New synthetic route to ethynyl-dUTP: A means to avoid formation of acetyl and chloro vinyl base-modified triphosphates that could poison SELEX experiments

5-Ethynyl-2′-deoxyuridine is a common base-modified nucleoside analogue that has served in various applications including selection experiments for potent aptamers and in biosensing. The synthesis of the corresponding triphosphates involves a mild acidic deprotection step. Herein, we show that this deprotection leads to the formation of other nucleoside analogs which are easily converted to triphosphates. The modified nucleoside triphosphates are excellent substrates for numerous DNA polymerases under both primer extension and PCR conditions and could thus poison selection experiments by blocking sites that need to be further modified. The formation of these nucleoside analogs can be circumvented by application of a new synthetic route that is described herein.     

Incorporation of single nucleoside triphosphates by isolated cell nuclei

Manganese ions can form insoluble complexes or coprecipitates with nucleoside triphosphates at high pH. We have demonstrated that nucleoside triphosphates found in precipitated complexes exhibit properties similar to those of poliribo-nucleotides in all steps of the Schmidt-Tannhausers procedure: insoluble in perchloric acid, trichloroacetic acid, and ethanol and soluble in 0.2 n NaOH. These observations could be applied to experimentation on RNA synthesis from nucleoside triphosphates.     

Brain nucleoside recycling

The rate limiting reactions of nucleotide synthesis are modulated by intracellular fluctuations of nucleoside triphosphate concentrations. This topic has been mostly studied at the level of the de novo nucleotide synthesis from simple precursors. However, there are districts, such as brain, which rely more heavily on the salvage of preformed purine and pyrimidine rings, mainly in the form of nucleosides. This raises the following question: how do these districts maintain the right balance between the purine and pyrimidine pools? We believe that it is now safe to state that a cross talk exists between the extra- and intracellular metabolism of purine and pyrimidine nucleosides in the brain. The extracellular space is the major site of nucleoside generation through successive dephosphorylations of released triphosphates, whereas brain cytosol is the major site of multiple phosphorylations of uptaken nucleosides at their 5′-position. Modulation of both extracellular nucleoside generation by membrane bound ectonucleotidases, and intracellular nucleoside phosphorylation by cytosolic kinases might contribute to maintain the right extra- and intracellular purine and pyrimidine nucleotide balance in the brain.     

Gene ytkD of Bacillus subtilis encodes an atypical nucleoside triphosphatase member of the Nudix hydrolase superfamily

Gene ytkD of Bacillus subtilis, a member of the Nudix hydrolase superfamily, has been cloned and expressed in Escherichia coli. The purified protein has been characterized as a nucleoside triphosphatase active on all of the canonical ribo- and deoxyribonucleoside triphosphates. Whereas all other nucleoside triphosphatase members of the superfamily release inorganic pyrophosphate and the cognate nucleoside monophosphate, YtkD hydrolyses nucleoside triphosphates in a stepwise fashion through the diphosphate to the monophosphate, releasing two molecules of inorganic orthophosphate. Contrary to a previous report, our enzymological and genetic studies indicate that ytkD is not an orthologue of E. coli mutT.     

Different responses to some stimulators of prolyl hydroxylase activities in various rat organs.

Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) of soluble fraction (105 000 X g supernatant) of rat granulation tissues was markedly enhanced by addition of nucleoside triphosphates to the assay medium. But the stimulatory activities of nucleoside triphosphates were very different in fractions derived from tissues of rat. In skin, lung or whole fetal tissues other than granuloma, GTP enhanced the enzymatic activity by 3-4 fold. On the other hand, in kidney, liver and spleen tissues it brought about no enhancement. The same results were obtained even if ATP regenerating system was added in the assay medium. The stimulatory effect of nucleoside triphosphates was not seen with the soluble fraction of liver, but it appeared with the enzyme fraction purified by affinity column chromatography. The same phenomenon was observed by addition of bovine serum albumin instead of nucleoside triphosphates as stimulator. We discuss the possible reasons as to why the responses of the enzyme to stimulators were quite different among various tissues.     

In vitro packaging of bacteriophage T7 DNA requires ATP.

Removal of nucleoside triphosphates from extracts prepared from bacteriophage T7-infected Escherichia coli results in a stringent requirement for added ATP to form infective phage particles by in vitro packaging of bacteriophage T7 DNA. Optimal packaging efficiency was achieved at a concentration of about 1.25 mM. Other nucleoside triphosphates could be substituted for ATP, but none of the common nucleoside triphosphates was as effective as ATP in promoting in vitro encapsulation.     

