ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-¹⁴C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-¹⁴C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.     

Effects of Cycloheximide upon Formation of Ribonucleic Acid Cytidylic and Uridylic Acids

Concentrations of cycloheximide as low as 3 μg/ml inhibited incorporation of labeled orotic acid or uridine into RNA cytidylic acid of soybean (Glycine max) hypocotyl sections. Even lower concentrations of this well known protein synthesis inhibitor interfered with conversion of labeled cytidine into RNA uridylic acid. Both cycloheximide and puromycin inhibited absorption of ³H-phenylalanine and its incorporation into protein, but puromycin did not significantly affect the labeling patterns of RNA cytidylic and uridylic acids when orotic acid-6-¹⁴C was fed. Results give further support to the hypothesis that cycloheximide inhibits the interconversion of uridine and cytidine nucleotides, presumably by acting as a glutamine antagonist in the glutamine-dependent reaction catalyzed by cytidine triphosphate synthetase.     

Pyrimidine biosynthesis and its regulation in the developing rat brain.

—Measurements of the incorporation of [¹⁴C]NaHCO₃ into orotic acid, uridine nucleotides and RNA in tissue minces establish the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in rat brain. Selective inhibition of the incorporation of various radiolabelled precursors into orotic acid by uridine demonstrates the operation of a feedback control mechanism in brain minces and indicates carbamoylphosphate synthetase to be the site of inhibition; purine nucleosides were similarly found to inhibit the de novo biosynthesis of pyrimidines. The activity of the orotate pathway, as assessed by the rate of incorporation of [¹⁴C]NaHCO₃ into orotic acid, was found to be very high in fetal brain and to decline rapidly with neurological development; the mature rat brain exhibits less than 1% of the activity of the fetal brain at 18 days of gestation. Comparative studies on the ability of minces of the brain and several extraneural tissues to utilize [¹⁴C]NaHCO₃ and [¹⁴C]aspartate as precursors of orotic acid lead us to speculate that variations in the ability of tissues to synthesize orotic acid de novo are determined by similar variations in their ability to synthesize carbamoylphosphate.     

DNA and RNA Syntheses by Intraerythrocytic Stages of Plasmodium knowlesi*

Incorporation of the nucleic acid precursors, orotic acid, adenosine, thymidine, and uridine, was studied in various stages of intraerythrocytic Plasmodium knowlesi from infected rhesus monkeys. Incubation of the parasitized erythrocytes with the precursors was for 3 hr periods using a plasma-free culture medium. The samples containing primarily rings, early trophozoites, or late trophozoites incorporated orotic acid, adenosine, and uridine into RNA; however, these stages exhibited negligible or very low levels of incorporation of any of the precursors into DNA. The sample containing late trophozoite and schizont stages incorporated orotic acid, adenosine, and uridine into RNA, and orotic acid, adenosine, and very low levels of thymidine into DNA. These results indicate that DNA synthesis (the S phase of the cell cycle) occurs very close to the time of nuclear division, and that either the G1 or G2 phase is very short in P. knowlesi. It was also observed that adenosine and orotic acid, 2 precursors which are incorporated into both DNA and RNA, are utilized differently by the intraerythrocytic parasites. Incorporation of orotic acid into RNA and DNA and adenosine incorporation into DNA were continuous for the entire incubation period, whereas incorporation of adenosine into RNA was very low during the last 2 hr of each period. It was further demonstrated that the parasites utilized exogenous uridine for synthesis of RNA, and that the older parasite stages incorporated thymidine into DNA.     

