Rate Of Ribosomal RNA Synthesis Research Articles

Glucose strongly stimulates rate of the rRNA synthesis in Ehrlich ascites carcinoma cells.

A change in glucose concentration in an Ehrlich ascites carcinoma cell suspension from 0.1 mM to 20 mM causes a more than 50-fold stimulation of the rate of ribosomal RNA synthesis. Such an effect occurs without any dramatic effect on the rate of protein synthesis in cells. This process parallels a decrease in intracellular pH which may be a reason of Na +/H +-antiport activation.

RDNA transcription and cardiac hypertrophy

A common result of the various stimuli that alter the growth rates of cells is a parallel alteration of the rate of ribosome biogenesis. In many cell types, this mandates an altered rate of ribosomal RNA synthesis. Preliminary studies indicate that rDNA transcription may in fact be regulated at more than one level or step. Our understanding of the process of transcription and the mechanisms by which it is regulated are discussed.

Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture

Contractile activity of neonatal cardiac myocytes stimulated hypertrophic growth as compared with nonbeating cells that were depolarized with 50 mM KCl. Growth of contracting myocytes was associated with an increased rRNA content as measured by the total RNA/DNA ratio. The fractional rates of rRNA synthesis (K8) and rRNA degradation were determined in contracting and nonbeating myocytes to assess their relative contributions in increasing rRNA content during growth. The values for K8 were calculated from the specific radioactivity of 3'-[3H]UMP in 18 and 28 S rRNA after purification by hybridization to cloned rDNA. The cellular [3H]UTP pool served as the precursor for rRNA synthesis in myocytes that were labeled with 50 microM [3H]uridine. K8 values for 18 and 28 S rRNA in contracting myocytes were accelerated by 59 and 53%, respectively, after 3 days as compared with nonbeating myocytes. Calculations of the rate of cellular rRNA synthesis, which took into account the increased content of myocyte rRNA, revealed that synthesis of both 18 and 28 S rRNA was accelerated 2-fold after 2 days of contraction. The derived values for degradation of 18 and 28 S rRNA were increased marginally in contracting myocytes, but cellular rRNA degradation rates averaged 57% higher. The difference between cellular rates of rRNA synthesis and degradation in contracting myocytes accounted for the 30% increase in rRNA content. These data demonstrated that increased rRNA content in contracting myocytes resulted from acceleration of the fractional rate of rRNA synthesis.

Glucocorticoids modify the rate of ribosomal RNA synthesis in rat thymus cells by regulating the polymerase elongation rate

The mechanism by which glucocorticoids inhibit RNA polymerase A activity, and hence rRNA synthesis, in rat thymus cells has been investigated. Studies of the intranuclear distribution of RNA polymerase A between chromatin bound (“engaged”) and unbound (“free”) forms revealed that the steroid-mediated inhibition of the activity of the “engaged” form of the enzyme was not accompanied by significant changes in “free” pool activity. In the presence of rifamycin AF/0-13, an inhibitor of re-initiation of RNA polymerase A, the rate of [ 3H]UMP incorporation into RNA was slower in nuclei from steroid-treated cells than in those from control cells, although in both conditions similar plateau levels of UMP incorporation were attained. Direct measurements of the numbers of transcribing RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis was the result of a decrease in enzyme elongation rate; no significant change was observed in the number of transcribing enzymes. The steroid-induced inhibition of pre-rRNA synthesis was selectively abolished by mild proteolysis of nuclei, suggesting the involvement of a labile, regulatory glucocorticoid-induced protein. It is concluded that glucocorticoid treatment of rat thymus cells decreases 45S rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A, possibly by modification of the enzyme.

