Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent snucleotide pools for nucleic acid synthesis

Novikoff rat hepatoma cells (subline NlSl-67) in suspension culture incorporate ³H-5-uridine into the acid-soluble nucleotide pool more rapidly than into RNA, resulting in the accumulation of labeled UTP in the cells. When labeled uridine is removed from the medium after 20 minutes or 4.75 hours of labeling, the rate of incorporation of label from the nucleotide pool into RNA decreases to less than 10% of the original rate within five to ten minutes, in spite of the presence of a large pool of labeled UTP in the cells, and incorporation ceases completely if an excess of unlabeled uridine is present during the chase. Upon addition of ¹⁴C-uridine to ³H-uridine pulse-labeled, chased cells, the ¹⁴C begins to be incorporated into RNA without delay and at a rate predetermined by the concentration of ¹⁴C-uridine in the medium and without affecting the fate of the free ³H-nucleotides labeled during the pulse-period. The results are interpreted to indicate that uridine is incorporated into at least two different pools, only one of which serves as primary source of nucleotides for RNA synthesis. During active synthesis of RNA, the latter pool of free nucleotides is very small and rapidly exhausted when uridine is removed from the medium. However, UTP accumulates in this pool when cells are labeled at 4–6°, since at this temperature RNA synthesis is blocked while uridine is still phosphorylated by the cells, and the UTP is rapidly incorporated into RNA during a subsequent ten-minute chase at 37°. From these types of experiments it is estimated that only 20–25% of the total uridine nucleotides formed in the cells from uridine in the medium is directly available for RNA synthesis and that the remainder becomes available only at a slow rate. Evidence is presented which suggests that one uridine nucleotide pool is located in the cytoplasm and another in the nucleus and that mainly the nuclear pool supplies nucleotides for RNA synthesis. The size of the latter pool is under strict regulatory control, since preincubation of the cells with 0.5 mM unlabeled uridine has little or no effect on the subsequent incorporation of ³H-uridine, although it results in an increase of the overall cellular uridine nucleotide content to at least 5 mM. Other results indicate that adenosine is also incorporated into two independent nucleotide pools, whereas the cells normally appear to possess a single thymidine nucleotide pool.     

RIBOSOME SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The cellular site of synthesis of ribosomal RNA in Tetrahymena pyriformis was studied by analyzing the purified nuclear and cytoplasmic RNA from cells pulse labeled with uridine-³H. The results of studies using zonal centrifugation in sucrose density gradients show that the ribosomal RNA is synthesized in the nucleus as a large precursor molecule sedimenting at 35S. The 35S molecule undergoes rapid transformation through two main nuclear intermediates, sedimenting at about 30S and 26S. The smaller ribosomal RNA (17S) appears first in the cytoplasm and it seems to be absent from the nucleus. The apparent delay in the appearance of the larger ribosomal RNA (26S) in the cytoplasm is due to the presence of a larger pool of its precursors in the nucleus as indicated by pulse-chase experiments. The newly synthesized ribosomal RNA's appear in the cytoplasm as discrete 60S and 45S ribonucleoprotein particles, before their incorporation into the polysomes.     

Nucleocytoplasmic transport and processing of small nuclear RNA precursors.

We have analyzed the structures and locations of small nuclear RNA (snRNA) precursors at various stages in their synthesis and maturation. In the nuclei of pulse-labeled Xenopus laevis oocytes, we detected snRNAs that were longer than their mature forms at their 3' ends by up to 10 nucleotides. Analysis of the 5' caps of these RNAs and pulse-chase experiments showed that these nuclear snRNAs were precursors of the cytoplasmic pre-snRNAs that have been observed in the past. Synthesis of pre-snRNAs was not abolished by wheat germ agglutinin, which inhibits export of the pre-snRNAs from the nucleus, indicating that synthesis of these RNAs is not obligatorily coupled to their export. Newly synthesized U1 RNAs could be exported from the nucleus regardless of the length of the 3' extension, but pre-U1 RNAs that were elongated at their 3' ends by more than about 10 nucleotides were poor substrates for trimming in the cytoplasm. The structure at the 3' end was critical for subsequent transport of the RNA back to the nucleus. This requirement ensures that truncated and incompletely processed U1 RNAs are excluded from the nucleus.     