The effect of feeding with a tryptophan-free amino acid mixture on rat-liver polysomes and ribosomal ribonucleic acid

1. Starving rats were given complete and tryptophan-deficient amino acid mixtures by stomach tube and were killed from 1 to 7hr. later. The polysome profile in the livers of rats fed with the tryptophan-deficient mixture showed a shift in distribution such that the large aggregates were decreased and the small aggregates were increased, particularly dimers. This polysome shift was reversed when the complete amino acid mixture was given by stomach tube 2hr. after administering the tryptophan-free amino acid mixture. 2. After removal of liver polysomes by centrifugation, some smaller ribosomal aggregates (oligosomes) remaining in suspension were harvested by prolonged centrifugation of the supernatant fluid. A large increase in the dimer population of this fraction was observed in the rats receiving the incomplete mixture. 3. When the polysome and oligosome fractions were incubated with cell sap, an energy-generating system and labelled amino acids dl-[1-(14)C]leucine and l-[Me-(14)C]tryptophan were incorporated into the cell fractions in the ratio 4.5:1. Preparations of polysomes and oligosomes from rats fed with the tryptophan-free amino acid mixture showed a decreased amino acid-incorporating activity compared with particulate preparations made from rats fed with the complete mixture. 4. The yield of free ribosomes prepared from the unfractionated liver microsomes by treatment with iso-octane was 40-50% greater in rats fed with the amino acid mixture deficient in tryptophan. 5. A post-microsomal fraction was prepared from cell sap and was shown to consist of ribosomal sub-units. When the animals were fed with the tryptophan-deficient mixture, there was an increase in content of this post-microsomal fraction and in the ratio 30s RNA/19s RNA. Rats were also given [5-(3)H]orotic acid at the time of feeding with the amino acids. Lack of tryptophan in the mixture caused a decrease in the specific activity of both RNA fractions which affected the 30s RNA more extensively than the 19s RNA. 6. These changes in the distribution and quantity of the cellular components engaged in protein synthesis are discussed in relation to RNA metabolism and amino acid-incorporating activity of the liver cell and their response to feeding with the tryptophan-free amino acid mixture.     

An improved method for the isolation of polysomes from synchronous macroplasmodia of Physarum polycephalum

An improved method for the isolation of polysomes from synchronous macroplasmodia of Physarum polycephalum is described. The procedure involves preincubation and homogenization of the macroplasmodia in high ionic strength media supplemented with high concentrations of Mg²⁺ and ethylene glycol bis (β-aminoethyl ether) N,N′-tetraacetic acid. The polysomes recovered from plasmodial postmitochondrial lysates or heavy particle fractions prepared in this way are sensitive to RNase and EDTA and more than 90% of the total single ribosome population is present as polysomes. The polysomes obtained could also be utilized as a beginning point for the isolation of poly(A)-containing RNA which showed a mass average sedimentation value of 18 S. The development of a satisfactory procedure for the isolation of intact polysomes and poly(A)-containing RNA from macroplasmodia should facilitate studies on mRNA metabolism and translational activity during a naturally synchronous mitotic division cycle.     