Silver bands in chronic granulocytic leukemia: I. Increased banding associated with blastic transformation

We have tested the proposal that the level of silver banding in leukemic cells of Ph 1+ chronic granulocytic leukemia (CGL) patients increases as the disease progresses. Blood and/or bone marrow cells from 14 patients were cultured for 24 hr before banding. In all but one case, there were two populations of mitoses, those with silver bands on their nucleolar organizing regions (NORs) and those without. The percentage of cells that banded was higher, on average, in cultures from 7 patients in blastic transformation (80%) than in 8 chronic cases (36%) or in one accelerated phase (49%). Also, the mean number of NORs stained in banded cells was higher in blastic phase (6.9) compared with chronic phase cells (4.4). Hyperdiploid cell lines were present in four cases of myeloblastic transformation. All such cells were silver banded, and the mean fraction of NORs banded in them was relatively high. An increase in silver banding with time was shown in two of the patients. It seems that silver banding does increase in CGL cells as the disease progresses. This may arise either through an increase in the rate of ribosomal RNA synthesis in leukemic cells present in the blastic phase or possibly by a decrease in the rate of degradation (or processing) of newly synthesized rRNA.

Phosphorylation of RNA polymerases: specific association of protein kinase NII with RNA polymerase I.

The activities of the three DNA-dependent RNA polymerases from a rapidly growing rat tumour, Morris hepatoma 3924A, and from rat liver were examined. The activity of RNA polymerase I was higher in the tumour than in the liver. The enhanced capacity for RNA synthesis was a result of a higher concentration of polymerase I in the tumour as well as of an activation of this enzyme in vivo. The possibility that the high specific activity of the hepatoma polymerase I resulted from phosphorylation was investigated. Two major cyclic-AMP-independent nuclear casein kinases (NI and NII) were identified; the activity of protein kinase NII in the tumour was ten times that in liver. Protein kinase NII was capable of activating and phosphorylating RNA polymerase I in vitro. This kinase could also stimulate RNA polymerase II activity, although to a lesser extent than RNA polymerase I. RNA polymerase III was not affected by protein kinase NII. Protein kinase NII was tightly associated with polymerase I and was found even in purified preparations of the polymerase. Antibodies against both RNA polymerase I and protein kinase NII were present in sera of patients with certain rheumatic autoimmune diseases. These results imply that RNA polymerase I and protein kinase NII are in close association in vivo as well as in vitro and that polymerase phosphorylation may regulate the rate of ribosomal RNA synthesis in the cell.

Regulation of ribosomal RNA synthesis in Drosophila melanogaster cells with different contents of sex chromosomes and nucleolar organizers

The quantity of ribosomal RNA genes and the rate of ribosomal RNA synthesis has been studied in established cell lines of Drosophila melanogaster with diploid, polyploid and hemizygous karyotypes and in larval diploid cells carrying 1, 2, 3 or 4 nucleolar organizer regions. The rate of rRNA synthesis has been shown to depend on the number of sex chromosomes rather than ribosomal genes or nucleolar organizers.

Chromatin structure along the ribosomal DNA of Dictyostelium: Regional differences and changes accompanying cell differentiation

The ribosomal genes of Dictyostelium discoideum are extrachromosomal palindromic DNA molecules situated in the nucleolus. Each molecule comprises ribosomal RNA coding regions and non-transcribed spacer regions. We used both biochemical and electron microscopic approaches to investigate the structure of transcribing and non-transcribing chromatin. Nucleoli from exponentially growing cells were digested with micrococcal nuclease, and the resulting DNA fragments were separated by gel electrophoresis and transferred to DBM paper. They were hybridized with cloned EcoRI fragments derived from different parts of the ribosomal gene. Probes of the coding region showed a smear, while probes of the non-transcribed regions gave pronounced banding patterns more complex than typical nucleosome repeats, but not due solely to sequence-specific cutting by micrococcal nuclease. The DNA of the coding region was digested more quickly than that of the non-transcribed ones. When nucleoli were digested with restriction enzymes, sites within the coding region were accessible and sites in the non-transcribed region were protected. The structure of ribosomal chromatin in differentiating cells, in which the rate of ribosomal RNA synthesis is reduced, was examined using essentially the same methods. The coding region, probed by hybridization to micrococcal digests, then showed a typical DNA repeat pattern indicating that this region had become condensed into nucleosomes, and its accessibility to restriction enzymes was very much reduced. On electron micrographs of lysed nucleoli from exponentially growing cells, two types of chromatin were observed, one with a beaded nucleosomal appearance, the other with putative RNA polymerase molecules attached to fibres indistinguishable from free DNA adsorbed to the same grid. The combined results suggest that whereas regions that are not transcribed are packaged with proteins that protect them from nuclease digestion, actively transcribing ribosomal genes are associated with few macromolecular constituents apart from those required for transcription and its regulation.

