Accumulation Of Ribosomal RNA Research Articles

Synthesis of heterogeneous nuclear RNA in full-grown oocytes of xenopus laevis (Daudin)

At various times following injection of either 3H-GTP or 32PO 4 into full-grown (stage 6) Xenopus laevis oocytes, RNA has been extracted and fractionated on polyacrylamide gels. Based on size, base composition and incorporation data, we have defined the kinetics of synthesis and accumulation of ribosomal RNA (40S, 28S, 18S), heterogeneous RNA of high molecular weight (>40S) and heterogeneous RNA migrating with molecular weights of from 4S to 40S. Nuclear isolations have been performed to determine the cellular distribution of these classes of RNA as a function of time. Evidence is presented which shows that stage 6 oocytes synthesize RNA which by virtue of its size, base composition, rapid turnover and nuclear location is equivalent to the heterogeneous nuclear RNA observed in somatic cells. In addition, the data suggest synthesis of a class of nuclear RNA with a half-life of several hours. A small fraction (5%) of the nuclear RNA is stable, enters the cytoplasm and may represent RNA added to the stockpile of maternal transcripts known to be present in stage 6 oocytes.

Control of Ribosome Synthesis in Escherichia coli : Analysis of an Energy Source Shift-Down

The rate of ribosome synthesis and accumulation in Escherichia coli during the transition after an energy source shift-down was analyzed. The shift was imposed on cultures of stringent and relaxed strains growing in glucose minimal medium by the addition of the glucose analogue alpha-methylglucoside. In the stringent strain, ribosome synthesis was almost instantaneously reduced after the shift, whereas the relaxed strain exhibited a more gradual response. The rate of messenger ribonucleic acid (mRNA) synthesis was affected similarly, though to a smaller extent. A comparison of the rates of synthesis and accumulation of ribosomal RNA (rRNA) and ribosomal proteins showed that far more ribosomal components were synthesized after the shift than were accumulated, indicating that a substantial part of the rRNA made after the shift was unstable. A new method was used to measure relative rates of rRNA synthesis and to estimate the transcription time for the rRNA operon under different conditions. In steady states of growth with growth rates ranging from 0.75 to 2.3 doublings/h, as well as during the transition after a shift-down, the transcription time of the rRNA operon was constant. The rate of synthesis of rRNA correlated during this transition - in contrast to the rate of accumulation (M. T. Hansen et al., J. Bacteriol. 122: 585-591, 1975) - with the ppGpp pool in the same way as has been observed during partial amino acid starvation.

Cytochemical and Biochemical Analysis of Development ofUrechisOocytes

The echiuroid marine worm Urechis caupo is uniquely suited for the study of oogenesis. A relatively large quantity of oocytes at various developmental stages can be obtained and subjected to coordinated cytochemical and biochemical analysis Oocytes at the cluster, early diplotene, mid-diplotene, and diffuse diplotene or lampbrush stages are active in the synthesis and accumulation of ribosomal RNA, several proteins, carbohydrates, lipids, and also, perhaps, yolk constituents. Only cortical granule formation, which occurs during later stages of oogenesis, appears to be stage specific. Ribosomal RNA genes are also transcribed in the nucleolus of the mature oocytes or unfertilized eggs. However, the rate of production in these eggs appears to be regulated at the level of maturation of rRNA precursor molecules.

Ribosome accumulation in 3-methylcholanthrene-induced liver growth in adult male rats

One injection of 20 mg/kg body weight of 3-methylcholanthrene results in liver growth, as evidenced by increases in liver wet weight, total protein, and total RNA. DNA does not increase. Total ribosomal RNA also accumulates, but changes in free and membrane-bound ribosome fractions are proportionately different from changes in total ribosomal RNA. Within 12 hr after injection, there is an increase in free ribosomal RNA which then decreases to control levels between 48 and 72 hr. In contrast, membrane-bound ribosomal RNA shows no immediate change, but between 48 and 72 hr the membrane-bound ribosomal RNA increases markedly above control levels, at the same time that free ribosomal RNA is decreasing to control levels. Thus, ribosomal RNA accumulation observed after 3-methylcholanthrene injection is not due to a coordinate increase in free and membrane-bound ribosomal RNA.

Studies on messenger and ribosomal RNA synthesis in plant tissue cultures induced to undergo synchronus cell division.

