Dietary nucleotide requirements of the mosquito, Culex pipiens

Dietary nucleic acid, or certain constituents thereof, is essential for full development of the mosquito Culex pipiens. Yeast RNA, whole or hydrolysed, is fully effective. Sperm DNA is totally ineffective on its own, but becomes fully effective when supplemented with uridylic acid. RNA can be replaced with only partial success by a mixture of its component nucleotides (adenylic, guanylic, cytidylic, and uridylic acids), but with the addition of thymidylic acid (characteristic of DNA but present in RNA only as a minor component of transfer RNA), the resulting five-nucleotide mixture is a fully adequate replacement for yeast RNA. Single and multiple deletions from this five-nucleotide mixture showed a minimal requirement for three nucleotides: adenylic acid (a purine ribonucleotide) and thymidylic acid (a pyrimidine deoxyribonucleotide) were both specifically required; as the third nucleotide, either cytidylic or uridylic acid (both pyrimidine ribonucleotides) was equally satisfactory. Mixtures of the five nucleosides or five deoxynucleotides corresponding in base composition to the effective five-nucleotide mixture were only partially effective substitutes.     

Phagostimulation of larval Culexpipiens L. by nucleic acid nucleotides, nucleosides and bases

ABSTRACT. The influence of nucleic acid constituents on the rate of ingestion of charcoal powder filtered from ambient water by larvae of Culex pipiens L. was examined. All nucleotides tested stimulated ingestion to some extent. Various mono-, di- and tri-phosphates of adenosine were most effective and at concentrations of 1 mM stimulated ingestion nearly as well as yeast extract, a powerful phagostimu-lant. Guanylic, thymidylic, cytidylic and uridylic acids were less stimulatory, the latter two even at 10 mM. Cyclic AMP and deoxyadenylic acid were less effective than other adenine nucleotides. The nucleosides adenosine, guanosine and uridine were almost as effective as their corresponding nucleotides (adenylic, guanylic and uridylic acids); thymidine was less effective than thymidylic acids, whereas cytidine was non-stimulatory. Adenine, guanine, uracil and cytosine, the bases of the ribose nucleotides, were non-phagostimulatory, whereas thymine, base of the deoxynucleotide, thymidylic acid, caused low but significantly increased ingestion. These findings are compared with the reported phagostimulation by nucleic acid constituents of certain plant feeding insects and with the stimulation of engorgement of the blood meal by many blood feeding insects.     

Synthetic polynucleotides as model substrates for ribosomal RNA processing

A nuclear exoribonuclease from Novikoff ascites cells was used to study the hydrolysis of single-stranded heteropolymers containing [¹⁴C]adenylic acid and either uridylic acid or cytidylic acid and heteropolymers of [¹⁴C]adenylic acid and one of the corresponding 2′-$\text{O}$-methylated nucleotides. The results of these studies indicate that both the rate and extent of hydrolysis are greatly inhibited by the presence of 2′-$\text{O}$-methylated nucleotides. Restriction of exonuclease activity by 2′-$\text{O}$-methylated nucleotides provides a possible mechanism for rRNA processing.     

Further Nutritional Requirements of Paramecium aurelia

SYNOPSIS. An acetone-insoluble yeast fraction required for axenic growth of P. aurelia , stock 51, variety 4, sensitive , after fractionation contained at least 3 essential components: (1) one soluble in perchloric acid and completely replaceable by a mixture of ribosides or ribotides; (2) one inactivated after digestion with trypsin, chymotrypsin or papain. Proteose-Peptone restored activity to this preparation, which suggests a peptide requirement; and (3) one not yet characterized.
As for the purine and pyrimidines, these combinations, in decreasing order of activity, supported growth: guanosine + cytidine, guanosine + uridine, guanylic acid + cytidylic acid, and guanylic acid + uridylic acid. Each combination was maximally effective when the molar purine: pyrimidine ratio was ∼ 0.4. On a molar basis, the minimal riboside combinations were ∼ 1.3 × more active than the ribotides.
Sparing of the purine and pyrimidine requirement was also investigated. In the presence of limiting amounts of guanylic acid, the following compounds, in decreasing order of activity, had sparing activity: deoxyguanosine, inosine, xanthosine, adenylic acid, and guanine. Adenine, adenosine, and deoxyadenosine were inhibitory under the test conditions. The requirement for cytidylic acid was spared by deoxycytidine, uridine, uridylic acid, deoxyuridine, thymidine, thymine, and uracil, in descending order.     

