Studies on compartmentation of uridine 5'-triphosphate during synthesis of cytoplasmic and plastid ribosomal RNA's in Euglena gracilis

• 1.1. Compartmentation of uridine 5'-triphosphate (UTP) was studied during synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and plastid ribosomal RNA (pl-rRNA) in photoorganotrophically grown cells of Euglena gracilis Z.
• 2.2. Using the approach of Wiegers et al. (1976) the steady state specific radioactivity of UTP was compared with that ofcyt-20S rRNA, cyt-25S rRNA, pl-16S rRNA and pl-23S rRNA under low and at 100-fold higher specific radioactivity of exogenously fed pHl-uracil.
• 3.3. The equal steady state specific radioactivities of all rRNAs at both feeding conditions argue against compartmentation of UTP during their synthesis.
• 4.4. At high specific radioactivity of exogenous [³H]-uracil the salvage-derived labelled UMP was shown to be diluted 15,000-fold by unlabelled UMP formed de novo, whereas this dilution factor was 100-fold lower at low specific radioactivity of [³H]-uracil indicating inhibition of the de novo synthesis of UMP.
• 5.5. Transport is suggested of uridine nucleotides into chloroplasts by the 15-fold higher specific radioactivity of intracellular [³H]-uracil than that of UTP as well as UMP residues in pl-rRNA.

     

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.)

Summary Ovaries ofC. erythrocephala synthesize large amounts of poly(A)⁺ and poly(A)^– RNA during early and middle stages of oogenesis as shown by labelling with³H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)⁺ RNA and the poly(A)⁺ RNA present in sufficient amounts for optical density measurements (steady state poly(A)⁺ RNA) was observed. During early and mid-oogenesis, in the poly(A)^– RNA fraction, 4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)⁺ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)⁺ RNA were not obvious. However, different sizes of labelled poly(A)⁺ RNA species were detected in 0–2h old preblastoderm embryos, after injection of³H-uridine into females either 3–4 days (stage 3–4 of oogenesis) or 24 h before oviposition (stage 5–6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)⁺ RNA fraction represents about 2–3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)⁺ RNA varied from about 30 to 200 nucleotides.     

RNA transcription and translation in sea urchin oocytes and eggs

The steady-state concentrations and absolute rates of synthesis of ribosomal RNA (rRNA) molecules were measured in oocytes, eggs, embryos, and larvae of the Hawaiian sea urchin Tripneustes gratilla. The steady-state concentration per genome of the RNA precursor sequences measured by hybridization to a cloned rDNA fragment was approximately 100- to 300-fold greater in the RNA obtained from oocytes and eggs than in the RNA extracted from embryos and larvae. Since the rate of processing of the rRNA precursor at different stages is not greatly different, the rates of rRNA synthesis must be considerably greater in oocytes than in embryo cells. The absolute rate of RNA synthesis in oocytes and embryos was determined from the incorporation of [³H]guanosine into cellular GTP pools and into both precursor and mature rRNA species. The data indicate an approximately 40-fold higher rate of rRNA synthesis in oocytes than that measured in embryos or previously in larvae (J. Griffith and T. Humphreys, 1979, Biochemistry18, 2178–2185). Together these results indicate that the ribosomal genes are transcribed much more rapidly during sea urchin oogenesis than during embryogenesis or larval stages.     

A ribosomal RNA operon from Pseudomonas stutzeri Zobell

A ribosomal RNA operon from the marine bacterium, Pseudomonas stutzeri Zobell, was cloned and characterized by Southern hybridization and sequence analysis. The 16S rRNA, 23S rRNA, 5S rRNA and 2 tRNA genes (alanine and isoleucine) were identified by homology with sequences in GenBank. The rRNA gene exhibited typical eubacterial organization (16S-tRNAs-23S-5S). A putative ribosomal promoter and anti-terminator regions were also identified and described. Significant differences in spacing of the anti-terminator regulatory elements were observed between P. stutzeri Zobell and Escherichia coli.     

