Evidence for Two Metabolically Distinct Types of Ribonucleic Acid in Chromatin and Nucleoli

Patterns of radioisotope incorporation are useful characteristics in describing cellular RNA fractions, and have indicated a distinctive "nuclear" RNA. In order to characterize the RNA fractions of the two nuclear components, nucleoli and chromatin, and to determine thereby the precise localization of the RNA typical of isolated nuclei, time-courses of P³² incorporation into nucleolar, chromosomal, and cytoplasmic RNA of Drosophila salivary glands have been determined from autoradiograms. Two experiments are reported which cover 12 and 18 hour periods, including an initial 2 hour feeding on P³². Concentrations of RNA-P³² (identified by ribonuclease digestion) were determined by grain counts. After 1 hour only the nucleolar RNA is labelled. Activity is detectible in chromosomal and cytoplasmic RNA after the 2nd hour. The nucleolar fraction reaches its maximum activity shortly after transfer of the larvae to non-radioactive food, the other fractions several hours later. Maximum activities persist in the chromosomal and cytoplasmic fractions; nucleolar activity decreases after the 9th hour. The observed differences in times at which incorporation begins and maximum activities are reached, and in maintenance of maximum activities indicate that chromosomal and nucleolar RNA are distinct fractions. The metabolic characteristics which have been ascribed to "nuclear" RNA apply only to the nucleolar fraction.     

THE ROLE OF THE NUCLEOLUS : I. Tritiated Cytidine Activity in Liver Parenchymal Cells of Thioacetamide-Treated Rats

Male rats of the Sherman strain were fed for 2 weeks a diet of ground purina rat chow containing 0.04 per cent thioacetamide. Animals were injected intraperitoneally with tritiated cytidine, 200µc/100 gm body weight, and sacrificed in pairs, a control and a thioacetamide-treated rat, at prescribed intervals. Liver tissues were preserved with the freeze-substitution method and postfixed in anhydrous OsO₄. Other samples were fixed directly with an acetic acid-ethanol mixture (1:3). AR-10 stripping film was applied to 2- and 4-µ sections and exposed for appropriate lengths of time. Nuclear and nucleolar volumes were obtained by direct measurement. Cytoplasmic volumes were obtained with the aid of Chalkley ratios. Nucleolar and cytoplasmic RNA concentrations were calculated from cytophotometric extinction (E_{540 mµ}) measurements. Data were expressed as grains/unit area, grains/unit area/concentration (or specific activity) and grains/total structure. In the liver parenchymal cells of thioacetamide-treated rats, the nucleolus shows vast increases in volume, RNA content, and grain count/total structure, 14-fold, 25-fold, and over 30-fold, respectively. The nucleus increases 2-fold in volume and about 3-fold in total grain count. Cytoplasmic volume increases only 20 per cent and displays a total grain count about equal to that in the control. The time course of incorporation curves for nucleolus and non-nucleolar nucleus (NNN) contain 2 distinct turnover fractions, rapid and slow. Both fractions were increased after thioacetamide treatment but remained proportional to those of controls. The unique stimulated RNA turnover in the nucleus and nucleolus, coupled to a "normal" turnover in the cytoplasm, suggests that this nuclear-nucleolar loss of label does not represent an exclusive passage of formed nuclear RNA to the cytoplasm.     

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C¹⁴ and uridine-H³ were used as ribonucleic acid (RNA) precursors, thymidine-H³ as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C¹⁴ as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.     

Zellwachstum und RNS-Synthese im Ei-Nährzellverband von Ophryotrocha puerilis

Summary During its growth phase, the oocyte is associated with a single highly polyploid nurse cell. Although the nurse cell contains high amounts of cytoplasmic ribonucleic acid, no RNA seems to be transferred to the oocyte. Autoradiographs prepared after pulse labelling with ³H-uridine indicate that the nuclei of both cell types are actively engaged in RNA synthesis during the whole period of oogenesis. Chromosomal RNA synthesis is most intense in oogonia before onset of the growth period. It still proceeds, although at a lower rate, after termination of oocyte growth when all RNA is lost from the nucleoli. Nucleolar RNA synthesis, on the other hand, is strictly correlated with the growth phase proper. Grain counts on chromatin and nucleoli of both egg cells and nurse cells at all stages of oogenesis indicate that nucleolar and chromosomal RNA synthesis are independent of each other to a large extent. It is thought that the type of RNA synthesized within the nucleolus is essentially ribosomal while the chromosomes are primarily engaged in the formation of messenger RNA.     