Metabolism of cytidine and uridine in bean leaves

The metabolism of cytidine-2-¹⁴C and uridine-2-¹⁴C was studied in discs cut from leaflets of bean plants (Phaseolus vulgaris L.). Cytidine was degraded to carbon dioxide and incorporated into RNA at about the same rates as was uridine. Both nucleosides were converted into the same soluble nucleotides, principally uridine diphosphate glucose, suggesting that cytidine was rapidly deaminated to uridine and then metabolized along the same pathways. However, cytidine was converted to cytidine diphosphate and cytidine triphosphate more effectively than was uridine. Cytidine also was converted into cytidylic acid of RNA much more extensively and into RNA uridylic acid less extensively than was uridine. Azaserine, an antagonist of reactions involving glutamine (including the conversion of uridine triphosphate to cytidine triphosphate), inhibited the conversion of cytidine into RNA uridylic acid with less effect on its incorporation into cytidylic acid. On the other hand, it inhibited the conversion of orotic acid into RNA cytidylic acid much more than into uridylic acid. The results suggest that cytidine is in part metabolized by direct conversion to uridine and in part by conversion to cytidine triphosphate through reactions not involving uridine nucleotides.     

Free nucleotides in bromodeoxyuridine-treated neuroblastoma cells

Free nucleotides in bromodeoxyuridine-treated neuroblastoma cells were investigated. The separation and purification of nucleotides were carried out by anion exchange and paper chromatography. After bromodeoxyuridine treatment, the distribution of free nucleotides with the same base, when referred to total nucleotides, shifted toward a distribution pattern closer to that of mature neuronal cells. There was an enhancement of adenylic nucleotides and a decrease of uridylic and cytidylic nucleotides. The guanylic nucleotides displayed no changes. This nucleotide distribution is in agreement with other biochemical data from differentiated neuroblastoma cells.Dr. Ciesielski-Treska is stagiaire de Recherche à l'INSERMDr. Wintzerith is Chargée de Recherche au CNRSThis paper is part of the Doctorat d'Etat thesis of A. Dierich.     

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.     

Effect of acetylcholine on incorporation of 14C-precursors into uridylic and cytidylic nucleotides of the RNA of digestive glands

The incorporation rate of [2-14C]orotic acid and [2-14C]uridine into the cytidylic RNA nucleotides is significantly lower than into the uridylic ones. In the liver it was twice as low as in the stomach mucosa or in pancreas of albino rats. The administration of acetylcholine in combination with proserine has no influence on the RNA content and its nucleotide composition in the tissues. The administered drugs however caused changes in the relation of the incorporation rates of both labels into uridylic and cytidylic RNA nucleotides, which evidences for the uridylic nucleotide synthesis. In the liver such changes are not detected, but utilization of the labeled uridine is shown to be more intensive for the cytidylic RNA nucleotides synthesis.     

Purification and properties of a new ribonuclease from Aspergillus saitoi.

From a commercial digestive produced from Aspergillus saitoi, a ribonuclease [EC 3.1.4.23] having a molecular weight of 12,500 has been isolated in addition to the RNase reported previously, which had a molecular weight of 38,000. The enzyme was found to be homogeneous by chromatography on DEAE-cellulose, disc electrophoresis on polyacrylamide gel, and ultracentrifugation. The NH2-terminal amino acid was identified as glutamic acid. The amino acid composition indicated the presence of about 13 tyrosyl residues, 3 histidyl residues, and 2 half-cystine residues. The pH optimum of the RNase was 4.5, using RNA as a substrate. The enzyme was stable on heating at 70 degrees for 5 min from pH 2 to 10. It hydrolysed RNA completely to mononucleotides via 2', 3'-cyclic nucleotides. The rates of release of nucleotides and 2', 3'-cyclic nucleotides were in the order: guanylic acid is greater than adenylic acid is greater than cytidylic acid is greater than uridylic acid.     