AUTORADIOGRAPHIC STUDIES OF NUCLEIC ACIDS AND PROTEINS DURING MEIOSIS IN LILIUM LONGIFLORUM

Taylor , J. Herbert (Columbia U., New York, N. Y.) Autoradiographic studies of nucleic acids and proteins during meiosis in Lilium longiflorum. Amer. Jour. Bot. 46(7): 477–484. Illus. 1959.—A study was made of the incorporation of glycine-C¹⁴, orotic acid-C¹⁴ and cytidine-H³ into nucleic acids and proteins of sporogenous and tapetal cells of lily anthers preceding and during meiosis. Methods for differential extraction of nucleic acids from tissue sections, which had been frozen, dehydrated by alcohol-substitution, and fixed in hot alcohol, were tested by chromatographic analysis of extracts. Both acid and enzyme hydrolysis were shown to be useful for quantitative or, at least, semi-quantitative work. DNA synthesis was shown to occur only during premeiotic interphase in sporogenous cells, but at two intervals in tapetal nuclei, once when the microsporocytes are in zygotene and again during pachytene. Each time the synthetic period was followed by a normal mitosis. Accumulation of RNA in microsporocytes occurred at stages up to late leptotene. After this period, labeled RNA accumulated almost exclusively in their nuclei and at a slower rate than in earlier stages. DNA synthesis, as measured by incorporation of glycine-C¹⁴ and orotic acid-C¹⁴, gave the same results and confirm earlier results with inorganic phosphate-P³². For RNA, glycine-C¹⁴ and orotic acid-C¹⁴ gave different results. When glycine-C¹⁴ was the source of label, incorporation of C¹⁴ in RNA stopped during DNA synthesis in sporogenous cells. Glycine-C¹⁴ was not utilized to a significant extent at any time by tapetal cells for RNA synthesis, but extensively for DNA and protein synthesis. Orotic acid-C¹⁴ was incorporated into RNA of both tapetum and sporogenous cells at various periods in development apparently including the interval of DNA synthesis. Protein synthesis as measured by incorporation of glycine is relatively rapid during premeiotic interphase and leptotene. It continues during the remainder of prophase, but at a much reduced rate. In tapetal cells the rate is rapid in the nuclei during periods of DNA synthesis, but even faster in both cytoplasm and nucleus after divisions are completed and the microsporocytes are in late prophase and division stages. This period of synthesis is perhaps necessary for the postmeiotic functioning of tapetum when it appears to secrete the wall materials for the microspores.     

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-¹⁴C]orotic acid and inorganic [³²P]phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.     

Pyrimidine biosynthesis in rat brain

—Studies on the incorporation of [¹⁴C]NaHCO₃ into both orotic acid and RNA in tissue slices reveal the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in the rat brain. A comparison of the rates of incorporation of bicarbonate into orotic acid and RNA in tissue slices of brain and liver indicate the brain to be one-fourth to one-half as active as the liver in the de novo biosynthesis of pyrimidines. The results of this study favor the proposal that the adult rat brain can meet its needs for pyrimidines through de novo synthesis and is not dependent upon salvage activity and an extraneural supply of pyrimidines.     

The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA

The control of protein synthesis in oocytes of Xenopus laevis has been investigated by injecting oocytes with mRNA and polysomes followed by labeling with ¹⁴C-amino acid mixtures. Contrary to previous reports in which injected oocytes were labeled with ³H-histidine, injected globin mRNA is found to decrease amino acid incorporation into endogenous proteins competitively at all concentrations tested. No increase in overall amino acid incorporation is detected when more mRNA is supplied. Similar results are obtained after labeling injected oocytes with leucine, methionine, proline or valine individually. An explanation is presented for the conflicting results obtained when histidine is used as a label.When reticulocyte polysomes are injected, rather than purified globin mRNA, incorporation of amino acids into endogenous proteins remains roughly constant and overall incorporation increases. Similarly, when encephalomyocarditis viral RNA is injected together with either globin mRNA or reticulocyte polysomes, the globin mRNA causes decreased amino acid incorporation into encephalomyocarditis proteins, but the polysomes do not do so. The results demonstrate that different types of mRNA compete for a strictly limited translational capacity which is saturated in the normal oocyte. The limiting component is present in polysomes and is not message-specific. The constraint on protein synthesis in the amphibian oocyte cannot be fully explained by masked mRNA.     

Metabolism of tissue cells in suspension. Synthesis of ribonucleic acid by liver cells in suspension

1. Liver cells in suspension are shown to incorporate several RNA precursors into their RNA. 2. The incorporation of [³²P]phosphate and [¹⁴C]adenine into the RNA of the cell suspension is usually of the same order as that in the perfused (or unperfused) liver slices. However, the initial lag in the incorporation of adenine into the RNA of the cell suspensions is much longer than that obtained for the tissue slices, and the optimum incorporation of adenine in the former, unlike that in the latter, needs exogenous glucose and probably a high concentration of phosphate. 3. The cell suspensions also differ from the tissue slices in being unable to incorporate [¹⁴C]orotic acid into their RNA, and resemble tumour tissues in incorporating uracil into their RNA at a rate significantly higher than that obtained with the tissue slices. 4. The above differences in the metabolic behaviour of liver-cell suspensions and tissue slices are considered to be due to the different levels of organization of the liver cells in the two tissue preparations.     

