Degradation of pre-messenger RNA in bovine thyroid slices; effect of thyrotropin

Summary Bovine thyroid RNA labeled by incubation of slices in the presence of ³²P-orthophosphate were fractionated by a two-step procedure. Total RNA were extracted by gel filtration on AcA 22 in the presence of pronase and separated by Sepharose 2B chromatography. A small fraction of heavily-labeled RNA (giant RNA) was obtained in the void volume (peak I); the major fraction of RNA (smaller than 45 S) was retarded on the column (peak II) and had a low specific radioactivity. Labeled and total RNA of peak I and labeled RNA species of peak II had a DNA-like nucleotide composition and were polyadenylated. In contrast, the nucleotide composition of total RNA of peak II was similar to that of ribosomal RNA and had a very low poly (adenylic acid) content. Pulse-chase experiments showed a precursor-product relationship between the two RNA fractions. These data indicate that labeled RNA of peak I and peak II likely correspond to newly-synthetized pre-mRNA and mRNA, respectively. Thyrotropin induced a decrease in the amount of ³²P-labeled pre-mRNA and a proportional increase of ³²P-labeled mRNA suggesting a stimulatory effect of the hormone on the degradation of pre-mRNA.Abbreviations SDS sodium dodecyl sulfate - TIPNS triisopropylnaphthalene disulfonic acid, sodium salt - TSH thyrotropin-stimulating hormone     

Incorporation of tritiated adenosine into mouse ovum RNA

The total RNA of ovulated mouse ova has been examined by polyacrylamide gel electrophoresis. The amount of RNA present in the two main peaks observed, 28 S and 18 S ribosomal RNA, has been estimated as 0.20 ng.The RNA of ovulated mouse ova was labeled by exposure of growing mouse oocytes to adenosine-8-³H in vivo. For this purpose a small volume of a concentrated solution of the precursor was injected into the ovarian bursa, and ova were collected by superovulation at various subsequent times. The major growth phase of the oocyte is known to lie between 20 and 6 days before ovulation. Significant incorporation into egg RNA was observed when bursal injection was performed between 19 and 7 days, but not between 5 days and 1 day before ovulation.The types of labeled RNA in ova ovulated at five intervals between 19 and 7 days after bursal injection of adenosine-8-³H or uridine-5,6-³H were analyzed by polyacrylamide gel electrophoresis. The distribution of label on the gels demonstrated that the bulk of the label appeared in ribosomal RNA and transfer RNA. In addition labeled heterogeneous RNA was estimated to represent 10–15% of the total incorporation.     

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).     

Synthesis of messenger ribonucleic acid in excised pea-seedling root segments. Separation of the messenger from microsomes by electrophoresis

1. Electrophoresis on cellulose acetate membrane in a tris-pyrophosphate buffer was used to separate microsomal fractions into three components: (1) the lipoprotein; (2) the nucleoprotein (termed the beta-band); (3) traces of free RNA (termed the alpha-band). In tris buffer containing Mg(2+) the alpha-band was not obtained. 2. The incorporation of uridine and phosphate into RNA by excised pea-seedling root segments was studied by using this electrophoretic technique. 3. It was shown that after a short (;pulse') incubation in the radioactive precursor and a longer (;chase') incubation in the non-radioactive precursor most of the incorporation was into the RNA of the alpha-band and little into that of the beta-band. Previous work showed that in roots of whole seedlings the incorporation is mostly into the ribosomal RNA, corresponding to the material in the beta-band. 4. A pulse-labelled RNA has also been found; this seems to be a cell fraction distinct from the microsomes or ribosomes. 5. The apparent base compositions of labelled RNA in the alpha-band and small amounts of labelled RNA in the beta-band and of unfractionated RNA were very different from the composition of ribosomal or transfer RNA, and somewhat like that of DNA. 6. It is suggested that the excised root segment synthesizes a messenger-RNA fraction labelled after a pulse incubation and a distinct messenger RNA labelled after a pulse and chase incubation, but no ribosomal or transfer RNA. The system is thus similar to the ;step-down' culture conditions in bacteria.     