Three new Nudix hydrolases from Escherichia coli

Three members of the Nudix (nucleoside diphosphate X) hydrolase superfamily have been cloned from Escherichia coli MG1655 and expressed. The proteins have been purified and identified as enzymes active on nucleoside diphosphate derivatives with the following specificities. Orf141 (yfaO) is a nucleoside triphosphatase preferring pyrimidine deoxynucleoside triphosphates. Orf153 (ymfB) is a nonspecific nucleoside tri- and diphosphatase and atypically releases inorganic orthophosphate from triphosphates instead of pyrophosphate. Orf191 (yffH) is a highly active GDP-mannose pyrophosphatase. All three enzymes require a divalent cation for activity and are optimally active at alkaline pH, characteristic of the Nudix hydrolase superfamily. The question of whether or not Orf1.9 (wcaH) is a bona fide member of the Nudix hydrolase superfamily is discussed.     

Regulation of RNA synthesis in higher plant cells by the action of a nucleoside triphosphatase

The influence of nucleoside triphosphates in relation to divalent cations on RNA synthesis of cells from a suspension culture from parsley was investigated. The data obtained from experiments with isolated nuclei and with an in vitro system with highly purified RNA polymerase I were compared with a chromatin-bound nucleoside triphosphatase activity within the nucleus. The results might suggest a regulatory role of the nucleoside triphosphatase activity in RNA synthesis.Abbreviations NTP nucleoside triphosphates - NTPase nucleoside triphosphatase     

Cyanine-dye-modified 2′-deoxyuridine-5′-triphosphates: Synthesis,applications, and linker effect on substrate properties for Taq DNA polymerase

A number of fluorescently labeled nucleoside triphosphates containing electroneutral indodicarbocyanine dye (Cy) have been synthesized. The dye has been attached to the C5 position of pyrimidine through the transalkene linkers of different structure. The synthesized labeled nucleoside triphosphates have been tested as substrates for Taq polymerase in PCR using the “TB-biochip” test system.     

Presence of Nucleoside Triphosphate Phosphohydrolase Activity in Purified Virions of Reovirus

A nucleoside triphosphate phosphohydrolase activity has been discovered in reovirus virions. This activity converts nucleoside triphosphates to nucleoside diphosphates in vitro. Properties of this enzyme are presented, with evidence that this activity is an integral part of the virion core.     

Effect of Nucleotides and Other Inhibitors on the Inactivation of Glutamate Decarboxylase

Abstract: The effects of 17 nucleotides and nucleotide analogs and 11 other compounds on the glutamate-promoted inactivation of brain glutamate decarboxylase were examined. Among the nucleotides, the major determinant of potency was the polyphosphate chain, Glutamate-promoted inactivation was strongly enhanced by low concentrations (<100 μM) of adenosine tetraphosphate and all eight nucleoside triphosphates tested. Nucleoside diphosphates enhanced inactivation, but were much less effective than the nucleoside triphosphates; nucleoside monophosphates were not effective. Modification of the polyphosphate chain of the nucleoside triphosphates also affected potency; adenylylimidodiphosphate and α,β-methylene ATP were about as effective as nucleoside diphosphates, but α,β-methylene ATP was nearly as effective as ATP. The nucleoside base had only a small effect on potency; purine nucleotides were more potent than pyrimidine nucleotides, and one nucleotide with a tricyclic base, 1, N⁶-etheno ATP, was as effective as the purine nucleoside triphosphates. The 2'-hydroxyl group of ribose was unimportant, since deoxy ATP was as effective as ATP. Three nonnucleotide polyanions were strong promoters of inactivation; inositol hexasulfate and 5-phosphorylribose 1-pyrophosphate were at least as effective as ATP; inositol hexaphosphate (phytate) was as effective as the nucleoside diphosphates. These results suggest that a major determinant of potency was a strong negative charge on the molecule. Negative charge was not sufficient, however, since fructose 1,6-bisphosphate did not promote inactivation. Inactivation by all of these compounds was slow, requiring more than 20 min for full effect. Two competitive inhibitors, chloride and glutarate, acted immediately and also reduced rather than enhanced glutamate-promoted inactivation.     

Effect of nucleotides on potential and pH changes across the thylakoid membrane of spinach chloroplasts.

With appropriate preparations of spinish chloroplasts we observe three distinct effects of the nucleotides: 1. An accelaration of the dark decay of the light induced 520 nm absorbance change after ATP addition. 2. An acidification of the internal space of the thylakoids after ATP addition in darkness. 3. A dark ATPase activity which is regulated by the deltapH across the membrane. We conclude that the effect of the nucleoside triphosphates on the 520 nm signal is linked to a change of the proton conductivity of the membrane, induced by the formation of a deltapH across the membrane in consequence of the dark ATPase activity. The mode of action of the nucleoside diphosphates in the presence of inorganic phosphate on the 520 nm signal is discussed. It is proposed that the effects observed are linked to the hydrolysis of the newly formed nucleoside triphosphates.     

