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PubMed1994

Cloning of the Nilaparvata lugens reovirus genome: conserved terminal nucleotide sequences and nucleotide sequence of genome segment S10.

The segmented double-stranded RNA genome of Nilaparvata lugens reovirus (NLRV) was cloned, and the nucleotide sequence of genome segment S10 and terminal nucleotide sequences of the rest of the segments were determined. Genome segment S10 of NLRV consisted of 1430 nucleotides with a single open reading frame extending for 1293 nucleotides from nucleotide 46. It encoded a polypeptide of 431 amino acids with an M(r) of 49.4K, which was a non-structural protein. The plus-strand RNA of all genome segments had the same conserved trinucleotide 5' AGU- and hexanucleotide-GUUGUC 3' in the 5'- and 3'-terminal regions, respectively. These conserved terminal sequences resembled those found in the segments of the members of the genus Fijivirus.

Amino Acid Sequence↗
PubMed1976

8-Alkylaminoadenyl nucleotides as probes of dehydrogenase interactions with nucleotide analogs of different glycosyl conformation.

The nucleotides 8-amino-, 8-methylamino-, and 8-dimethylaminoadenylic acid have been synthesized and their preferred conformations about the glycosyl bond in qaueous solution have been determined by 1H nuclear magnetic resonance spectroscopy. Paramagnetic relaxation studies, nuclear Overhauser enhancement measurements, chemical shifts, and coupling constant comparisons indicate that their is rotation about the glycosyl bond and that preference for either the anti or syn conformation depends on the extent of alkyl substitution on the 8-amino group. The primary and secondary amines 8-amino- and 8-methylaminoadenylic acid adopt a perferential anti conformation about the glycosyl bond, while the tertiary amine 8-dimethylaminoadenylic acid exists predominantly in the syn form. These three analogs provide a system to study interactions of a dehydrogenase with coenzyme inhibitors having different glycosyl conformer populations. All three analogs are competitive inhibitors of NADH in reaction with chicken muscle lactate dehydrogenase, and the Ki values show little dependence on the nature of the amino substitution. This demonstrates that the distribution of conformations about the nucleotide glycosyl bond does not effect the competition of the nucleotide for lactate dehydrogenase apoenzyme. Several models for enzyme-coenzyme binding are discussed. The available data cannot distinguish whether the enzyme binds nucleotide in both the anti and syn conformations or in purely the anti conformation. However, at some stage of the enzyme-coenzyme interaction, there appears to be a strong stabilization of the nucleotide in the anti conformation about the glycosyl bond.

Animals↗
PubMed1982

Divalent cation-nucleotide complex at the exchangeable nucleotide binding site of tubulin.

Tubulin was first treated with alkaline phosphatase-agarose to vacate the exchangeable nucleotide binding site and then tested for manganese binding sites by Mn(II) EPR. Buttlaire et al. ((1980) J. Biol. Chem. 255, 2164-2168) have shown that high affinity manganese binding occurs at a single site normally occupied by magnesium. We report that the number of high affinity manganese binding sites per mol of tubulin depends on the number of occupied exchangeable nucleotide binding sites. Thus, removal of nucleotides results in a loss of high affinity manganese binding sites. The EPR spectra of manganese bound to tubulin and to GTP are found to be qualitatively similar. These data indicate that high affinity manganese binding is the result of the formation of a metal-nucleotide complex at the exchangeable nucleotide binding site. In addition it was found that zinc, cobalt, and magnesium bind with approximately equal affinity to this site whereas calcium binds only weakly.

Alkaline Phosphatase↗
PubMed1982

Different classes of nucleotide binding sites in the (Na+ + K+)-ATPase studied by affinity labeling and nucleotide-dependent SH-group modifications.

The ATP analog 6-[(3-carboxy-4-nitrophenyl)thiol]-9-beta-D-ribofuranosylpurine 5'-triphosphate (Nbs6ITP) is slowly hydrolyzed at pH 7.4 by the (Na+ + K+)-ATPase, whereas it binds covalently at pH 8.5 and inhibits the enzyme irreversibly. Time courses of irreversible inhibition could only be fitted to a model in which the enzyme can exist in two slowly interchangeable states, one of which is enzymatically active and binds Nbs6ITP first reversibly and then covalently. Arguments that the covalent binding occurs at a low affinity nucleotide binding site are: (a) similarity of the Ki Nbs6ITP for the reversible and the irreversible inhibition and of K0.5 for ATP protection; (b) stoichiometry of covalent Nbs6ITP binding per alpha subunit of 0.8; and (c) change of complex substrate dependence of the enzyme to a Michaelis-Menten type after Nbs6ITP modification. This change in kinetics and the finding that the Nbs6ITP inactivation at a low affinity nucleotide binding site is increased by micromolar ADP concentrations indicates that the (Na+ + K+)-ATPase contains two different nucleotide binding sites. Since studies of nucleotide effects on enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) did not confirm the hypothesis of an SH-group in a nucleotide binding site, Nbs6ITP may bind to another functional group, e.g. to an OH-group of tyrosine.