STUDIES ON THE ORIGIN OF PYRIMIDINES FOR BIOSYNTHESIS OF NEURAL RNA IN THE RAT

Uridine was far superior to orotic acid in labelling the RNA in incubated slices of rat brain. On the other hand, uridine and orotic acid were equally effective in labelling the RNA of hepatic or renal slices In rats in vivo, uridine, but not orotic acid, labelled brain RNA, and the cerebellar RNA contained the most label. In contrast, both uridine and orotic acid labelled hepatic RNA. Only when surgical intervention prevented peripheral metabolism of orotic acid, thereby raising its concentration in the plasma, did neural tissue utilize this precursor for limited biosynthesis of RNA. However, among the tissues studied, the preference for uridine over orotic acid for RNA synthesis was unique to neural tissue.     

The effect of kinetin on the incorporation of labelled orotate into various fractions of ribonucleic acid of excised radish leaf discs

Summary Discs from senescing radish leaves were floated either on water or on a solution of kinetin and incubated in the dark. The kinetin-treated discs were fed with orotic acid-5-T and the control discs with orotic acid-6-C¹⁴. The two lots of discs were combined and the RNA extracted by the phenol method. The H³:C¹⁴ ratio of the RNA after fractionation on sucrose density gradient was compared with the H³:C¹⁴ ratio of RNA extracted from two lots of control discs, one fed with orotic acid-5-T and the other with orotic acid-6-C¹⁴ and then extracted together. In this way it was found that 21 hours treatment with kinetin causes a small but consistent stimulation of labelled precursor incorporation into all classes of RNA investigated.     

The stability of rat liver ribonucleic acid in vivo after methylation with methyl methanesulphonate or dimethylnitrosamine

1. RNA was isolated from rat liver at selected times after the intraperitoneal injection of either [¹⁴C]methyl methanesulphonate (50mg/kg) or [¹⁴C]dimethylnitrosamine (2mg/kg). These doses were chosen to minimize effects due to toxicity. 2. Two methods of extraction and purification of RNA were used and an analysis of the radioactivity present was made by column chromatography of acid hydrolysates of the purified RNA. 3. The extent of methylation of guanine, the principal site of alkylation in rat liver RNA, was determined at times up to 14 days after injection. Although dimethylnitrosamine is a potent liver carcinogen and methyl methanesulphonate is not carcinogenic to rat liver, the rate of disappearance of 7-methylguanine from RNA was similar for both compounds, with a half-life of about 3.5 days. 4. An estimate of the biological half-life of rRNA was made by using [³H]orotic acid. A half-life of 5 days was obtained and this was not affected by injecting animals with unlabelled methyl methanesulphonate at the same dosage of 50mg/kg used in the studies of RNA methylation. 5. After administration of labelled orotic acid, reutilization of labelled RNA degradation products probably results in an overestimation of the biological half-life for rRNA. It is suggested that non-toxic doses of methylating agents such as methyl methanesulphonate and dimethylnitrosamine may prove to be a more effective way of accurately estimating the biological turnover of RNA species.     

Action of ribonuclease upon the polysomes of cultured mouse cells

Summary Pancreatic ribonuclease (RNase) and³H-uridine were used to study certain compositional and ontogenetic features of the polysomes of strain L mouse cells. Growing cells were exposed to the radioactive nucleoside,³H-uridine, for brief defined periods, and the sensitivity of the polysomes to digestion by RNase was determined. The RNase-resistant RNA of polysomes is shown to be primarily ribosomal, and the RNase-sensitive material formed during brief pulse labeling studies is largely messenger RNA. Actinomycin D inhibition of RNA synthesis was used to confirm this identification. The technique described here was used to investigate the effects of hydrocortisone on polysome formation. The hormone (10⁻⁶ m) lessens the extent of the nucleoside incorporation into polysomal and total RNA and delays the appearance of newly synthesized 18 S and 28 S rRNA into cytoplasmic polysomes. This work was partially supported by grants from the United States Public Health Service (GM 10866), from the National Science Foundation (GB 13924), and from The University of Kansas General Research Fund.     