Program of early development in the mammal: Changes in absolute rates of synthesis of ribosomal proteins during oogenesis and early embryogenesis in the mouse

The absolute rates of synthesis of specific ribosomal proteins have been determined during growth and meiotic maturation of mouse oocytes, as well as during early embryogenesis in the mouse. These measurements were made possible by the development of a high-resolution twodimensional gel electrophoresis procedure capable of resolving basic proteins with isoelectric points between 9.1 and 10.2. Mouse ribosomal proteins were separated on such gels and observed rates of incorporation of [³⁵S]methionine into each of 12 representative ribosomal proteins were converted into absolute rates of synthesis (femtograms or moles synthesized/hour/oocyte or embryo) by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and embryos (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341–359). Ribosomal proteins were synthesized at all stages of oogenesis and early embryogenesis examined and, while equimolar amounts of ribosomal proteins were found in ribosomes, they were always synthesized in nonequimolar amounts during development. Rates of synthesis of individual ribosomal proteins differed from each other by more than an order of magnitude in some cases. Synthesis of ribosomal proteins accounted for 1.5, 1.5, and 1.1% of total protein synthesis during growth of the oocyte, in the fully grown oocyte, and in the unfertilized egg, respectively. During meiotic maturation of mouse oocytes the absolute rate of synthesis of ribosomal proteins decreased about 40%, from 620 to 370 fg/hr/cell, as compared to a 23% decrease in the rate of total protein synthesis during the same period. On the other hand, during early embryogenesis the absolute rates of synthesis of each of the 12 ribosomal proteins examined increased substantially as compared with those of the unfertilized egg, such that at the eight-cell stage of embryogenesis synthesis of ribosomal proteins (4.17 pg/hr/embryo) accounted for about 8.1% of the total protein synthesis in the embryo. Consequently, while the absolute rate of total protein synthesis increased about 1.5-fold during development from an unfertilized mouse egg to an eight-cell compacted embryo, the absolute rate of ribosomal protein synthesis increased more than 11-fold during the same period. These results seem to reflect the differences reported for the patterns of ribosomal RNA synthesis during early development of mammalian, as compared to nonmammalian, animal species. The results are compared with those obtained using oocytes and embryos fromXenopus laevis.     

Dosage of 5 S and ribosomal genes during oogenesis of Drosophila melanogaster

During growth, the Drosophila egg chamber increases its DNA content over a thousandfold, mainly by polyploidization of the nurse cell nuclei. We wanted to determine if 5 S and ribosomal genes are replicated to the same extent as the remaining DNA. Egg chambers were mass fractionated to represent different size classes and, therefore, different stages of oogenesis. Nucleic acids were extracted from each class of egg chambers, and after removal and quantitation of the RNA, the content of 5 S and ribosomal genes in the different DNA fractions was assayed by filter hybridization. Diploid DNA and DNA from polytene salivary gland cells served as references. It was concluded that: (1) Ribosomal genes become underreplicated as oogenesis proceeds, but to a much lower extent than in polytene chromosomes of salivary glands of the same organism. (2) By contrast, 5 S genes are equally replicated in egg chambers of all stages of oogenesis. (3) Notwithstanding the large increase in DNA content of egg chambers during oogenesis, the increase in total RNA content (mostly ribosomal RNA) is over 15 times as large.     

Very long-lived messenger RNA in ovaries of Xenopus laevis

It is possible to label with radioactivity newly synthesized ovarian RNA after intraperitoneal injection of [³H]guanosine and [³H]uridine into immature Xenopus laevis, if ovaries in which only previtellogenic stage 1 oocytes are present. Following the amount of radioactivity in the ovarian pool of acid-soluble precursors indicates a complete clearance of acid-soluble radioactivity within 15–20 days after injection. Incorporation of radioactivity into total RNA (which is almost exclusively 4 and 5S RNAs at this stage) and poly(A)⁺ RNA ceases between 15 and 20 days after injection, but the total amount of radioactivity in these RNA fractions does not decline appreciably over the next 18 months. During this time, the ovary grows and develops since stage 6 oocytes eventually appear and there is a 10- to 20-fold increase in total RNA content, which changes in composition from almost exclusively (95%) 4 and 5S RNAs to mainly (75%) 18 and 28S RNAs. Thus, despite continued growth and development, radioactive RNA molecules synthesized during previtellogenesis survive for lengths of time commensurate with the length of oogenesis (1–2 years). Although very limited (<7%) reincorporation of radioactivity into RNA is detected, it cannot alone account for the stability of the label in poly(A)⁺ RNA. These results are interpreted as indicative of synthesis during previtellogenesis of tRNA, 5SrRNA, and messenger RNA molecules which are very long-lived.     