RNA polymerase I in hepatoma 3924A: Mechanism of enhanced activity relative to liver

Two major cyclic nucleotide-independent protein kinases, NI and NII, have been identified in Morris hepatoma 3924A and rat liver. When expressed per unit DNA, the activities of protein kinase NI and NII were 1.3 and 12 times greater, respectively, in the hepatoma than in liver. Protein kinase NII, but not NI, was capable of phosphorylating and activating the DNA-dependent RNA polymerases I and II. Phosphorylation of RNA polymerase I was accompanied by an increase in average size of the RNA synthesized in vitro, whereas, phosphorylation of RNA polymerase II was concomitant with an elevation in the number of RNA chains initiated. RNA polymerase I polypeptides of M r 120,000, 65,000 and 25,000 were phosphorylated by protein kinase NII; RNA polymerase II polypeptides of M r 214,000, 140,000 and 21,000 were modified by this kinase. In contrast to the purified hepatoma enzyme, RNA polymerase I activity in nuclear lysates was not affected by addition of protein kinase NII. In vitro phosphorylation of the tumor lysate followed by immunoprecipitation of RNA polymerase I polypeptides indicated little or no phosphate transfer to the 65,000 M r polypeptide of the enzyme. These data suggested that the tumor enzyme, particularly the 65,000 M r polypeptide, was highly phosphorylated in vivo, but becomes dephosphorylated during purification. Unlike the tumor enzyme, RNA polymerase I in the liver lysate responded to protein kinase addition; phosphorylation of the liver polymerase I polypeptides of M r 120,000, 65,000 and 25,000 was observed. These observations indicate that the liver enzyme is not completely phosphorylated (activated) in vivo and that the relatively rapid rate of ribosomal RNA synthesis in the rapidly growing hepatoma may result, at least in part, from a polymerase I which is maximally phosphorylated.

Persistent macrosegregated nucleoli in the siphonous green algae Avrainvillea, Cladocephalus and Blastophysa and possible implications concerning ribosomal RNA synthesis, nuclear cycles and life histories

Macrosegregation of nucleoli, a feature generally associated with a temporarily reduced rate of ribosomal RNA synthesis, occurs in three genera of siphonous green algae, Avrainvillea, Cladocephalus and Blastophysa. This condition persists throughout the vegetative phase of the life history and, thus, may indicate a peculiar rRNA cycle.Nuclear cycles in siphonous green algae are discussed and types of nuclei found in the major groups are compared.

Unco-ordinate regulation of ribosomal RNA and ribosomal protein synthesis during L 6E 9 myoblast differentiation

Using the rat myoblast L 6E 9 cell line, we have shown previously that a five- to tenfold reduction in ribosome synthesis occurs concomitant with the differentiation of myoblasts into myotubes (Krauter et al., 1979). Regulation of ribosome synthesis, which occurs under physiological conditions, has been shown to occur at the transcriptional level. This cell line provides an excellent model with which to study whether all ribosomal components (RNAs and protein) are co-ordinately or independently regulated. The rate of synthesis and degradation of individual ribosomal proteins has been studied by a combination of pulse labeling and two-dimensional gel electrophoresis. We have found that ribosomal protein synthesis is unco-ordinately regulated with respect to the rate of ribosomal RNA synthesis. In spite of the five- to tenfold decrease in rRNA synthesis in myotubes, ribosomal proteins are synthesized at the same rate in myoblasts and myotubes. The rate of ribosomal protein accumulation, however, reflects the rate of rRNA synthesis. Myotubes accumulated ribosomal proteins fivefold more slowly than myoblasts. The ribosomal proteins synthesized in excess of rRNA in myotubes are degraded with half-lives of one hour or less. On the contrary, the proteins which assemble onto mature ribosomes are stable for at least 24 hours. From these results, we conclude that in L 6E 9 cells the limiting step in ribosome assembly is the transcription of the 45 S precursor rRNA.