Messenger and ribosomal RNA metabolism was tudied in a plant tissue culture system: cells from the quiescent tubers of Helianthus tuberosus (Jerusalem artichoke) were induced to divide synchronously and dedeifferentiate by excision and culture of explants in nutrient medium. Large accumulations of ribosomal RNA and protein started early in the 20-h lag-period preceding the first division. In pulse-labelling experiments, two types of polydisperse messenger-like RNAs were detected one with and one without a poly(adenylic acid) sequence. In the first 2 h of culture the two polydisperse RNA fractions were the predominant types of RNA synthesised. Ribosomal RNA synthesis was very low during the first 2 h, but acclerated later. Low concentrations of actinomycin-D strongly inhibited ribosomal RNA synthesis, but had little effect on the synthesis of transfer RNA or either type of polydisperse messenger-like RNA. In explants cultured with low concentrations of actinomycin-D ribosomal RNA accumulation was completely inhibited, but cell division and protein accumulation occurred, though at a reduced rate. It is concluded that the synthesis and accumulation of new ribosomal RNA which normally occur during culture are not required for the induction of cell division or for protein accumulation, i.e. the ribosomal RNA existing in the quiescent tuver cells can support protein accumulation and cell division induced by excision and culture. The quiescent tuber tissue is also shown to contain significant amounts of messenger-like RNA.

Regulation of RNA synthesis in plant cell culture: Delayed synthesis of ribosomal RNA during transition from the stationary phase to active growth

Ribosomal RNA synthesis was studied during the early phases of growth activation in a cell suspension culture derived from peanut ( Arachis hypogaea, L.) cotyledon. Upon dilution from stationary phase, these cells show a characteristic lag of 3 days before the commencement of cell division. An analysis of the nature of RNA synthesized during this early period of growth showed that the cells obtained immediately upon dilution from stationary phase synthesize primarily messenger RNA and essentially no ribosomal RNA. The synthesis of ribosomal RNA is delayed for about 24 hr after which it rises sharply resulting in a 2- to 3-fold accumulation of ribosomal RNA per cell during the subsequent 24-hr period. Both the messenger RNA and the ribosomal RNA were characterized by their cellular localization; by sucrose and CsCl gradient analyses, and by the determination of their base ratios. It would appear that a major facet of the lag phase in the cell growth is the diversion of a significant part of the RNA biosynthetic apparatus from the synthesis of messenger RNA to that of ribosomal RNA.

Effects of two strains of tobacco mosaic virus on growth and RNA content of tobacco leaves

Tobacco leaves 1.5 to 18 cm long were infected with tobacco mosaic virus (TMV) strains vulgare or flavum. Nucleic acids were extracted, and fractionated and assayed by gel electrophoresis. Leaf growth and contents of DNA, ribosomal RNA, chloroplast ribosomal RNA, transfer RNA, and TMV-RNA were measured at various times after infection. The younger the leaf infected, the longer the duration of synthesis of TMV-RNA, and the higher the final virus concentration reached. Synthesis of flavum-RNA was as great as that of vulgare-RNA in young leaves, but was much less than that of vulgare-RNA in old leaves. The growing, healthy leaf accumulated ribosomal RNA, chloroplast ribosomal RNA, and transfer RNA. The maximum contents of these per leaf were reached as or just before the leaf reached its maximum length of 15–20 cm. Thereafter there was a loss of these components. TMV-infection of a 1.5 cm-long leaf inhibited its growth and nucleic acid accumulation. Infection of a 5 cm-long leaf had no effect on the subsequent rises in ribosomal RNA and transfer RNA contents, but chloroplast ribosomal RNA accumulation was inhibited. The effects of TMV-strains vulgare and flavum on young leaves were similar. Infection of older leaves, at or later than the time of maximum ribosomal RNA content, led to contents of ribosomal and transfer RNA higher than in control leaves, as the rate of loss of these two components was reduced, especially by strain flavum. Under no conditions was a degradation of cytoplasmic ribosomal RNA found as a result of viral infection, but chloroplast ribosomal RNA was quickly degraded in flavum-infected leaves.

A simple and sensitive method for quantitative measurement of cellular RNA synthesis

A quantitative technique is described for measuring the rate of cellular RNA synthesis by analysis of the incorporation of 3H-8-adenosine into ATP precursor pools and into RNA. This technique is unique in that quantitative measurements of RNA synthesis require only very small amounts of biological material and can be performed with a minimum of technical manipulation. Comparison of measurements of the rate of synthesis of ribosomal RNA in chick fibroblasts growing in tissue culture obtained using this technique with chemical measurements of accumulation of ribosomal RNA demonstrated that this technique could give an accurate measure of RNA synthesis in animal cells.

