THE BASE COMPOSITION OF NUCLEIC ACIDS IN CHROMOSOMES, PUFFS, NUCLEOLI, AND CYTOPLASM OF CHIRONOMUS SALIVARY GLAND CELLS

The base composition of RNA from individually isolated giant chromosomes, puffed chromosome segments, nucleoli, and samples of cytoplasm from Chironomus salivary gland cells was determined by microelectrophoresis. Data on the adenine: guanine quotient of the chromosomal DNA were also obtained. The results show that: 1) Chromosomal, nucleolar, and cytoplasmic RNA's differ significantly from each other in base composition. 2) Nucleolar and cytoplasmic RNA's, in spite of the difference, show great similarities with regard to the base composition and are both rich in adenine and uracil. 3) The RNA extracted from chromosome I differs significantly from the RNA's extracted from different segments of chromosome IV, and the latter differ significantly from each other. 4) The values for the RNA: DNA quotients of chromosome segments parallel the development of synthetically active genes, so-called Balbiani rings. 5) The chromosomal RNA does not show a base symmetry in any of the investigated cases, nor is the content of guanine + cytosine the same as that for DNA. The first of these two facts excludes the possibility that the chromosomal RNA is a complete copy of both strands of the chromosomal DNA. In spite of the difference in guanine + cytosine content between the two nucleic acids the RNA may still partly or completely be a single strand copy depending upon how representative the DNA values are for the synthetically active DNA.     

CHANGES IN THE BASE COMPOSITION OF NUCLEAR RIBONUCLEIC ACID OF NEURONS DURING A SHORT PERIOD OF ENHANCED PROTEIN PRODUCTION

Nuclei from isolated nerve cells were sampled by microdissection. The content and composition of the nuclear RNA was studied and compared with that of the cytoplasmic RNA of Deiters' nerve cells of rabbits. Analyses were made of control nerve cells and of cells in which an enhanced RNA and protein production had been induced by chemical means, tricyano-amino-propene, for 60 minutes. The nuclear RNA content of the control nerve cells was 56 µµg, i.e. 3 per cent of the total RNA content of the nerve cell. The base ratios were: adenine 21.3, guanine 26.6, cytosine 30.8, uracil 21.3. Purine-pyrimidine analyses showed that the nuclear RNA differed significantly from the cytoplasmic RNA in having higher adenine and uracil values. The guanine and cytosine values were high, however, and the ratio G/C was 0.86 as compared with 1.16 for the cytoplasmic RNA. The composition of the nuclear RNA was interpreted as reflecting the extraordinarily strong development of the nucleolus in these neurons. During the 60 minutes of enhanced neuronal RNA production (+25 per cent) the guanine value increased and the uracil value decreased significantly in the nuclear RNA. In the cytoplasmic RNA the guanine value also increased although not so much as the nuclear guanine. The cytoplasmic cytosine value decreased. The result indicated that the production of the characteristic cytoplasmic RNA had been influenced by the change in the nuclear RNA     

COMPOSITION OF RIBONUCLEIC ACID FROM VARIOUS PARTS OF SPIDER OOCYTES

Microphoretic purine-pyrimidine analyses of the ribonucleic acid (RNA) in nucleoli, nucleoplasm, cytoplasm, and yolk nuclei of spider oocytes have been carried out. The material necessary for the analyses was isolated by micromanipulation. Determinations of the amounts of RNA in the different parts of the cell were also performed. No differences between the composition of RNA in the nucleolus and the cytoplasm could be disclosed. Nucleoplasmic RNA was, on the other hand, distinctly different from that in the nucleolus and in the cytoplasm. The difference lies in the content of adenine, which is highest in nucleoplasmic RNA. The few analyses carried out on yolk nuclei showed their RNA to be variable in composition with a tendency to high purine values. The cytoplasm contains about 99 per cent of the total RNA in these cells, the nucleoplasm about 1 per cent, and the nucleolus not more than 0.3 per cent, although the highest concentrations are found in these latter structures. When considered in the light of other recent findings the results are compatible with the view that nucleolar RNA is the precursor of cytoplasmic RNA.     