Ribonucleic Acid Synthesis During the Differentiation of Sporangia in the Water Mold Achlya

The role of ribonucleic acid (RNA) synthesis in the development of sporangia in the saprolegniaceous mold Achlya was studied. Methods were developed for growing and treating large populations of mycelia so that the hyphal tips would differentiate into sporangia with considerable synchrony. Under the starvation conditions imposed for the differentiation of sporangia, net RNA, deoxyribonucleic acid (DNA), and protein synthesis ceased. However, incorporation of radioactive precursors into RNA continued at a high rate throughout the period of differentiation, showing that the enzymatic mechanism for RNA synthesis was still in an active state. Actinomycin D inhibited the differentiation of sporangia and the incorporation of labeled precursors into RNA. The level of actinomycin used did not inhibit the normal outgrowth and branching of the mycelia that occurred during differentiation. Thus, DNA-dependent RNA synthesis was required for the differentiation of sporangia. Sucrose gradient analysis of newly synthesized RNA showed that only the ribosomal and soluble fractions of RNA were labeled during vegetative growth. During the differentiation of sporangia, ribosomal and soluble RNA fractions were also labeled, and, in addition, a heterodisperse fraction of labeled RNA which was heavier than ribosomal RNA appeared; this fraction was not evident in the newly synthesized RNA from vegetative mycelia.     

Factors causing release of ribosomal subunits from isolated nuclei of regenerating rat liver in vitro.

Incubation medium II causes release of ribosomal subunits from isolated prelabeled nuclei of regenerating rat liver in vitro (Sato, T., Ishikawa, K. and Ogato, K. (1976) Biochim. Biophys. Acta 000, 000-000). The effects of individual components of this medium on release of subunits were studied and the following results were obtained. 1. Dialyzed cytosol was effective in causing release of total labeled RNA, but its effect on release of labeled ribosomal subunits was rather lower than that of low molecular yeast RNA. Spermidine inhibited the release of total labeled RNA as well as that of labeled ribosomal subunits. 2. Low molecular yeast RNA was the most effective component for inducing release of labeled ribosomal subunits. Homologous ribosomal RNA was as effective as yeast RNA. Cytoplasmic ribosomes, prepared by washing with solution of high salt concentration, and their subunits were also effective. 3. Transfer RNA was not so effective as yeast RNA and ribosomal RNA and even after heat treatment it had little effect. 4. Among the homopolyribonucleotides tested, polyuridylic acid had a strong effect but polyadenylic acid, polycytidylic acid and polyinosinic acid had no effect. 5. The effects of yeast RNA and polyuridylic acid in causing release of labeled ribosomal subunits were dependent upon their concentrations in the reaction mixture. The characteristics of the factors which cause release of labeled ribosomal subunits in vitro are discussed on the basis of the results.     

Effects of camptothecin, an inhibitor of DNA topoisomerase I on ribosomal gene structure and function in TG cells.

The effects of camptothecin treatment and topoisomerase I inhibition on ribosomal gene structure and function were investigated in TG cells, a human tumour cell line. 90- and 180-min treatments with 25 microM camptothecin resulted in an increased DNA fragmentation and decreased activity of topoisomerase I in cell extracts. After 180-min treatment, the incorporation of labelled uridine into total cell RNA was reduced to 39% and the ribosomal RNA synthesis to 10%, as compared to values of control cells. At the ultrastructural level, the nucleolar components appeared to be segregated; after selective DNA staining, with osmium-amine complex, a part of the nucleolar chromatin of treated cells showed the presence of thin, extended DNA filaments, superimposable to those present in control cells.     

DNA fiber replication in chromosomes of a higher plant (Pisum sativum).