Regulation of DNA-like RNA and the apparent activation of ribosomal RNA synthesis in sea urchin embryos: Quantitative measurements of newly synthesized RNA

Summary RNA synthesis was analyzed in sea urchin embryos in order to determine the contribution which changes in synthesis of heterogeneous DNA-like RNA and of ribosomal RNA make to the changes in TNA metabolism which occur during embryogenesis. The base composition and sedimentation behavior of RNA molecules incorporating radioactivity during a short incubation of embryos with radioactive RNA precursors indicate that heterogeneous, DNA-like RNA was the predominant class of RNA synthesized in embryos at all stages of development. During longer incubations with radioactive precursors, radioactivity preferentially accumulated in ribosomal RNA. This preferential accumulation of newly synthesized rRNA was much more extensive at later than at early stages of development. However, accumulation of newly synthesized 28 S ribosomal RNA could be demonstrated as early as the first half of blastula stage when rRNA was extensively purified from DNA-like RNA by sucrose gradient sedimentation and MAK chromatography. In order to interpret these developmental changes in the relative accumulation of ribosomal and DNA-like RNA, the amounts of newly synthesized RNA accumulated during cleavage, blastula, and pluteus stages were quantitatively estimated by measurement of the incorporation of radioactive precursors into RNA and simultaneous determination of the specific activity of the nucleotide triphosphate precursor pools. The results indicated that the accumulation of newly synthesized DNA-like RNA per nucleus was quantitatively much greater at cleavage and blastula stages than at pluteus stage, whereas the accumulation of newly synthesized rRNA per nucleus at blastula and pluteus stages were similar. Therefore, decreases in the quantitative accumulation of DNA-like RNA account entirely for the change in the relative accumulation of ribosomal RNA and DNA-like RNA which occurs during development of sea urchin embryos.

Ribosomal protein synthesis during amino acid starvation and chloramphenicol treatment

The production of the individual species of ribosomal protein during the residual protein synthesis occurring upon amino acid starvation or upon chloramphenicol treatment has been investigated in rel − and rel + strains of Escherichia coli. The starved rel + strain, which devotes a very small proportion of its residual protein synthesis to ribosomal protein, produces the individual species of ribosomal protein in approximately the same relative proportions as during exponential growth. The starved rel − strain, which devotes a relatively large amount of its residual protein synthesis to ribosomal protein, produces one or a few of the individual species of ribosomal protein in very high relative proportions. These protein species are associated predominantly with the 50 s rather than the 30 s ribosomal subunit. Chloramphenicol treatment of a rel + strain, like amino acid starvation of a rel − strain, brings about the accumulation of ribosomal RNA and causes an increased proportion of the residual protein synthesis to be devoted to ribosomal protein. However, in contrast to the amino acid-starved rel − strain, the chloramphenicol-treated rel + strain produces the individual species of ribosomal protein in approximately the same relative proportions as during exponential growth. This is interpreted to mean that the unbalanced synthesis of the different individual species of ribosomal protein during amino acid starvation of rel − bacteria is a consequence of the rel − allele of the rel gene and not due merely to the unbalanced accumulation of ribosomal RNA.

Effect of purine and pyrimidine analogues on growth and RNA metabolism in the soybean hypocotyl-the selective action of 5-fluorouracil.

The effects of several base analogues and cycloheximide on RNA synthesis, protein synthesis, and cell elongation were studied in excised soybean hypocotyl. None of the pyrimidine analogues tested affected growth or protein synthesis; only 5-fluorouracil appreciably inhibited RNA synthesis. 8-Azaguanine and 6-methylpurine markedly inhibited RNA and protein synthesis and cell elongation. Cycloheximide effectively inhibited both cell elongation and protein synthesis. The results show that 5-fluorouracil selectively inhibited ribosomal and soluble RNA synthesis without affecting the synthesis of D-RNA. These results indicate that the requirement for RNA synthesis to support continued protein synthesis and cell elongation is restricted to the synthesis of D-RNA.5-Fluorouracil was incorporated into all classes of RNA in a form believed to be 5-fluorouridylic acid. Cycloheximide markedly inhibited the accumulation of ribosomal RNA, but the results indicate that CH did not inhibit, per se, the synthesis of ribosomal RNA. The accumulation of newly synthesized D-RNA was only slightly affected by cycloheximide. These results show that the inhibition of cell elongation by cycloheximide correlates with the inhibition of protein synthesis, but not with the effect on RNA metabolism.