A STUDY OF NUCLEOLAR VACUOLES IN CULTURED TOBACCO CELLS USING RADIOAUTOGRAPHY, ACTINOMYCIN D, AND ELECTRON MICROSCOPY

Previously it has been found that in tobacco callus cells nucleolar vacuoles repeatedly form and contract. In this study, nucleolar vacuoles were investigated by using radioautography, actinomycin D, and electron microscopy. It was found, from grain counts of nucleoli labeled with uridine-³H, that nucleoli containing vacuoles had more than three times as many grains/µ² of nucleolar substance as did nucleolei without vacuoles. Treatment of tobacco callus cells with various concentrations of actinomycin D caused the percentage of cells containing nucleolar vacuoles to decrease; with the highest concentration the percentage of these cells dropped from the normal level of about 70% to less than 10%. However, after removal of actinomycin D the cells regained nucleolar vacuoles up to the control level. When radioautography was used with actinomycin D, it was found that the actinomycin D inhibited the uptake of uridine-³H, i.e. inhibited RNA synthesis, in those nucleoli which lost their nucleolar vacuoles. In addition, after removal of the cells from actinomycin D, it was found that as the cells regained nucleolar vacuoles the nucleoli also began to incorporate uridine-³H. Electron micrographs showed the nucleoli to be composed of a compact, finely fibrous central portion surrounded by a layer of dense particles 100–150 A in diameter. Nucleolar vacuoles occurred in the fibrous central portion. Dense particles similar to those in the outer layer of the nucleoli were found scattered throughout the vacuoles and in a dense layer at their outer edge. These data suggest that in cultured tobacco callus cells the formation and contraction of nucleolar vacuoles is closely related to RNA synthesis in the nucleolus.     

Rapid turnover of non-histone chromosomal proteins in skeletal muscle

Incorporation of ³H-leucine into histones and non-histone chromosomal proteins was investigated in liver, a tissue in which proteins generally turn over rapidly, and in muscle, a tissue in which proteins turn over slowly. Incorporation into histones was low in both tissues. Incorporation into non-histone chromosomal proteins which, in liver, proceeded at about the same rate as into soluble cytoplasmic proteins was, in muscle, considerably more rapid than into any other cytoplasmic or nuclear protein fraction investigated. The significance of the relatively high incorporation rate into the non-histone chromosomal proteins in muscle is not known. However, autoradiographic experiments suggest that in muscle all nuclei display a high rate of incorporation into these proteins, and gel electrophoretic experiments indicate that a high rate of turnover is characteristic of many of the proteins comprising this fraction.     

Nucleolar RNA Synthesis during Meiosis of Lily Microsporocytes

SEVERAL authors have reported a decrease in nucleolar incorporation of ³H-uridine into RNA in male gametocytes of maize, locusts and mammals during meiotic prophase^1–4, but the inactive nucleolus often persists. In the microsporocytes of Liliutn henryi the cytoplasmic ribosomes reportedly decrease in number during the extended meiotic prophase as the cellular RNA concentration also decreases⁵. Stern (personal communication) has also observed a decrease in RNA content in meiotic cells of Lilium longiflorum. We have examined the RNA synthetic activities of lily microsporocytes to see if the large nucleolus present is engaged in the synthesis of ribosomal RNA.     