Ribosomal precursor particles were extracted from the yeast Saccharomyces carlsbergensis and analysed. After a brief labelling of yeast protoplasts with ³H-uridine, three basic ribonucleoprotein components were detected, sedimenting at approx. 90S, 66S and 43S in sucrose gradients containing magnesium. The 90S particles contained the 37S ribosomal precursor RNA as a major component and a small though variable amount of 29S ribosomal precursor RNA. The 66S and 43S particles contained 29S and 18S ribosomal precursor RNA, respectively. Kinetic data indicate a precursor-product relationship between the 90S particles and the two other ribonucleoprotein components, consistent with the conversion: 90S → 66S + 43S. The 90S and 66S preribosomes appeared to be present exclusively in the nucleus, whereas the 43S particles were mainly present in the cytoplasmic fraction. Apparently, the final maturation step in the formation of the 40S ribosomal subunits takes place in the cytoplasm. The 90S and 66S precursor particles have a relatively higher ratio of protein to RNA than the mature large ribosomal subunits, as judged from their buoyant densities in CsCl gradients. This finding suggests that also in a primitive eukaryotic organism, like yeast, ribosome maturation involves, in addition to a decrease in the size of the RNA components, an even stronger decrease in the amount of associated protein. In contrast, the 43S particles appeared to have the same buoyant density as the 40S ribosomal subunits.     

Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system.     

Blood gas analysis: effect of air bubbles in syringe and delay in estimation.

Two common sources of error in blood pH and blood gas analysis were studied. The effect of delay in estimation was studied in 10 volunteers and 40 patients. Syringes were stored at 0 degree C, (crushed ice), 4 degrees C (refrigerator) and 22 degrees C (room temperature). The pressure of oxygen (PO2) fell significantly by 20 minutes at 4 degrees C and 22 degrees C but did not change significantly at 0 degree C for up to 30 minutes. Blood pH, pressure of carbon dioxide (PCO2), and base excess did not change significantly for up to 30 minutes at 4 degrees C and 22 degrees C and up to 60 minutes at 0 degrees C. The effect of air bubbles in the syringe was studied by leaving a single bubble or froth in contact with the blood for one to five minutes in 40 patients. Po2 rose significantly after two minutes'' contact with froth and two minutes'' contact with the air bubble, and PCO2 fell significantly after three minutes'' contact with the air bubble. Size of the bubble had little effect on rates of change. Blood pH, bicarbonate, TCO2, and base excess did not change significantly after up to five minutes'' contact. For accurate estimation of PO2 and PCO2 it is necessary to avoid frothing, to expel all air bubbles within two minutes, and to inject the sample into the machine within 10 minutes or store the syringe in crushed ice. The requirements for blood pH and base excess measurement are less exacting.     

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.     

Labeling of RNA in young and adult rat brain: Evidence for different RNA processing

The labeling of RNA in young and adult rat brain has been studied by measuring in vitro (tissue slices incubation) the incorporation of labeled uridine into RNA of total tissue and of the various subcellular fractions purified from cerebral hemispheres of 1- and 10-month-old rats. Gel electrophoretic analysis of the newly synthesized nuclear and microsomal RNA was also accomplished. An active metabolism of RNA in adult animals was found; moreover, distinct differences in ribosomal RNA processing in cerebral hemispheres of 1- and 10-month-old rats, with a more rapid processing in the brain of adult animals, were obtained.     

Einbau von Uridin und Orotsäure in plastidäre ribosomale RNA von Chlorella nach Antibiotica-Behandlung

Zusammenfassung Chlorella pyrenoidosa inkorporiert unter normalen Anzuchtbedingungen kurzfristig angebotenes Uridin fast ausschließlich in plastidäre ribosomale RNA. Es lassen sich rasch markierte Ribosomen und deren Untereinheiten von 70 S, 50 S und 30 S nachweisen. Diese Markierung wird durch Rifampin in geringen Konzentrationen bereits nach wenigen Minuten unterbunden. Auf das Zellwachstum hat Rifampin bei heterotropher Anzucht dagegen auch in höheren Konzentrationen keinen Einfluß. Chloramphenicol hemmt den kurzfristigen Uridin-Einbau in ribosomale Partikeln von 70 S, 50 S und 30 S, nur geringfügig dagegen denjenigen in ribosomale RNA. Auch die Wirkung des Chloramphenicols tritt rasch ein. Cycloheximid beeinflußt den Kurzzeit-Einbau von Uridin in ribosomale Partikeln und in RNA nicht, wenn die Inkubationszeit 60 min nicht überschreitet.Die Markierung der Nucleinsäuren von Chlorella mit 6-(14C)-Orotsäure zeigt vergleichbare Empfindlichkeiten gegen die drei Antibiotica wie der Einbau von 6-(14C)-Uridin und 5-(3H)-Uridin.