Cytoplasmic and nuclear pyrimidine ribonucleotide pools in HeLa cells

A comparison of the rate of approach to equilibrium U:C labeling for two species of RNA whose base composition is known has been described previously as a method to determine the similarity or difference in the pyrimidine ribonucleotide pools from which the RNA species are synthesized. The procedure is now utilized to examine the pools used for the synthesis of HeLa cell nuclear RNA, mitochondrial RNA and poliovirus RNA. The results indicate that poliovirus RNA molecules derive from a pyrimidine precursor pool indistinguishable from that used for the synthesis of both heterogeneous nuclear RNA and 45 S ribosomal RNA in the nucleus, but that a separate and distinct pool is used for the synthesis of mitochondrial RNA. In addition, a comparison of the radioactivity ultimately found in UMP and CMP in a single species of RNA labeled in the presence or absence of actinomycin D shows that the conversion of uridine to cytidine nucleotides is significantly slowed in the presence of the drug. Direct measurement of the ratios of radioactivity in these RNA precursors in the acid-soluble pool confirm this observation.     

Nuclear retention elements of U3 small nucleolar RNA

The processing and methylation of precursor rRNA is mediated by the box C/D small nucleolar RNAs (snoRNAs). These snoRNAs differ from most cellular RNAs in that they are not exported to the cytoplasm. Instead, these RNAs are actively retained in the nucleus where they assemble with proteins into mature small nucleolar ribonucleoprotein particles and are targeted to their intranuclear site of action, the nucleolus. In this study, we have identified the cis-acting sequences responsible for the nuclear retention of U3 box C/D snoRNA by analyzing the nucleocytoplasmic distributions of an extensive panel of U3 RNA variants after injection of the RNAs into Xenopus oocyte nuclei. Our data indicate the importance of two conserved sequence motifs in retaining U3 RNA in the nucleus. The first motif is comprised of the conserved box C' and box D sequences that characterize the box C/D family. The second motif contains conserved box sequences B and C. Either motif is sufficient for nuclear retention, but disruption of both motifs leads to mislocalization of the RNAs to the cytoplasm. Variant RNAs that are not retained also lack 5' cap hypermethylation and fail to associate with fibrillarin. Furthermore, our results indicate that nuclear retention of U3 RNA does not simply reflect its nucleolar localization. A fragment of U3 containing the box B/C motif is not localized to nucleoli but retained in coiled bodies. Thus, nuclear retention and nucleolar localization are distinct processes with differing sequence requirements.     