THE BASE COMPOSITION OF RIBONUCLEIC ACID IN LAMPBRUSH CHROMOSOMES, NUCLEOLI, NUCLEAR SAP, AND CYTOPLASM OF TRITURUS OOCYTES

The base composition of RNA's extracted from chromosomes, nucleoli, nuclear sap, and cytoplasm of Triturus oocytes has been determined by microelectrophoresis. The chromosomal RNA has a content of guanine+cytosine equal to that of DNA, but there is no complementarity in the composition as for DNA. Nuclear sap contains a highly variable RNA with a tendency towards high uracil values. Nucleolar and cytoplasmic RNA's are similar in composition and both are of the guanine-cytosine rich type. The chromosomes and nucleoli contain roughly equivalent amounts of RNA, somewhat less than is present in the nuclear sap. The RNA/DNA ratio of the whole chromosomes is about 10. However, the ratio in the synthetically active regions, the loops, is much higher, since the loops contain all the chromosomal RNA but only a small fraction of the DNA.     

THE BASE COMPOSITION OF NUCLEIC ACIDS IN CHROMOSOMES, PUFFS, NUCLEOLI, AND CYTOPLASM OF CHIRONOMUS SALIVARY GLAND CELLS

The base composition of RNA from individually isolated giant chromosomes, puffed chromosome segments, nucleoli, and samples of cytoplasm from Chironomus salivary gland cells was determined by microelectrophoresis. Data on the adenine: guanine quotient of the chromosomal DNA were also obtained. The results show that: 1) Chromosomal, nucleolar, and cytoplasmic RNA's differ significantly from each other in base composition. 2) Nucleolar and cytoplasmic RNA's, in spite of the difference, show great similarities with regard to the base composition and are both rich in adenine and uracil. 3) The RNA extracted from chromosome I differs significantly from the RNA's extracted from different segments of chromosome IV, and the latter differ significantly from each other. 4) The values for the RNA: DNA quotients of chromosome segments parallel the development of synthetically active genes, so-called Balbiani rings. 5) The chromosomal RNA does not show a base symmetry in any of the investigated cases, nor is the content of guanine + cytosine the same as that for DNA. The first of these two facts excludes the possibility that the chromosomal RNA is a complete copy of both strands of the chromosomal DNA. In spite of the difference in guanine + cytosine content between the two nucleic acids the RNA may still partly or completely be a single strand copy depending upon how representative the DNA values are for the synthetically active DNA.     

Developmental changes in ribosomal ribonucleic Acid and fraction I protein in wheat leaves

In light-grown wheat (Triticum aestivum L.) seedlings, the amount of chloroplast and cytoplasmic ribosomal RNA increased to a maximum in the first leaf near the end of its growth and declined by about 60% in the following 3 days. While total ribosomal RNA was declining, labeled uracil was still incorporated into cytoplasmic ribosomal RNA, but the rate of incorporation into chloroplast ribosomal RNA fell by more than 80%, as did the incorporation of labeled leucine into fraction I protein. Either there is greater replacement of cytoplasmic ribosomal RNA than chloroplast ribosomal RNA in mature leaves, or chloroplasts are able to repress the incorporation of exogenous precursor when there is no net synthesis of RNA.     

Autoradiographic study of RNA synthesis during meiotic prophase in the human oocyte

The incorporation of 3H-uridine in oogonia and oocytes during meiotic prophase I was studied in three human fetuses 13, 18, and 19 weeks old. Following a 40- or 60-min pulse, intense nuclear and nucleolar labeling was observed in oogonia. During the preleptotene chromosome condensation stage, the heteropycnotic masses were unlabeled, while numerous silver grains were seen on the filaments persisting around these masses. During leptotene, chromosomal and nucleolar RNA synthesis was significant, but less than that in the oogonia. The rate of incorporation declined rapidly during zygotene and fell to a very low level at early pachytene. Throughout pachytene no nucleolar RNA synthesis was observed. Chromosomal RNA synthesis progressively recovered during middle pachytene, was of moderate intensity at late pachytene, and increased again at early diplotene. Nucleolar RNA synthesis was very intense at early diplotene, at the same time as nucleolar size and basophilia increased.     