---

Incorporation of uridine and of orotate into chloroplast ribosome RNA of Chlorella after treatment with antibiotics

Summary Normal grown cells of Chlorella pyrenoidosa incorporate uridine exclusively into chloroplast ribosomal RNA after short time labeling. With sucrose gradient separation, labeled ribosomal particles of 70 S, 50 S and 30 S can be shown. This labeling is prevented by rifampin in low concentrations after a few minutes. At the same concentration of the antibiotic and also with 10-fold higher concentration, no effect on heterotrophic cell growth is observed. This indicates clearly that mitochondria cannot be influenced by rifampin. Chloramphenicol also inhibits the formation of uridine labeled ribosomal particles of 70 S, 50 S and 30 S. In the presence of this antibiotic, some labeled ribosomal RNA is formed. Also the effect of chloramphenicol can be shown after short incubation periods. Cycloheximide treatment of the cells within 30 and 60 min and up to the 10-fold concentration of protein synthesis inhibition (Morris, 1967) results in no effect on labeling of ribosomal RNA and of ribosomal particles in Chlorella with uridine. Only after prolonged treatment of the cells with cycloheximide is some effect on uridine incorporation observed.The comparison of the incorporation patterns of 6-(14C)-orotate, (6-14C)-uridine and 5-(3H)-uridine into nucleic acids in the presence of rifampin, chloramphenicol and cycloheximide shows some similarities. After 60 min incubation with the precursors, the incorporation is reduced by all three antibiotics. In rifampin treated cells, orotate and both uridines are preferentially incorporated into DNA. With chloramphenicol, the relative incorporation of orotate and of uridine into the 5 S and the 16 S RNA is higher as compared with the 23 S RNA. Cycloheximide results in an increase in the relative incorporation of orotate as well of uridine into DNA. The similarities of the effects of the three antibotics indicate that the preferential incorporation of uridine into chloroplast ribosomes of Chlorella is not due to a compartmentation of the uridine-UMP-pathway.

---

Abkürzungen BisMSB bis(O-Methylstyryl)-Benzol - PPO 2,5-Diphenyloxazol - MAK-Säule Säule aus methyliertem Albumin mit Kieselgur     

RNA synthesis in the ovary and oocytes of the starfish

Qualitative studies on the in vitro uptake and incorporation of tritiated uridine into RNA of the somatic and germinal elements of the starfish ovary were carried out prior to and during hormone-induced oocyte maturation and spawning.Autoradiography of nonhormone-treated ovaries indicated that the outer ovarian wall contained the highest concentration of label, with lesser amounts in the follicle cells and least in the oocytes. Oocytes and follicle cells localized at the periphery of the ovary were labeled first, and both cells became progressively labeled throughout the ovary with time; the label first appeared localized in the nucleolus of the oocyte.Sucrose gradient analysis of the separated cellular components of prelabeled hormone-treated ovaries indicated that RNA synthesis occurred in all segments of the ovary and that the spawned oocyte fraction was the least active. Synthesis of ribosomal RNA was detectable after a lag period of approximately 4 hr. Oocytes incubated in ³H-uridine during and subsequent to 1-methyladenine-induced spawning and maturation synthesized 15–19 S and low molecular weight RNA but not ribosomal RNA. Synthesis of the 15–19 S RNA was inhibited with ethidium bromide and to a limited extent by actinomycin D. Isolated mitochondrial fractions contained most of the labeled 15–19 S RNA. These data suggest the mitochondrial origin of most, if not all, of this intermediate-weight RNA. On the basis of these studies, it appears that starfish oocytes and follicle cells are metabolically active at the transitional period from growth to maturational stages in oocytes. Synthesis of RNA furthermore apparently continues in the cytoplasm subsequent to germinal vesicle breakdown and spawning.     