Studies on the secondary structure of nuclear ribonucleic acids

Rat liver nuclei were separated into two fractions, the nucleolus and the nucleoplasm, by a combined salt-enzymic extraction. RNA was purified from both sources and analysed on sucrose density gradients. In both fractions a prominent heterogeneous RNA class with mean sedimentation coefficient of 18S was found. This material was analysed by measuring the rate of reaction with formaldehyde, the ultraviolet absorbance-temperature profile, the spectrophotometric observation of conformational changes as a function of pH, the spectrophotometric titration of uracil and guanine residues and the effect of both temperature and ionic strength on the spectrophotometric titration of cytosine residues. Nucleoplasmic 18S RNA fraction exhibited, on heating and also by adjustment of the pH to 2.5, a hyperchromicity of about 16%, close to that observed, in control experiments, for ribosomal RNA (22%). Titration of cytosine residues in solutions containing 1mm-NaCl and 0.1m-NaCl yielded pK values equal to 4.41 and 3.84 respectively. These results suggest that this RNA fraction is composed of structurally complex polymers. The hypochromicity of the nucleolar 18S RNA fraction determined by heating or adjusting the pH to 2.5, was not greater than 6% of the initial value. The rate of reaction with formaldehyde was 88% of that observed for the hydrolysed 18S fraction which suggested only 12% hydrogen bonding. pK values for uracil and guanine residues were 10.1 and 10.05 respectively. Titration of cytosine residues yielded a pK of 4.10, which was found to be independent of temperature and ionic strength variations.     

Cross section calculations for electron scattering from DNA and RNA bases

Differential and integral cross sections for elastic electron collisions with uracil, cytosine, guanine, adenine and thymine have been calculated using the independent atom method with a static-polarization model potential for incident energies ranging from 50 to 4000 eV. Total cross sections for single electron-impact ionization of selected DNA and RNA bases have also been calculated with the binary-encounter-Bethe model from the ionization threshold up to 5000 eV. Cross sections within the investigated energy range, can be related to the molecular symmetry, the number of target electrons and molecular size; elastic and ionization processes are most efficient for guanine and adenine molecules, while the lowest cross sections were obtained for the uracil molecule. The ionization cross sections for cytosine, thymine, adenine and guanine are compared with those recently obtained with a semi-classical and binary-encounter-Bethe formalisms. No theoretical and experimental data for elastic electron scattering from DNA and RNA bases are available, but comparisons with calculations for molecules of similar size and geometry allows the validity of the theoretical approach to be verified.     

Presence in the composition of bacterial DNA of covalently bound RNA fragments

The nucleotide composition of chromosome and plasmid DNA free of hybrid RNA isolated from resting Escherichia coli cells preliminary cultivated with the help of [14C] uracil has been studied. It has been established that DNA contains [14C]uracil side by side with adenine, thymine, guanine and cytosine. It confirms the presence of RNA fragments in the composition of bacterial DNA which are connected with it covalently.     

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.     

The RNA i-Motif in the Primordial RNA World

The primordial RNA world is a hypothetical era prior to the appearance of protein and DNA, when RNA molecules were the sole building blocks for early forms of life on Earth. A critical concern with the RNA-world hypothesis is the instability of the cytosine nucleobase compared to the other three bases (adenine, guanine, and uracil). The author proposes that cytosine residues could have stably existed in the primordial world in the RNA i-motif, a four-stranded quadruplex structure formed by base-pairing of protonated and unprotonated cytosine residues under acidic conditions. The i-motif structure not only increases the lifetime of cytosine residues by slowing their deamination rate, but could also allow RNA polymers to bind to certain ligands (e.g., anions) to perform critical functions. Future studies focused on determining the rate of cytosine deamination in RNA i-motifs over a range of pH, temperature, and pressure conditions, and on interrogating the interactions between ligands and RNA i-motifs, could uncover new evidence of the origin of life on Earth.