THE CYTOPLASMIC CONTROL OF NUCLEAR ACTIVITY IN ANIMAL DEVELOPMENT

1.This article reviews the occurrence, mechanism, and functional significance of the cytoplasmic regulation of nuclear activity during cell differentiation and especially during early animal development. 2.Nuclei from brain, and from other kinds of adult cell normally inactive in DNA synthesis, are rapidly induced to commence DNA synthesis by components or properties of intact egg cytoplasm. The components of egg cytoplasm which induce DNA synthesis are not species-specific and they are likely to include DNA polymerase. It is known that DNA polymerase exists in egg cytoplasm before it becomes associated with nuclei in which it is effective. The induction of DNA synthesis in brain nuclei by living egg cytoplasm is always preceded by a pronounced nuclear swelling, a dispersion of chromosomes or chromatin, and the entry of cytoplasmic protein into the nucleus. 3.RNA synthesis can be experimentally induced or repressed by living cytoplasm. The cytoplasm of unfertilized and fertilized eggs appears to contain components which can reversibly and independently repress the synthesis of ribosomal RNA, transfer RNA, and heterogeneous RNA. RNA synthesis can be induced by introducing nuclei inactive in this respect into the cytoplasm of cells very active in RNA synthesis. The induction and repression of RNA synthesis is preceded by a marked swelling of the nucleus and the dispersion of its chromosome material. 4.The cytoplasmic control of chromosome condensation before division has been demonstrated by introducing sperm or adult brain nuclei into the cytoplasm of oocytes undergoing meiotic maturation. 5.The evidence that regional differences in the composition of eggs and other cells are associated with changes in nuclear and gene activity is reviewed in Section 111. While it is certain that these regional differences are of great importance in cell differentiation, evidence that they have a direct effect on nuclear activity has been obtained in a few instances only. In some species it has been shown that the cytoplasmic components related to germ-cell differentiation include RNA and, frequently, granules. 6.It is concluded that whenever nuclei are introduced experimentally into the cytoplasm of another cell, they very quickly assume, in nearly every respect, the nuclear activity characteristic of the host cell. In many instances, altered function has been demonstrated in nuclei which subsequently support normal development. The induced nuclear changes are therefore regarded as normal and it is believed that they are achieved through the same mechanism as that by which the host cell nucleus originally came to function in its characteristic way. Examples are cited to show that changes in gene activity very frequently arise immediately after mitosis. The changes induced experimentally in transplanted nuclei resemble in very many respects those undergone by nuclei which are naturally reconstituted after mitosis, and it is argued that the two processes are functionally equivalent, It is suggested that during telophase of mitosis, chromosomes are reprogrammed in respect of potential gene activity by association with cytoplasmic proteins. Inter-phase nuclei seem not to show changes of gene activity except when they undergo a pronounced enlargement after entering a new cytoplasmic environment.     

Effect of various precursors on the synthesis of adenine and uracil nucleotides in the rat heart (author's transl)

The dynamics of cardiac adenine and uracil nucleotides, following a subcutaneous injection of isoproterenol, was studied on the rat in vivo. The effect of continuous supply of adenosine, uridine, or ribose on the level of ATP and UTP was investigated on control rats and on isoproterenol-treated animals. The precursors were administered by continuous infusion (1 ml.h-1) into the superior caval vein. 1. ATP and UTP levels were decreased within one hour after a single dose of isoproterenol (5 mg.kg-1) (Fig. 1). 2. Then, the level of ATP rose slowly toward the control value. The normal level was not reached within 48 h (Fig. 1). 3. On the contrary, the initial drop in UTP concentration was followed by a rapid restoration. The control value was reached in 3 h, and then the UTP pool was increased to 180% of the normal level, 12 h after isoproterenol application. 4. As previously shown by other authors, the restoration of ATP was accelerated by a continuous supply of adenosine (37 micromoles per hour) or ribose (170 micromoles per hour) (Fig. 2). 5. The infusion of ribose (170 micromoles per hour) or uridine (41 micromoles per hour) completely suppressed the initial decrease in UTP level caused by beta-receptor stimulation. The further enlargement of the UTP pool was greatly enhanced by ribose or uridine (Fig. 3). 6. The infusion of adenosine was also positive on UTP regeneration. On the contrary, uridine had no effect on the ATP pool (Fig. 3). 7. When supplied to non-treated animals, all precursors caused an enhancement of the UTP level. Adenosine and ribose increased the ATP pool (Fig. 2 and 3). These results contribute to the comparison of the efficiency of the various pathways of cardiac nucleotide synthesis. They show that both de novo synthesis and salvage pathways are limited by the amount of precursors. The increase in UTP synthesis caused by ribose is consistent with the theory put forward for purines (ZIMMER et GERLACH, 1974) that phosphoribosyl-pyrophosphate availability limits the efficiency of de novo synthesis of nucleotides; it demonstrates that this concept is also true for de novo synthesis of pyrimidine nucleotides.     

Re-utilization of pyrimidine nucleotides during rat liver regeneration.