Compartmentation of uridine 5′-triphosphate occurs during synthesis of cytoplasmic and mitochondrial ribosomal RNAs of cultured tomato cells but not during synthesis of cytoplasmic ribosomal and transfer RNAs

Compartmentation of uridine 5-triphosphate (UTP) was studied during the nucleolar synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and the synthesis of cytoplasmic transfer RNA (cyt-tRNA) in the nuclear matrix as well as the synthesis of mitochondrial ribosomal RNA (mt-rRNA) in tomato (Lycopersicon esculentum Mill. cv. Lukullus) cell-suspension culture using the approach of Wiegers et al. (Eur. J. Biochem. 64, 535–540, 1976). Before measurements were made, it was ensured that: (i) there was steady-state labeling of all RNAs studied as well as UTP; (ii) there was stability of cyt-tRNA and cyt-rRNA; (iii) there was no label randomization through degradation of [³H]uridine; (iv) there were significant differences in the specific radioactivity of UTP, the final immediate precursor of RNA, after supplying the cells with two different exogenous [³H]uridine concentrations.By comparing the steady-state specific radioactivity of UTP with that of cyt-tRNA and cyt-18S rRNA during constant [³H]uridine supply, we found that the three molecules had equal specific radioactivities which, however, differed significantly from that of the mt-rRNA. With a 20-fold higher uridine concentration, i.e. a 20-fold lower specific radioactivity of exogenous [³H]uridine, the specific radioactivity of cyt-rRNA, cyt-tRNA and UTP decreased proportionally whereas that of mt-RNA increased. These results argue against different UTP pools during synthesis of cyt-rRNA and cyt-tRNA, but indicate compartmentation of UTP during rRNA synthesis in the nucleus and the mitochondria of tomato cells.Abbreviations CMP cytidine 5-monophosphate - cyt-rRNA cytoplasmic ribosomal RNA - cyt-tRNA cytoplasmic transfer RNA - mt-rRNA mitochondrial rRNA - NC nitrocellulose - PAGE polyacrylamide gel electrophoresis - TLC thin-layer chromatography - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP uridine 5-diphosphate - UMP uridine 5-monophosphate - UTP uridine 5-triphosphate     

Cell cycle analysis in asynchronous cultures using the BUdR-Hoechst technique.

During synchronized germination of spores of Dictyostelium discoideum, protein synthesis begins almost concomitantly with syntheses of messenger-like RNA (mlRNA) and 4–5S RNA (presumably tRNA) in the swollen spore stage and the initiation of ribosomal RNA (rRNA) synthesis is somewhat delayed. DNA synthesis occurs in the early stages of the amoeba emergence phase. Cycloheximide (200 μg/ml) blocked spore germination as well as total protein synthesis, whereas actinomycin D (60 μg/ml) did not affect either. This concentration of actinomycin D selectively inhibited formation of rRNA but did not influence the synthesis of mlRNA. Examinations of RNA labeled with [¹⁴C]uracil during germination indicated that polysomes initially detectable in the course of the germination process contain ¹⁴C-labeled mlRNA. It was concluded that at least some of mRNA synthesized during germination of D. discoideum spores is involved in protein synthesis required for the germination.     

Quantitative Measurement of Rates of 5S RNA and Transfer RNA Synthesis in Sea Urchin Embryos

Embryonic differentiation is believed to be due to a programmed expression of genes, which includes their time of activation, sequence of appearance, and amount transcribed into the immediate gene product, RNA. Differential synthesis of the major RNA classes, such as the ribosomal RNAs (28S, 18S, 5S) and transfer RNA (tRNA), characterizes many animal developing systems, including the sea urchin embryological system. Previous work has shown that the genes for 5S RNA and tRNA are active during early cleavage in sea urchin embryos. The present study focused on quantitatively measuring and comparing the rate of 5S RNA and tRNA synthesis in cleavage, early blastula, and early pluteus embryos of Arbacia punctulata. At each stage, embryos were labeled for 3 h with [8-³H]-guanosine. Total cellular RNA was extracted using the cold (4°C)-phenol-sodium dodecyl sulfate method and purified (LiCl-soluble) RNA preparations were fractionated by electrophoresis on 10% polyacrylamide gels. The amount of 5S RNA and tRNA synthesized at each stage was calculated from the radioactivity coincident with the 5S RNA and with the tRNA absorbance peaks (A_{260 nm}) on each gel, from the known guanosine monophosphate (GMP) compositions of sea urchin 5S RNA and tRNA and from the average specific radioactivity of the GTP precursor pool during each 3 h labeling period. The results showed that on a per embryo basis the rates of 5S RNA and tRNA synthesis increased slightly (about 1.4-fold) from cleavage through pluteus stages, while on a per cell basis the rates declined severalfold (about 3-fold) during embryogenesis. The rates of 5S RNA and tRNA synthesis determined here parallel previously-reported levels of RNA polymerase III in sea urchin embryos, suggesting that cellular levels of RNA polymerase III may exert some positive control over 5S RNA and tRNA synthesis during sea urchin embryogenesis.     