Possible Interference by an Acid-stable Enzyme during the Extraction of Nucleoside Di- and Triphosphates from Higher Plant Tissues

Acid extracts from tissues of two solanaceous plants were found to contain a heat-labile, nondialyzable factor which hydrolyzes nucleoside di- and triphosphates to nucleoside monophosphates. This acid-resistant factor shows optimal ATP-hydrolyzing activity at pH 5, whereas practically no activity was detected below pH 3 and above pH 9. It does not hydrolyze sugar phosphates, nucleoside monophosphates, uridine diphosphoglucose, and phosphoenolpyruvate. In order to estimate quantitatively the amount of nucleoside di- and triphosphates in a plant extract, care must be taken to circumvent possible interference by this factor. This is achieved by carefully maintaining the extract below pH 3.     

A 31P-NMR study of the interaction of Mg2+ ions with nucleoside diphosphates.

The interaction of Mg2+ with nucleoside disphosphates : ADP, GDP, CDP and UDP has been studied by phosphorus magnetic resonance spectroscopy in aqueous solution. The results show that these four nucleotides behave similarly, the Mg2+ ion binds to the alpha but not to the beta phosphate moiety. The strength of the interaction of Mg2+ ions with nucleoside diphosphates is weaker than with nucleoside triphosphates. The association of Mg2+ on the phosphate chain is stronger in a neutral than in an acid medium.     

The substrate specificity of dynein from Tetrahymena cilia

The substrate specificity of the 22S dynein ATPase from Tetrahymena cilia was investigated. The 22S dynein exhibited a high specificity for ATP in terms of both apparent Km and Vmax: naturally occurring nucleoside triphosphates other than ATP were hydrolyzed slowly with an apparent Km of 0.25-1 mM, a sharp contrast to that of ATP hydrolysis (1-4 microM). Pyrophosphate was a poor inhibitor for the dynein ATPase, indicating weak affinity. Since dynein binds ATP tightly and hydrolyzes it at a high rate, a method to determine a trace amount of ATP in the presence of other nucleoside triphosphates has been developed by taking advantage of this enzymatic characteristic of dynein. The effect of P1,P5-di(adenosine-5'-)-pentaphosphate (Ap5A) on the 22S dynein ATPase was also investigated. Ap5A acted as a weak competitive inhibitor of the ciliary 22S dynein ATPase and the nonlinearity of the double-reciprocal plot of the ATPase was confirmed in the presence of Ap5A.     

Accumulation of pyrophosphate correlates with the increased level of nucleoside triphosphates in Escherichia coli

We measured the concentration of nucleoside triphosphates and inorganic pyrophosphate in Escherichia coli in conditions where nucleotide synthesis or nucleic acid synthesis was inhibited. The inhibitors that brought about an accumulation of some of the four ribonucleoside triphosphates also increased the pyrophosphate level. In a pyrimidine auxotrophic strain uracil starvation led to simultaneous accumulation of ATP and pyrophosphate, and they both rapidly returned to normal level when starvation was relieved. These results indicate the possible involvement of pyrophosphate in the reactions leading to the accumulation of nucleoside triphosphates.     

Enzyme activities associated with an invertebrate iridovirus: nucleotide phosphohydrolase activity associated with iridescent virus type 6 (CIV).

A nucleoside triphosphate phosphohydrolase activity is firmly associated with a purified invertebrate iridovirus, iridescent virus type 6. The enzyme activity hydrolyzes all the nucleoside triphosphates, but has a high preference for ATP. The products of the reaction are nucleoside diphosphates. Conditions for nucleoside triphosphate phosphohydrolase activity are described.     

A procedure for the isolation and determination of deoxyribose derivatives

A method of determining deoxyribose derivatives in biological material is described. It has high sensitivity, and is particularly useful in that it can be applied to a large range of tissues for which the other available assays are unsuitable. This is because the method is applicable to complex mixtures of nucleotides in which such substances as ribonucleotides are present in very large excess over deoxyribonucleotides, and it is not necessary to equilibrate the nucleotide-precursor pool with radioactive phosphate. The method has mainly been developed with the object of determining deoxyribonucleoside triphosphates, but it can be used to assay ribonucleoside triphosphates, as well as the mono- and diphosphates of both types of nucleoside. The procedure used involves three basic techniques: (1) periodate oxidation and methylamine-induced cleavage of the sugar ring to destroy 2'- and 3'-unsubstituted ribonucleosides; (2) column chromatography to separate the deoxyribonucleotides from each other and from other substances, such as the products of the periodate oxidation; (3) fluorimetric determination of deoxyribose after labilization of the pyrimidine-glycosidic bond by bromination of the heterocyclic ring. Each of these three procedures can be used independently, in conjunction with other analytical procedures.