Ribosomal RNA synthesis in pre- and post-gastrula-stage sea urchin embryos

The rate of ribosomal RNA synthesis has been measured in sea urchin blastulae and gastrulae Quantitation was effected by measuring the kinetics of incorporation of methyl-labeled methionine into S-adenosylmethionine and transfer of methyl groups to characteristic 2′- O-methylated dinucleotide and trinucleotide locations in ribosomal RNA. We ascertained that this methylation represented de novo synthesis, through a comparison of the labeling of these oligonucleotides with [ 32P]orthophosphate. The early blastula and late gastrula of Strongylocentrotus purpuratus both synthesize 40 molecules of nuclear preribosomal RNA per minute per cell. In Lytechinus pictus the blastula synthesizes 16 and the gastrula 23 molecules per minute per cell. Therefore neither are the ribosomal genes quiescent prior to gastrulation nor is an enhancement of their transcription a necessary concomitant to gastrulation. There does, however, appear to be an increase in the rate of processing of nuclear preribosomal RNA to cytoplasmic ribosomes just prior to or concomitant with gastrulation.

Regulation of RNA synthesis in Tetrahymena pyriformis: secretion of regulatory factors.

The rate of ribosomal RNA synthesis varies greatly with the population density in both exponentially and synchronously growing populations of Tetrahymena pyriformis. Shortly after inoculation of the population - at relatively low cell densities - a gene-dose effect dominates the picture, and a doubling in the gene number is immediately followed by a doubling in the rate of RNA synthesis. However, also other mechanisms are controlling the rate of RNA synthesis. Generally one finds high rates of RNA synthesis in the lag phase of newly inoculated cells, decreasing rate of RNA synthesis during most of the exponential growth phase and very low rate of synthesis in stationary phase cells. We now have results which show that the repression of RNA synthesis in densely populated cultures is caused by a dialysable factor, which is secreted by the cells. If cells are inoculated on a medium which contains this factor the high initial rate of RNA synthesis normally observed is prevented, but the cells multiply and grow with normal generation time until normal stationary-phase population densities are reached.

Does the rate of ribosomal RNA synthesis vary depending on the number of nucleoli in a nucleus?

Cultured kidney cells of Xenopus laevis were pulse-labeled with [ 3H]uridine for 10, 20 and 30 min during their logarithmic growth phase and then processed for autoradiography. The labeled cells were assigned into two categories, one- and two-nucleolated cells, and the rate of ribosomal RNA (rRNA) synthesis was measured by counting the number of grains in nucleoli. The results obtained revealed that a two-nucleolated cell incorporated significantly much more radioactivity into its nucleoli than did a one-nucleolated partner for all the periods examined. Cells of these different nucleolar types, however, contained essentially the same amount of rDNA (DNA complementary to rRNA) as estimated by in situ hybridization with [ 125I]rRNA. Although it remains to be proved that the observed increase in incorporation represents the increased rate of rRNA synthesis in two-nucleolated cells, the present findings seem to be very interesting, since they might indicate that the activity of rRNA genes is in some way regulated or affected by their spatial relationship in a cell nucleus.

Nucleolar size in parallel with ribosomal RNA synthesis at diapause termination in the eggs of Bombyx mori.

The eggs of Bombyx mori, both in diapause and nondiapause, were subjected to cytological examination of nucleoli and measurement of RNA precursor incorporation (2 hours) into ribosomal RNA. In diapause eggs, the nucleoli were very small and the rate of ribosomal RNA synthesis was the lowest of the samples tested. Most cells in diapause possessed nuclei with one nucleolus. In contrast, the eggs activated from diapause by long chilling attained the largest size of nucleoli and the highest rate of ribosomal RNA synthesis. A significant proportion of the cell nuclei still had only one nucleolus at this stage. Three days after activation, the eggs exhibited intermediate levels in both the size of nucleoli and the rate of ribosomal RNA sythesis. At this stage, about half of the egg cell nuclei had two nucleoli.