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

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-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 α-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 α-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.     

Serum,insulin and phorbol esters stimulate rRNA and tRNA gene expression in both dividing and nondividing Drosophila cells

The expression of genes that code for the large ribosomal RNAs (rRNAs) and tRNAs can be regulated by calcium, serum, insulin and a tumor-promoting phorbol ester, TPA. These effectors can rapidly alter rRNA and tRNA synthesis in dividing and nondividing Drosophila cells. In an in vitro assay system of the nondividing cells of the male accessory glands, calcium, insulin and TPA were shown to increase both rRNA and tRNA synthesis. Exposure of actively dividing Drosophila culture cells to differing serum concentrations or TPA also altered rRNA and tRNA synthesis. Nuclear run-on assays demonstrate that the exposure of these cells to increased serum concentrations coordinately alters RNA polymerase I loading on both 18S and 28S rDNA. These data indicate that calcium, growth factors and a tumor-promoter each can signal changes in ribosomal and tRNA gene expression.     

On the nature of RNA synthesized in pollen tubes ofNicotiana alata

The RNA formed in pollen tubes during 4 hours of growthin vitro was resolved by chromatography on methylated albumine on kieselguhr (MAK) into three principal fractions. Acoording to the labelling from uracil-¹⁴C about 11% was eluted with tRNA and 5 S RNA (low molecular weight RNA), 76% just after rRNA (D-RNA) and nearly 14% was recovered from the column by SDS at 35 °C (TB-RNA). In the presence of actinomycin D at concentration of 30 μg ml^-1 the synthesis of the three classes of RNA was inhibited by 71%, 97% and 70% respectively. On sucrose density gradient the radioactive low molecular weight RNA sedimented at 4 S-5 S which suggests that one or both of these RNA species are synthesized in pollen tubes. The D-RNA eluted from the MAK column is polydisperse in size exhibiting a wide range of sedimentation values up to about 35 S with a large peak at 9 S-10 S and two smaller peaks at 14 S-15 S and at about 23 S. The rapid labelling and the polydisperse rather low molecular weight character suggest that the D-RNA is a heterogeneous population of mRNA. The sedimentation profile of TB-RNA was similar to that of D-RNA. The RNA synthesized in the presence of³²PBO3-₄ or uracil-¹⁴C exhibited no radioactivity peaks corresponding to sedimentation peaks of rRNA.     

Degradation of maternal poly(A)-containing RNA during early embryogenesis of an insect (Smittia spec., chironomidae,diptera)

Summary Eggs of the chironomid midgeSmittia spec. were shown to contain maternal rRNA, tRNA and poly(A)-containing RNA. The ribonucleoprotein spectrum consisted of monosomes, ribosomal subunits, and subribosomal particles, whereas polysomes could be detected only in small amounts. Poly(A)-containing RNA was found in different regions of the RNP spectrum, mainly between 15 S and 60 S. After labelling maternal RNA by feeding tritiated uridine to the larvae, the radioactivity associated with poly(A)-containing RNA accounted for about 4% of the label in the total RNA extracted from newly deposited eggs. About half of the radioactivity in the poly(A)-containing RNA was lost between egg deposition and an advanced blastoderm stage. The loss was accompanied by both a decrease in the size of the poly(A)-containing RNA molecules and a shift of poly(A)-containing RNP particles to less dense regions in sucrose gradients. Comparison with poly(A)-containing RNA synthesized by the embryo indicates that the reduction in size of maternal poly(A)-containing RNA is not artifactual but reflects its degradation after the formation of blastoderm.