     

Regulation of uptake of purines, pyrimidines and amino acids by Candida utilis

Uptake of uracil by Candida utilis is increased by addition of leucine to a minimal medium in which organisms are growing. This response requires protein synthesis and has kinetics consistent with the induction of additional uracil transport by the amino acid or a derivative. Consequently, the contribution of exogenous radioactive uracil to the pyrimidine nucleotide pools increases so that RNA made after the amino acid is added is of greater specific radioactivity. Some other amino acids are as effective as leucine in increasing the incorporation of uracil into RNA. Growth with leucine present also increases to different extents the initial rates of uptake of adenine, cytosine, uridine, lysine, histidine, threonine, phenylalanine, aspartic acid and leucine itself. The action of leucine on lysine transport appears to involve induction. These effects are not restricted to leucine; growth with aspartic acid or phenylalanine in the medium gives similar results. Lysine, on the other hand, is without action on the uptake of leucine, aspartic acid, phenylalanine, threonine or uracil but decreases the initial rates of uptake of both histidine and lysine. We suggest that lysine represses its own transport. Similarly, there is a specific decrease in uracil uptake caused by growth with this pyrimidine. Thus in C. utilis there are complex interrelationships in the uptake of nitrogen-containing compounds.     

THE BASE COMPOSITION OF RIBONUCLEIC ACID IN LAMPBRUSH CHROMOSOMES, NUCLEOLI, NUCLEAR SAP, AND CYTOPLASM OF TRITURUS OOCYTES

The base composition of RNA's extracted from chromosomes, nucleoli, nuclear sap, and cytoplasm of Triturus oocytes has been determined by microelectrophoresis. The chromosomal RNA has a content of guanine+cytosine equal to that of DNA, but there is no complementarity in the composition as for DNA. Nuclear sap contains a highly variable RNA with a tendency towards high uracil values. Nucleolar and cytoplasmic RNA's are similar in composition and both are of the guanine-cytosine rich type. The chromosomes and nucleoli contain roughly equivalent amounts of RNA, somewhat less than is present in the nuclear sap. The RNA/DNA ratio of the whole chromosomes is about 10. However, the ratio in the synthetically active regions, the loops, is much higher, since the loops contain all the chromosomal RNA but only a small fraction of the DNA.     

Effect of uracil on colonial morphology ofNeisseria gonorrhoeae

The production of cells ofNeisseria gonorrhoeae that form type-T4 colonies in cultures started with cells that originally formed only type-T2 colonies was inhibited by calf thymus RNA. Guanosine and uracil were the only nucleic acid constituents that significantly reduced the T2 to T4 shift. Uracil gave the best results in degree of inhibition. It was found that some tritiated uracil was incorporated into the RNA of growing cells ofN. gonorrhoeae but that much more was incorporated into DNA probably after conversion to guanine and adenine. The data show that the shift from T2 to T4 can be progressively inhibited by increasing the concentration of uracil in the growth medium.     

The effect of a new antibiotic, cryptosporiopsin, on RNA synthesis in L-cells.

The effect of cryptosporiopsin on RNA synthesis in L-cells was studied as part of an investigation on the mechanism of action and potential toxicity of the antibiotic in mammalian cells. RNA synthesis in vitro was tested in intact isolated L-cell nuclei, in conjunction with selective inhibitors of nucleolar and nucleoplasmic RNA synthetic activities; It was found that only the nucleoplasmic activity (polymerase II), was inhibited by cryptosporiopsin and that the drug showed no effect on the activity of the nucleolar enzyme (polymerase I). RNA synthesis in vivo was tested using double labelling with I114-C]guanine and [3-H]-uridine in an attempt at discriminating between G+C nucleolar trna and high A+U nucleoplasmic RNA synthesis. Results revealed that the uptake of these precursors into both types of RNA was inhibited by cryptosporiopsin in intact cells. Measurements of the nucleotide pools in these cells indicated that the antibiotic affects uptak and phosphorylation of nucleosides and nucleotides, especially the production of ATP; These results suggest that the uptake inhibition observed in vivo could be due, at least in part, to energy and/or precursor shortage.     

A universal code for RNA recognition by PUF proteins

The design of proteins that can bind any RNA sequence of interest has many potential biological and medical applications. Here we have expanded the recognition of Pumilio and FBF homology protein (PUF) repeats beyond adenine, guanine and uracil and evolved them to specifically bind cytosine. These repeat sequences can be used to create PUF domains capable of selectively binding RNA targets of diverse sequence and structure.     