Nutrition of Myxococcus xanthus FBa and Some of Its Auxotrophic Mutants

A defined medium containing 15 amino acids plus salts was used to study the nutrition of Myxococcus xanthus FBa. The amino acids phenylalanine, leucine, isoleucine, valine, and methionine were essential for growth, whereas glycine, proline, asparagine, alanine, lysine, and threonine stimulated growth. An unusual pattern of requirement was found in the aromatic amino acids. Phenylalanine was essential and served as the precursor of tyrosine. Growth in the absence of tryptophan was adaptive, with cells reaching a growth rate equal to that of controls after a lag of about a week. (14)C-labeled ribose and glucose were not appreciably metabolized. Auxotrophs requiring purines and pyrimidines were isolated and were used to study the fate of externally supplied nucleic acid derivatives. Appropriate mutants could satisfy their requirements with free bases, nucleosides, and nucleotides, and could hydrolyze nucleic acids and use the products. However, studies using (14)C-ribose-labeled uridine (isolated from a Salmonella typhimurium pyrimidine auxotroph) showed that externally supplied nucleic acid derivatives were incorporated almost solely into the nucleic acids of the myxobacters, with little used either for energy-yielding oxidations or other cell anabolism.     

Partial characterization of ribonuclease (RNase) activity from the isolated and solubilized brush border of Hymenolepis diminuta

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.     

Nucleic acid analogs for high performance liquid chromatography

HPLC resins containing nucleic acid base derivatives were successfully prepared. These resins were found to give excellent complementary separation of nucleic acid base derivatives, nucleosides, nucleotides, and oligonucleotides. These resins may be useful for separation of components of nucleic acids and polynucleotides as a specific separation system, while ion-exchange and reverse-phase systems are non-specific separation systems.     

Characterization of Two Simian Virus 40-Specific RNA Molecules from Infected BS-C-1 Cells

Two discrete simian virus 40 (SV40) RNA species sedimenting at 19 and 16S, respectively, that are present in infected BS-C-1 cells were characterized with respect to the base composition and the ribonuclease T1 fingerprints. The base composition of the 19S SV40 RNA was found to be cytidylic acid (C), 23.0; adenylic acid (A), 28.3; guanylic acid (G), 23.9; and uridylic acid (U), 24.8; that of the 16S SV40 RNA was C, 19.3; A, 34.0; G, 22.0; and U, 24.7 mol%. Analysis of the ribonuclease T1 fingerprints indicated a difference in the base sequence of the 19 and 16S SV40 RNA. The presence of long sequences of adenylic acid residues (poly A) in these viral RNAs was confirmed.     

Nutrient media for plant-parasitic nematodes. VI. Nucleic acids content and nucleotide synthesis in Aphelenchoides rutgersi

The nucleic acids content of Aphelenchoides rutgersi, Hooper and Myers, was 0.9% DNA and 2.6% RNA dry weight. The DNA contained 29.5% adenine, 29.3% thymine, 22.5% guanine, and 18.8% cytosine, while the RNA was composed of 22.8% adenine, 23.0% uracil, 31.4% guanine, and 22.9% cytosine on a molar basis.The nematodes needed folic acid for reproduction regardless of the presence or absence of nucleic acid supplements in the culture medium. This was shown by including aminopterin, a folic acid antagonist in the culture medium. A 2-hr incubation of nematodes with glycine-¹⁴C (U) and orotic-5-³H acid resulted in the incorporation of ³H-label into both DNA and RNA. Only the RNA fraction contained a significant amount of ¹⁴C-label. When this RNA was fractionated, the adenine and guanine accounted for the ¹⁴C-label, while cytidylic and uridylic acids contained the ³H-label, thereby demonstrating purine and pyrimidine synthesis by A. rutgersi. The incorporation of orotic acid into the pyrimidines was 8 times higher than that of glycine into purines.     

Microbiology of Saturated Salt Solutions and Other Harsh Environments. II. Ribonucleotide Dependency in the Growth of a Salt-Habituated Penicillium notatum in Salt-Free Nutrient Media

A strain of Penicillium notatum has been found growing upon media containing saturated calcium acetate which upon passage through media containing saturated KC1 loses its ability to grow at normal rates in salt-free glucose-peptone medium. This salt-habituated form can be restored to a near-normal growth rate by adding yeast extract, RNA or RNA-hydrolysates, but not by DNA or its hydrolysate. The 4 RNA nucleotides in combination replace yeast extract whereas riboside and base combinations do not. Adenylic, guanylic or cytidilic acids, singly or in various combinations, are even more effective, but uridylic acid alone is not active at all. Ribonucleotide pairs including uridylic acid are less active, but 3 ribonucleotide combinations are highly effective, even in the presence of uridylic acid. Differential effects were evident in mycelial form and sporulation, as well as growth. In addition to a direct role in Penicillium RNA metabolism, the significance of ribonucleotides in energy metabolism or cell wall synthesis is mentioned as alternatives to explain their restorative effects.     

Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22.

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.     

De novo purine and pyrimidine biosynthesis during thymocyte concanavalin A stimulation

Measurement of the incorporation of [¹⁴C]formate and [¹⁴C]HCO₃⁻ enable us to characterize the activity of the synthetic pathways of purine and pyrimidine nucleotides in vitro throughout the process of the conA stimulation of rat thymocyte populations enriched for immunocompetent cells by isopycnic centrifugation. Our results show that de novo synthesis compensates for the total absence of exogenous nitrogenous bases and nucleosides in the culture medium. The magnitude of the proliferative response in media supplemented with dialysed fetal calf serum (FCS) was found to be the same as that observed when complete FCS was used in the culture medium. The induction of de novo synthesis (1) contributes to the expansion of the free nucleotide pool (the quantity of ATP measured in the perchloric acid (PCA)-soluble material of a same number of cells is increased by a factor of 10); (2) supplies the nucleotides necessary for nucleic acid synthesis (the total number of cells is increased by a factor of 3 after 4 days culture period). The activity of the metabolic pathways involved appears to be solicited by the dynamic requirements for thymocyte stimulation. For each step in the cellular activation a steady state of adenylic nucleotide synthesis and condensation into nucleic acids is established.     

Mutants affecting thymidine metabolism in Neurospora crassa

When (14)C-thymidine labeled only in the ring is administered to Neurospora crassa, the majority of the recovered label is found in the ribonucleic acid (RNA). Three mutants were isolated in which different steps are blocked in the pathway that converts the pyrimidine ring of thymidine to an RNA precursor. Evidence from genetic, nutritional, and accumulation studies with the three mutants shows the pathway to proceed as follows: thymidine --> thymine --> 5-hydroxymethyluracil --> 5-formyluracil --> uracil --> uridylic acid. A mutant strain in which the thymidine to thymine conversion is blocked is unable to metabolize thymidine appreciably by any route, including entry into nucleic acids. This suggests that Neurospora lacks a thymidine phosphorylating enzyme. A second mutation blocks the pathway at the 5-hydroxymethyluracil to 5-formyluracil step, whereas a third prevents utilization of uracil and all compounds preceding it in the pathway. The mutant isolation procedures yielded three other classes of mutations which are proposed to be affecting, respectively, regulation of the thymidine degradative pathway, transport of pyrimidine free bases, and transport of pyrimidine nucleosides.     

Changes in nucleolar and ribosomal RNA of the frog kidney after malignant transformation by the Lucke tumor herpesvirus

Malignant transformation of the frog kidney by the Lucke herpesvirus changes the nucleotide base composition of normal kidney nucleolar and ribosomal RNA. In the Lucke tumor there is a moderate decline in guanylic acid and a sharp decline in adenylic acid levels. Conversely, there is a sharp increase in cytidylic acid and uridylic acid levels in the tumor cells. However, there was an increase in the G + C content of nucleolar and ribosomal RNA over that obtained from the normal kidney cells. Nearly identical quantitative changes in the base composition of each RNA species were measured for the adult (spontaneous) mesonephric carcinoma and a Lucke-herpesvirus-induced pronephric tumor cell line; similar correspondence was obtained for the normal adult mesonephros and a normal pronephric cell line.     

Base composition of duck 10S RNA and its poly(A) segment

The base composition of the poly(A) segment of duck 10S RNA was determined to be 92% AMP and 8% GMP. The GMP was probably the result of contamination of poly(A) with other segments of the RNA. A comparison of the theoretical and determined base compositions of the whole 10S RNA molecule suggested that it contains, besides a coding sequence, two noncoding sequences only one of which is poly(A).Abbreviations AMP adenylic acid - GMP guanylic acid - CMP cytidylic acid - UMP uridylic acid - SDS sodium dodecyl sulfate - EDTA ethylene diamine tetraacetic acid - TCA trichloroacetic acid