Movement of ribosomes over messenger RNA in polysomes of rel + and rel - Escherichia coli strains

The in vitro movement of ribosomes over messenger RNA was studied in both the presence and the absence of protein synthesis. For this purpose, labeled polysomes were extracted from rel⁺ and rel^? strains of Escherichia coli grown in the presence of radioactive uracil and incubated in a cell-free system containing tRNA, amino acids, soluble enzymes and a source of energy. The gradual conversion of the labeled polysomes into monosomes and ribosomal subunits was followed by subjecting the reaction mixture to sucrose gradient sedimentation after various incubation times and measuring the radioactivity present in the three relevant ribosomal fractions.It was found that when the conditions of incubation allow protein synthesis to occur, polysomes extracted from rel⁺ and rel^? cells are converted mainly into free monosomes, which can be made to dissociate into subunits by high-sodium or low-magnesium ion concentrations. Under conditions in which protein synthesis cannot occur because a mutant aminoacyl-tRNA synthetase has been rendered inactive, polysome conversion still occurs, though to a reduced extent. When the products of such residual run-off are examined, however, a difference is manifest between polysomes extracted from rel⁺ and from rel^? strains: whereas the polysomes from the rel^? strain are still converted into free monosomes even in the absence of protein synthesis, the polysomes from the rel⁺ strain are now converted mainly into subunits. It can be inferred, therefore, that ribosomes from rel^? bacteria, but not those from rel⁺ bacteria, continue movement over messenger RNA in the absence of protein synthesis.Studies of mixed extracts from rel^? and rel⁺ bacteria have shown that the character of the run-off process does not depend on the source of tRNA and soluble enzymes; the proportions of monosomes and subunits among the run-off products formed in the absence of protein synthesis depend only on the source of the polysomes. It is suggested that the mutation of the rel gene alters the functional architecture of ribosomes.     

Direct counting of tissues containing radioactive cholesterol by a two-phase liquid scintillation method

A rapid and simple method for counting radioactivity in tissue samples containing [³H]- or [¹⁴C]-cholesterol is described.Up to 500 μl of the specimen to be counted (plasma, tissue homogenate) is measured into a counting vial. The lipids of the tissue are extracted into 15 ml of a toluene-based scintillation mixture containing 37.5% ethylene glycol monomethyl ether that is added to the same vial. With the addition of 1 ml water, two phases form: the upper toluene phase containing all cholesterol together with the scintillating phosphor and the lower water phase containing most of the quenching material. Bleaching to reduce color quenching is not necessary. Chemiluminescence is negligible. The counting efficiencies are appreciably higher than those obtained in aqueous one-phase scintillation systems but lower than those obtained with pure standards in one-phase pure toluene scintillation systems.     

Improvements in and Environmental Applications of Double-Vial Radiorespirometry for the Study of Microbial Mineralization

Several variations in the scintillation mixture and the filter paper arrangements for double-vial radiorespirometry were compared. Improved efficiencies (44%) and shorter response times were found by adding wetting agents and methanolic NaOH to the scintillation mixture in the filter paper. The scintillation chemicals used did not contain dioxane and were found to be nontoxic to the test microbiota in this system. Covering the inner reaction vial with aluminum foil minimized the reduction in counting efficiency when testing colored or dense environmental samples. Mineralization rates were determined with ¹⁴C-labeled glucose, acetate, and glutamate and [¹⁴C]cellulose- and [¹⁴C]lignin-labeled lignocellulose for composting cow manure, forest soil, and arctic lake sediment microbiota. This improved method can be used in a variety of procedures involving the measurement of microbial mineralizations of organic compounds. Since no liquid scintillation cocktail is used for counting, the radioactive wastes are aqueous or can be incinerated, making disposal easy.