Transport of nucleic acid bases against their concentration gradients through quaternized chitosan membrane

A water-insoluble anion exchange membrane was prepared by crosslinking with ethyleneglycol diglycidylether, a membrane made of quaternized chitosan and poly(vinyl alcohol). The transports of nucleic acid bases such as uracil, cytosine, adenine, and guanine were investigated as one side of the membrane in a diaphragm cell was acidic and the other basic. Uracil was transported against its concentration gradient from the basic side to the acidic side regardless of the pH on the basic side. Cytosine, adenine, and guanine were also transported against their concentration gradients, but the direction of their transport depended upon the pH on the basic side. In particular, the transport directions for adenine and guanine were switched during identical transport experiments. Mechanisms for the transport of these nucleic acid bases against their concentration gradients through the quaternized chitosan membrane are discussed.     

The Soluble Nucleotide Content of Germinating Wheat Embryos

Estimates are given of the amounts of adenine, uracil, guanine,and cytosine present in the soluble nucleotides of wheat embryosduring germination in the dark. After 48 hours at 25° Cthe nucleotide content per gramme of dry tissue reaches a maximumlevel when the content of bases is approximately 3.5 µmolesof uracil, 2.8 µmoles of adenine, 0.6 µmoles ofguanine, and 0.5 µmoles of cytosine. The soluble nucleotidecontent of embryos growing at 0 to 5° C is lower than thatof embryos of the same dry weight grown at 25° C. Initiallythe amount of adenine present is greater than the amount ofuracil but after 20 hours of germination at 25° C uracilbecomes the predominant base in the soluble nucleotide fraction.Ion-exchange resin chromatography was used to separate the chiefsoluble nucleotides present in the extract of embryos grownfor four days at 25° C. Uridine diphosphate glucose accountsfor the major part of the uracil and adenosine monophosphatefor most of the adenine. Nicotinamide-adenine dinucleotide wasdetected and identified and also a little uridine 5'-mono-phosphate.The possibility is discussed that most of the adenosine monophosphateis produced by degradation of reduced nicotinamide-adenine dinucleotidephosphate.     

Polymethylene derivatives of nucleic bases with ω-functional groups: VII. Cytotoxicity in the series of <Emphasis Type="Italic">N</Emphasis>-(2-oxocyclohexyl)-ω-oxoalkyl-substituted purines and pyrimidines

New polymethylene derivatives of the nucleic bases with β-diketo function in the ω-position have been synthesized by alkylation of uracil, thymine, cytosine, hypoxanthine, adenine, and N ²-isobutyryl guanine with 2-(ω-chloroalkanoyl)cyclohexanones. The physicochemical characteristics of compounds synthesized and their effect on tumor K562 and HCT116 cell lines have been studied.     

Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with uracil and uridine

Ribosome inactivating protein (RIP) catalyzes the cleavage of glycosidic bond formed between adenine and ribose sugar of ribosomal RNA to inactivate ribosomes. Previous structural studies have shown that RNA bases, adenine, guanine, and cytosine tend to bind to RIP in the substrate binding site. However, the mode of binding of uracil with RIP was not yet known. Here, we report crystal structures of two complexes of type 1 RIP from Momordica balsamina (MbRIP1) with base, uracil and nucleoside, uridine. The binding studies of MbRIP1 with uracil and uridine as estimated using fluorescence spectroscopy showed that the equilibrium dissociation constants (K_D) were 1.2 × 10⁻⁶ M and 1.4 × 10⁻⁷ M respectively. The corresponding values obtained using surface plasmon resonance (SPR) were found to be 1.4 × 10⁻⁶ M and 1.1 × 10⁻⁷ M, respectively. Structures of the complexes of MbRIP1 with uracil (Structure-1) and uridine (Structure-2) were determined at 1.70 and 1.98 Å resolutions respectively. Structure-1 showed that uracil bound to MbRIP1 at the substrate binding site but its mode of binding was significantly different from those of adenine, guanine and cytosine. However, the mode of binding of uridine was found to be similar to those of cytidine. As a result of binding of uracil to MbRIP1 at the substrate binding site, three water molecules were expelled while eight water molecules were expelled when uridine bound to MbRIP1.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)