GENETIC MODULATION OF RNA METABOLISM IN DROSOPHILA. I. INCREASED RATE OF RIBOSOMAL RNA SYNTHESIS

ABSTRACT It has been suggested that a particular Y chromosome which is rDNA-deficient (YbbSuVar-5) may be associated with an increased utilization of rDNA template in adult testes (Shermoen and Kiefer 1975). To extend the observations on this chromosome, experiments were designed to determine if the chromosome has an effect on rRNA synthesis in bobbed adults and on classic bobbed phenotypes (shortened and thinner scutellar bristles and delayed development). Specific activity measurements were made on rRNA extracted from adult males of the genotypes car bb/Ybb- and car bb/YbbSuVar-5, which are rDNA-deficient to the same extent, and from Samarkand+ isogenic (Sam+ iso), which is a wild-type stock. The resulting data demonstrated that the presence of the YbbSuVar-5 chromosome increases the rate of ribosomal RNA synthesis in adult flies. In addition, it was found that the presence of this particular Y chromosome restores wild-type bristle phenotype and development time. Appropriate genetic crosses indicate that the observed effects (increased rRNA synthesis, restoration of wild-type phenotype) are a function of this particular Y chromosome, and are not due to autosomal factors. The results of these experiments suggest that the rate of rRNA accumulation is under genetic control.

GENETIC MODULATION OF RNA METABOLISM IN DROSOPHILA. II. COORDINATE RATE CHANGE IN 4S, 5S, AND POLY-A ASSOCIATED RNA SYNTHESIS

It has been demonstrated that a particular rDNA-deficient Y chromosome (Y(bbSuVar-5)) increases the rate of ribosomal RNA synthesis in adult testes (Shermoen and Kiefer 1975) and in whole flies (Clark, Strausbaugh and Kiefer 1977). As an initial attempt to explore the molecular basis of this phenomenon, experiments were designed to determine if the rate increase was specific for rRNA as opposed to the other species of RNA. The genotypes used in these studies were car bb/Y(bb-), car bb/Y( bbSuVar-5), and Sam(+) iso. car bb/Y(bbSuVar-5 ) and car bb/Y(bb-) are deficient to the same extent in rDNA and Sam(+) iso is a wild-type stock. Following isotope incorporation, total RNA was extracted by a phenol:chloroform method and separated by polyacrylamide gel electrophoresis. The various RNA species were quantified by UV absorption and their radioactivity determined by gel fractionation and liquid scintillation counting. The resulting data permitted the calculation of a specific activity (i.e., dpm/microg RNA) which was defined as synthetic rate. Polyadenylated RNA was isolated using a poly-U sepharose column and similar rate calculations were made. The data from these studies indicate that the rate of synthesis of all species of RNA examined (28S + 18S, 5S, 4S transfer RNA and polyadenylated RNA) is increased by the presence of the Y(bbSuVar-5) chromosome. Genetic and molecular mechanisms are discussed.

Multiple pathways for primary processing of ribosomal RNA in Escherichia coli.

A comparison of isogenic RNase III+ and RNase III- strains of Escherichia coli shows that although both synthesize precursor and mature 16 S and 23 S ribosomal RNAs, the transient rRNA species of the RNase III- strain differ from those of the RNase III+ strain. The RNase III+ strain synthesizes p16 and p23 rRNA, whereas the RNase III- strain produces unstable 17 S, 18 S, "p23," 25 S and 30 S RNA molecules. The 30 S RNA, which is a primary transcript of the ribosomal RNA gene cluster, does not contribute significantly to any of the smaller RNAs, nor is m23 rRNA derived from 25 S but rather from "p23" RNA. Mature 16 S rRNA is derived from both 18 S and 17 S RNA, and 17 S RNA can be derived from 18 S. Additionally, an unstable RNA species about 300 bases long is missing in the RNase III- strain and another species which seems to be about 50 bases larger appears. Processing of the primary ribosomal RNA transcript in RNase III- strains of Escherichia coli is accomplished during its transcription by two independent pathways which are not so utilized in RNase III+ strains. One pathway yields 18 S and precursor 23 S RNAs which are processed to mature rRNAs; the second pathway yields 25 S RNA and perhaps 16 S rRNA. The second pathway, unlike the first, is inhibited by chloramphenicol treatment. At slow rates of ribosomal RNA synthesis, the nascent transcript is processed preferentially by the first pathway. We suggest that in the absence of RNase III, which is involved in the primary processing of rRNA in E. coli, other enzymes involved in primary and secondary processing of rRNA in RNase III+ cells can recognize their sites on the nascent rRNA transcript and accomplish the primary processing.