Kinetic studies on ribosomal proteins assembly in preribosomal particles and ribosomal subunits of mammalian cells.

Proteins were isolated from 80-S preribosomal particles and ribosomal subunits of murine L5178Y cells after short and longer periods of incubation with tritiated amino acids. The labeling patterns of ribosomal proteins were compared by two-dimensional polyacrylamide gel electrophoresis. The analysis of isotopic ratios in individual protein spots showed marked differences in the relative kinetics of protein appearance within nucleolar peribosomes and cytoplasmic subunits. Among the about 60 distinct proteins characterized in 80-S preribosomes, 9 ribosomal proteins appeared to incorporate radioactive amino acids more rapidly. These proteins become labeled gradually in the cytoplasmic ribosomal subunits. It was found that one non-ribosomal protein associated with 80-S preribosomes takes up label far more quickly than other preribosomal polypeptides. It is suggested that this set of proteins could associate early with newly transcribed pre-rRNA, more rapidly than others after their synthesis on polyribosomes, and could therefore play a role in the regulation of ribosome synthesis. In isolated 60-S and 40-S ribosomal subunits, we detected five proteins from the large subunit and four proteins from the small subunit which incorporate tritiated amino acids more quickly than the remainder. These proteins were shown to be absent or very faintly labeled in 80-S preribosomal particles, and would associate with ribosomal particles at later stages of the maturation process.     

Evidence for ribosomal RNA synthesis in pollen tubes in culture

The evidence is presented that pollen tubes ofNicotiana tabacum L. cultivated in shaken suspension do synthesize 5S, 18S and 28S RNA. Following incubation of pollen tubes in the presence of radioactive uracil or uridine, RNA was isolated from total pollen tube material after the removal of 4S RNA, from polysomes and from 80S ribosomal particles, and fractionated by density gradient centrifugation and MAK column chromatography. The results obtained further suggest a higher rate of 5S RNA synthesis with respect to 18S+28S RNA.     

Turnover of ribosomal RNA in mouse fibroblasts (3T3) in culture.

Growing and confluent cultures of mouse fibroblasts were labeled with ³H-uridine and chased with an excess of nonradioactive uridine to investigate the turnover of ribosomal RNA. Growing cultures did not turn over their 18S and 28S ribosomal RNA; however, confluent cultures did show ribosomal RNA (rRNA) turnover. If the cells were labeled while growing, and chased when confluent, 18S RNA displayed a two-component decay curve, while 28S RNA showed only single-component decay, similar in lifetime to the first component of the 18S RNA decay curve. If the cells were labeled while confluent, both the 18S and 28S RNA showed single-component decay curves with a lifetime possibly only slightly longer than the lifetime of the first component of the 18S RNA and the single component of the 28S RNA of the cultures labeled while growing.     

Ribosomal Ribonucleic Acid Synthesis and Maturation in the Blue-Green Alga Anacystis nidulans

Methods are described for preparation of pulse-labeled ribonucleic acid (RNA) from the blue-green alga Anacystis nidulans. Synthesis of labeled RNA was found to be in part dependent on concurrent photosynthesis and was inhibited by the antibiotic streptolydigin. Mature 23S ribosomal RNA (rRNA) appeared before mature 16S rRNA. Formation of either molecule was inhibited by chloramphenicol, and RNA species of lesser mobility accumulated. These species may be precursors of the mature forms. Maturation of 16S rRNA was also inhibited by streptolydigin. (The effect of this antibiotic on 23S rRNA maturation was not examined). In many respects, ribosomal RNA synthesis and maturation in this blue-green alga appear to follow the pattern already established for bacteria.