EVIDENCE FOR CYTOPLASMIC DNA IN ROOT CELLS OF NICOTIANA

Sterile root cultures from Nicotiana tabacum were grown with H³-thymidine added to the medium for various intervals. Incorporation of the labeled nucleoside into nuclear DNA occurred in a fraction of the nuclei which increased with time. In addition, the cytoplasm of all cells incorporated enough tritium to be readily detected by autoradiography. The tritium was not removed by hydrolysis in 1 N HCl at 60°C for 10 minutes, but was removed by digestion in a DNase solution which also removed nuclear DNA. The amount of tritium in the cytoplasm increased during the first 2 hours, but did not appear to increase significantly during the following 5 hours. If the roots were transferred to unlabeled medium after 2 hours, the label was diluted faster than expected by growth without turnover of the labeled component. If FUdR was added to the unlabeled medium, the depletion occurred faster during the first 6 hours, but later appeared to level off so that at 10 hours these cultures did not differ from those incubated without FUdR. However, the addition of an excess of unlabeled carrier had no effect on the rate of depletion of the cytoplasmic label. Actinomycin D, which inhibited the incorporation of H³-cytidine into RNA in the root tips, had no effect on the incorporation of H³-thymidine into the cytoplasmic component. However, Mitomycin C or a high concentration of deoxyadenosine inhibited the incorporation of H³-thymidine into the cytoplasmic component as well as into the nuclear DNA. It is concluded that H³-thymidine is incorporated into a cytoplasmic fraction which has the characteristics of DNA, with a measurable rate of turnover. This fraction is synthesized regardless of whether or not the nucleus is synthesizing DNA. Although the function of cytoplasmic fraction is not yet known, it does not appear to be that of supplying precursors for the synthesis of the nuclear DNA.     

Nucleolus size varies with sex,ploidy and gene dosage in insects

The nucleolus constitutes a cytologically visible phenotype for ribosomal DNA (rDNA). Nucleolar size, as determined by silver staining, is a good indicator of cell proliferation rate and biosynthetic activity. Nevertheless, the relationship between rDNA content and sexual dimorphism for nucleolar size is not well documented. In the present study, the impact of sex and ploidy level on nucleolar size is investigated in three haplo/diploid and three diplo/diploid species of insect. Nucleolar sizes are found to be proportional to ploidy level in the haplo/diploid hymenopterans Trypoxylon albitarse and Nasonia vitripennis. Conversely, in the ant Messor barbarus, nucleolar sizes are larger in haploid males (winged) than diploid females (apterous). Among the diplo/diploid species, evidence for gene dosage compensation on nucleolar activity is suggested by the absence of sex differences in Drosophila simulans, a species in which rDNA is limited to the X chromosome. By contrast, in the grasshopper Stenobothrus festivus, another species with rRNA genes restricted to the X chromosome, the size of the nucleolus is significantly larger in females than in males. Additionally, in the grasshopper Chorthippus parallelus, where rDNA is distributed evenly on several autosomes of males and females, the females also show larger nucleoli than males. In both grasshopper species, the magnitude of the female/male ratio for nucleolus area is very similar to the body size ratio, suggesting that body size, as well as sex, ploidy, gene dosage and physiological activity, may be an important determinant of nucleolus area.     

RNA metabolsim during vegetative growth and morphogenesis of the cellular slime mold Dictyostelium discoideum

During vegetative growth of the cellular slime mold Dictyostelium discoideum, RNA is rapidly labeled by radioactive precursor and both the 25 S and the 17 S ribosomal RNA species appear in the cytoplasm 6–7 min after the onset of labeling. Thirty minutes after further incorporation of radioactive RNA precursors has been blocked, less than 10% of the label in RNA is associated with the nuclear fraction. After aggregation of the slime mold amoebae, RNA appears in the cytoplasm at a reduced rate, the small ribosomal subunit appearing in the cytoplasmic fraction more slowly than the larger ribosomal subunit. Some labeled RNA remains in the nuclei of developing cells long after the incorporation of ³H-uridine is blocked.     