Proposed model of major sequential biochemical events of a trophic response

It appears that the induction of ornithine decarboxylase regulates the rate of ribosomal RNA synthesis as well as regulating the rate of synthesis of polyamines. Further, ornithine decarboxylase, in most cases, is induced after a significant activation of cAMP-dependent protein kinase. We propose a model for the process of hypertrophy based on studies of a considerable number of mammalian growth systems. The mechanism of parallel regulation of polyamines and RNA appears to be initiated by the direct effect of ornithine decarboxylase on RNA polymerase I.

Synthesis of polyadenylated messenger RNA during the cell cycle of Saccharomyces cerevisiae

The rates of ribosomal and polyadenylated messenger RNA synthesis were measured through the cell cycle of the budding yeast Saccharomyces cerevisiae. An exponentially growing culture was pulse-labelled with [2- 3H]adenine for 0.1 generation time, then fractionated according to cell volume by zonal centrifugation. A control experiment with a synchronously dividing culture showed that zonal fractionation corresponded to a fractionation by stage in the cell cycle. Nucleic acids were extracted from each fraction of the cell cycle from the zonal rotor, and DNA, ribosomal and polyadenylated messenger RNAs were isolated. DNA synthesis occurred between 0.4 and 0.8 of the cell cycle as fractionated on the zonal rotor. The rate of ribosomal RNA synthesis per cell was constant during G 1‡ doubled early in the period of DNA synthesis, then remained constant at the new level for the remainder of the cell cycle. The rate of processing of ribosomal RNA precursors to mature ribosomal RNAs declined at the time of the increase in rate of ribosomal RNA synthesis, but recovered later in the cycle. This could mean that the pool of ribosomal proteins is small, and that the rate of ribosomal protein synthesis increases later in the cell cycle than the rate of ribosomal RNA synthesis. The rate of synthesis of polyadenylated messenger RNA was constant during G 1 and the earliest part of the period of DNA synthesis, then doubled over the remainder of the period of DNA synthesis. After DNA synthesis, the rate of polyadenylated messenger RNA synthesis remained steady for the remainder of the cell cycle. It is suggested that gene number controls the rates of ribosomal and messenger RNA synthesis. When the number of genes doubles during the DNA synthesis period, the amount of transcription also quickly doubles. Repeat experiments suggested that the genes coding for ribosomal RNA are reproducibly replicated earlier, and over a shorter proportion of the DNA replication period than the genes coding for messenger RNAs. It is calculated that a minimum of 625 genes coding for messenger RNA per haploid genome, transcribed at the maximum rate throughout the cell cycle, would be required to give the observed cell-cycle pattern of messenger RNA synthesis. The total content of polyadenylated messenger RNA of cells at different stages of the cell cycle was measured by the formation of ribonuclease-resistant complexes between polyadenylic acid sequences in total UNA from non-radioactive yeast and [ 3H]polyuridylic acid. The amount of polyadenylated messenger RNA per cell rose after the doubling of the rate of polyadenylated messenger RNA synthesis, and by the end of the cell cycle was twice the level at the beginning of the cycle. The cell-cycle patterns of messenger and ribosomal RNA synthesis are discussed in relation to the control of balanced growth and doubling of all cellular components during the cell cycle, and in relation to known patterns of enzyme activity during the cell cycle of budding yeast.