The changes in the specific radioactivities of the pool of total acid-soluble uridine nucleotides and of uridine and cytidine components of total cellular and nuclear RNA were monitored in regenerating rat liver for 12 days after partial hepatectomy. Evidence is presented for the re-utilization of pyrimidine nucleotides derived from cytoplasmic RNA degradation for the synthesis of new RNA. The extent of recycling was assessed and the true rate of rRNA turnover determined more accurately. The reutilization of the uridine components of RNA was 7.0%/day during the proliferative and 3.2%/day during the post-proliferative phase, whereas that of the cytidine nucleotides was more pronounced (9.6%/day and 18.1%/day respectively). The results reveal the existence of partial compartmentalization of pyrimidine ribonucleoside triphosphate pools in the nucleus and cytoplasm of rat liver cells.     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

The action of alpha-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

alpha-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of alpha-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of alpha-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10-20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by alpha-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

The effect of ethionine on ribonucleic acid synthesis in rat liver.

1. By 1h after administration of ethionine to the female rat the appearance of newly synthesized 18SrRNA in the cytoplasm is completely inhibited. This is not caused by inhibition of RNA synthesis, for the synthesis of the large ribosomal precursor RNA (45S) and of tRNA continues. Cleavage of 45S RNA to 32S RNA also occurs, but there was no evidence for the accumulation of mature or immature rRNA in the nucleus. 2. The effect of ethionine on the maturation of rRNA was not mimicked by an inhibitor of protein synthesis (cycloheximide) or an inhibitor of polyamine synthesis [methylglyoxal bis(guanylhydrazone)]. 3. Unlike the ethionine-induced inhibition of protein synthesis, this effect was not prevented by concurrent administration of inosine. A similar effect could be induced in HeLa cells by incubation for 1h in a medium lacking methionine. The ATP concentration in these cells was normal. From these two observations it was concluded that the effect of etionine on rRNA maturation is not caused by an ethionine-induced lack of ATP. It is suggested that ethionine, by lowering the hepatic concentration of S-adenosylmethionine, prevents methylation of the ribosomal precursor. The methylation is essential for the correct maturation of the molecule; without methylation complete degradation occurs.     

STUDIES ON THE ORIGIN OF RIBOSOMES IN AMOEBA PROTEUS

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus).     

Autoradiographic studies on the distribution of labeled maternal RNA in early mouse embryos

Maternal RNA of mouse eggs and embryos was labeled by exposure of growing ovarian oocytes to 3H-uridine in vivo 8 to 16 days before ovulation and fertilization. Labeled embryos from the 1-cell stage to the blastocyst stage were collected, fixed, and autoradiographs of plastic sections prepared. The observed grain density was similar in the pronuclei and in the cytoplasm of 1-cell embryos. Knowing the volumes of nucleus and cytoplasm, it was determined that 3% of the maternal RNA was found in the pronuclei. It is suggested that some of this nuclear RNA may be stable small nuclear RNAs (e.g. U1 RNA) retained from the germinal vesicle stage through meiotic maturation. During the 2-cell stage and beyond, maternal RNA is degraded and labeled precursor is reincorporated into nuclear RNA, making it difficult to accurately quantitate the amount of nuclear maternal RNA. It is known that about one third of the total maternal RNA is lost between the 8-cell and blastocyst stages. It was found that cytoplasmic grain densities in inner and outer cells of the morula and blastocyst were not significantly different. Thus, the loss of maternal RNA does not proceed more rapidly in the differentiating trophoblast than in the inner cell mass.     

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.     

Balbiani ring RNA content and half-life in nucleus and cytoplasm of Chironomus tentans salivary gland cells

The content of RNA with an origin in the Balbiani rings 1 and 2 (BR 1+2) has been determined in chromosomes, nuclear sap and cytoplasm of Chironomus tentans salivary gland cells. Together with information on rate and completeness of export this permits an estimation of half-life of this RNA in cytoplasm and its residence time in the nucleus. The quantities in the BR, nuclear sap, and cytoplasm are roughly related as 110200. The 75 S RNA in the nuclear sap with an origin in the BR 1+2 must to a high extent be a precursor to the cytoplasmic 75 S RNA in vivo. The half-life of the cytoplasmic component is about 20 h and the half-life (residence time) for BR 1+2 RNA in the nuclear sap around one hour. The presence of a large pool of BR RNA in the sap explains the previously observed delay in its cytoplasmic appearance in vivo.     

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver.

With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 14C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.