Intracellular Amounts of Nucleic Acids and Protein During Pollen Grain Growth in Tradescantia

The rapid growth, large organelles, and synchronous development of T. paludosa pollen grains make them ideal subjects for cytochemical analysis. A microphotometric study of the nucleoli, chromosomes, and cytoplasm fixed at daily intervals during pollen grain maturation indicated that: 1. DNA (Feulgen) synthesis in the generative nucleus occurred during the first third of interphase, while the DNA content of the vegetative nucleus remained unchanged. 2. Throughout development, changes in RNA (azure B) content, in general, paralleled changes in protein (NYS¹, Millon) content in each organelle of the vegetative cell. Initially, the RNA and protein of all organelles increased up to mid interphase, when chromosomal and nucleolar fractions began to decline despite a continued increase in cytoplasmic RNA and protein. At least 24 hours before anthesis, the vegetative nucleolus had disappeared and chromosomal protein and RNA of the vegetative nucleus were apparently in rapid decline. Such a system offered an opportunity to study the role of the nucleus, especially the nucleolus, in RNA and protein metabolism in the cytoplasm, by noting what cytoplasmic processes could and could not continue at a time when nuclear mechanisms were absent or minimal. It was found that at least 2 fundamental processes continued during this period: both RNA and protein accumulated in the cytoplasm at a rapid rate. It was concluded that the nucleus is not the sole source of cytoplasmic RNA, for the data suggest that there are at least 2 separate and independent, or remotely dependent synthesizing systems, one nuclear and the other cytoplasmic. It is evident that nuclear influence on cytoplasmic synthesis need be neither direct nor immediate.     

BEHAVIOR OF NUCLEOLI AND CONTRACTING NUCLEOLAR VACUOLES IN TOBACCO CELLS GROWING IN MICROCULTURE

The ability to observe for extended periods of time individual tobacco cells growing in microculture has made it possible to describe the behavior of their nucleoli and contracting nucleolar vacuoles. Nucleoli typically disappeared in prophase and reappeared in telophase. If several nucleoli were present in telophase they generally fused to form only one or two during interphase. In one instance a nucleolus was seen to separate into two nucleoli prior to disappearance in late prophase. In aging and senescent cells the number of nucleoli or bodies similar to normal nucleoli often increased, and occasionally fragmentation of nucleoli was noted prior to death of cells. Budding of solid material from the nucleolus was also observed. The amount of nucleolar material decreased rapidly prior to death of tobacco cells. Nucleolar vacuoles were found to be a general and consistent component of tobacco cells in microculture. Nucleolar vacuoles typically formed and contracted repeatedly in interphase nuclei and apparently released a fluid material into the nucleus. Associated with the contraction of the nucleolar vacuoles was a corresponding decrease in diameter of the nucleolus. Nucleolar vacuoles were observed to occur in about 70% of the actively growing cells examined, whereas they were present in only 33% of the senescent or weakened cells. These data indicate a relationship between nucleolar vacuoles and the morphogenic status of the cells. Since it has been shown by others that the nucleolus is an active site of RNA metabolism, it is suggested that the contracting nucleolar vacuoles may be involved in the controlled release of a soluble product associated with RNA metabolism.     

THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS

The effect of protein synthesis inhibition by cycloheximide on nucleolar RNA synthesis and processing has been studied in HeLa cells. Synthesis of 45S RNA precursor falls rapidly after administration of the drug. However, the nucleolar content of 45S RNA remains relatively constant for at least 1 hr because the time required for cleavage of the precursor molecule into its products is lengthened after treatment with cycloheximide. The efficiency of transformation of 45S RNA to 32S RNA remains constant with approximately one molecule of the 32S RNA produced for each cleavage of a molecule of 45S RNA. However, shortly after the cessation of protein synthesis the formation of 18S RNA becomes abortive. The amount of 32S RNA present in the nucleolus remains relatively constant. After long periods of protein synthesis inhibition the 28S RNA continues to be synthesized and exported to the cytoplasm but at a greatly reduced rate. When the protein synthesis inhibitor is removed, a prompt, although partial, recovery in the synthesis rate of 45S RNA occurs. The various aspects of RNA synthesis regulation and processing are discussed.     

Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.