA of nucleoli and the nuclear residue 1 h after the administration of actinomycin D is the presence of 18-S RNA in the nuclear residue. The nuclear residue fraction is also a site of synthesis of rapidly sedimenting, rapidly labeled A-U-rich RNA as shown by (a) the continued labeling of the polydisperse rapidly sedimenting RNA after the inhibition of G-C-rich nucleolar RNA by actinomycin D and (b) the progressive increase in the A-U content of RNA in this fraction as the dose of actinomycin D is increased, and as time increases after treatment with actinomycin D. Studies on the rates of labeling of 18-S and 28-S RNA in the cytoplasm indicate that the specific activity of 18-S RNA is decreased less markedly than that of 28-S RNA following administration of actinomycin D (150–450 μg/kg body weight). These results provide support for the concept that 18-S RNA and 28-S RNA are synthesized at different sites in the nucleus, presumably in the strands of the nuclear ribonucleoprotein network and the nucleolus, respectively.

NUCLEAR PROTEINS OF NEOPLASTIC CELLS.

Publisher Summary Proteins and enzymes of the nucleus are important for the synthetic reactions involved in neoplastic cells and to the aberrations of growth that characterize these cells. It has been difficult to isolate and purify the proteins of the nucleus but in recent years, the enzymatic activities of some of these proteins have been elucidated. In studies on the nuclear proteins, it is important that the nuclei be isolated in such a manner that the nuclear proteins remain in the nucleus, and the proteins of the cytoplasm do not enter the nucleus or contaminate the preparation. The high rate of biosynthesis of both histones and acidic nuclear proteins in the growing tissues and neoplastic cells are associated with rapid biosynthesis of DNA. However, even in resting cells, there is biosynthesis of all nuclear proteins. Because the possibility exists that the individual substructures of the nucleus of tumor cells contain proteins important both to their structural and functional characteristics, it is essential to consider the kinds of structures from which the nuclear proteins may originate and the methodology for their isolation. The criteria generally used to evaluate the purity of nuclei are that the nuclei should be anatomically identical with those of the whole cell, the contents of the nuclei as they exist in the cell should all be present in the isolated product, and the isolated nuclei should not contain cytoplasmic constituents. The chapter describes the techniques for isolation of nuclei and isolation of nuclear components. Further, the enzymes of the nucleus, the acidic nuclear proteins, nuclear globulins, the nuclear ribonucleoproteins, acidic proteins of the deoxyribonucleoprotein complex, and the histones have also been discussed in the chapter.

Increased content of high molecular weight RNA fractions in nuclei and nucleoli of livers of thioacetamide-treated rats

Isolated nuclei of liver of thioacetamide-treated and control rats were fractionated by sequential extraction with salt solutions containing a ribonuclease inhibitor into 3 main fractions designated “nuclear sap”, deoxyribonucleoprotein, and residue or “nucleolar” fraction. The RNA of the nuclear fractions was isolated and characterized by means of base composition, distribution of 32P in nucleotides after pulse-labeling, sedimentation and distribution of isotope. Sedimentation profiles of RNA of nuclear fractions indicated that prolonged administration of thioacetamide resulted in a 6–8-fold increase in the amounts of 28-S, 35-S and 45-S RNA and a 10-fold increase in 55-S RNA. A hitherto undescribed peak of 55-S RNA was found in the RNA of nucleoli isolated from livers of thioacetamide-treated rats. There was an almost complete absence of 18-S RNA in nucleolar RNA. No significant differences were found in the sedimentation profiles of the “nuclear sap” and deoxyribonucleoprotein fractions compared to controls. The base composition and distribution of 32P in nucleotides of “newly-formed” RNA of the residue fraction indicated that thioacetamide administration produced increased biosynthesis and accumulation of G-C rich RNA. These results were correlated with the enlargement of nucleoli, increased levels of nuclear RNA and decreased levels of cytoplasmic ribosomal RNA following prolonged administration of thioacetamide.

Evidence that the granular and fibrillar components of nucleoli contain 28 and 6S RNA, respectively.

Abstract Electron microscopic studies of nucleoli were correlated with sucrose density gradient sedimentation analysis of nucleolar RNA in samples obtained from livers of control rats, rats treated with thioacetamide, rats treated with actinomycin D and rats treated with both compounds. By planimetric analysis the fibrillar elements comprised approximately 20 per cent of the nucleolar area in the controls; the percentages of the total nucleolar area containing fibrillar elements were markedly increased after administration of actinomycin D and markedly decreased after administration of thioacetamide. Corresponding changes were found in 6S RNA following administration of actinomycin D and thioacetamide. When both compounds were administered simultaneously, coalescence of the fibrillar elements was observed and the resulting spherules were apparently extruded from the nucleolus, leaving nucleoli largely composed of granular elements. In these nucleoli, the 6S RNA was almost absent. These data provide evidence that 6S RNA is present in the fibrillar element of the nucleolus and that the 28S RNA is present in granular elements.

EFFECTS OF THIOACETAMIDE ON LABELING OF RIBONUCLEIC ACID OF ISOLATED NUCLEOLI IN VITRO.

Abstract The conditions that were optimal for incorporation of [14C]uridine triphosphate into RNA of nucleoli of normal liver cells were found to be optimal for incorporation of [14C]uridine triphosphate into nucleoli of thioacetamide-treated rat livers. Maximum incorporation of isotope was found at 15–30 min after the incubation was begun. Thioacetamide produced a marked increase in the rate of uptake of total isotope into RNA and in the specific activity of the RNA despite the increased amount of RNA in the nucleoli obtained from thioacetamide-treated animals. These data suggest that thioacetamide may derepress the genomic segments involved in biosynthesis of nucleolar RNA.

STUDIES ON THE COMPOSITION OF NUCLEAR RESIDUAL PROTEINS FROM RAT LIVER AND WALKER 256 CARCINOSARCOMA.

Summary The results presented indicate that a main component of the nuclear “residual protein” of liver and Walker tumor is collagen and the remainder may result from incomplete extraction or denaturation of alkali-soluble nuclear proteins. These findings may account for the very low solubility of the “residual proteins” noted in previous studies [16, 17, 18].

Increased ribonuclease activity of nuclei of liver cells of thioacetamide-treated rats

Abstract An eight-fold increase in nuclear ribonuclease activity was found 24 to 48 hours after administration of thioacetamide in a dose of 50 mg/kg. This effect was not associated with an increased activity of cytoplasmic ribonuclease, or increase numbers of nuclei. Moreover, nuclei of regenerating livers did not have an increased ribonuclease activity. The possibility exists that nuclear ribonucleases hydrolyze nuclear precursors of ribosomes in thioacetamide-treated animals and the increased nucleolar synthesis of RNA reflects a loss of feedback controls.