Adenosine Triphosphate↗
PubMed1983

A second guanyl nucleotide-binding site associated with adenylate cyclase. Distinct nucleotides activate adenylate cyclase and permit ADP-ribosylation by cholera toxin.

There are two functionally and physically distinct types of guanyl nucleotide site associated with the adenylate cyclase system of pigeon erythrocytes. One is on the well known regulatory protein, N, that mediates the adenylate cyclase response to hormones, guanyl nucleotides and fluoride, and is the substrate for ADP-ribosylation by cholera toxin. We now describe a second site that must be occupied by GTP or an analog of GTP before N can be ADP-ribosylated. We call this second site S. It differs from the site on N in many respects. GTP appears to be rapidly hydrolyzed when it is bound to N but not when bound at S. GTP analogs such as guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) bind stably to both sites but the binding of GTP gamma S to N is more sensitive to EDTA and is more easily prevented by guanosine 5'-O-(2-thiodiphosphate). The nucleotide binding only to S is promoted by the cytosolic protein required by cholera toxin. Isoproterenol decreases GTP gamma S binding to S while indirectly increasing GTP gamma S binding to N. By adjusting the binding conditions, the nucleotides bound functionally to N and S can be varied independently and then the effect of ADP-ribosylation upon the adenylate cyclase activity can be seen to depend on the type of nucleotide bound to N. This activity rises, falls slightly, or remains at zero, if N is occupied by GTP, GTP gamma S, or guanosine 5'-O-(2-thiodiphosphate, respectively.

Adenosine Diphosphate Ribose↗
PubMed1993

Nucleotide binding to actin. Cation dependence of nucleotide dissociation and exchange rates.

We have reinvestigated nucleotide binding to actin in order to resolve conflicts regarding the mechanism of nucleotide dissociation and exchange. We present evidence that supports a mechanism for nucleotide binding to actin in which the tightly bound divalent cation (Ca2+ or Mg2+) directly interacts with the bound nucleotide. The dissociation rates of ATP or ADP from actin are limited by the dissociation of the high affinity divalent cation from actin and vary inversely with free Ca2+ or free Mg2+ concentration. The divalent cation concentration range over which attenuation of the ATP dissociation takes place is about 100-fold greater for Mg2+ than that for Ca2+ due to the much slower association rate constant for Mg2+ compared with Ca2+. The relative affinity for ATP versus ADP is 200:1 for Ca-actin in 100 microM free [Ca2+], and 4:1 for Mg-actin in 100 microM free [Mg2+]. Actin without a tightly bound divalent cation has about a 3-fold greater affinity for ATP than ADP. At constant free divalent cation concentration, the rate of nucleotide exchange on actin is described by competitive binding kinetics.

Actins↗
PubMed1996

Localization of modified nucleotides in Schizosaccharomyces pombe spliceosomal small nuclear RNAs: modified nucleotides are clustered in functionally important regions.

The specific and dynamic RNA:RNA interactions between pre-mRNA and small nuclear RNAs (snRNAs), especially U2, U5, and U6 snRNAs, form the catalytic core and are at the heart of the spliceosome formation. The functionally important regions in the snRNAs correspond to the highly modified regions in snRNAs from human, rat, and plant cells. To better understand the importance of the modifications of snRNAs, we identified and localized the modified nucleotides in the five spliceosomal snRNAs of Schizosaccharomyces pombe cells. Twenty-two modified nucleotides, including base methylations, 2'-O-methylations, and pseudouridines, were found in the five spliceosomal snRNAs. The conservation of modified nucleotides between human and S. pombe snRNAs is striking. In addition, most of the modified nucleotides are in or around positions that form hydrogen bonds with the pre-mRNA or with other snRNAs. The results are consistent with the suggestion that modified nucleotides are clustered around functionally important regions of the spliceosomal snRNAs. These data provide the basis for further functional studies on posttranscriptional modifications in spliceosomal snRNAs.

Base Sequence↗
PubMed1991

Interaction of La (III) and Tb (III) ions with purine nucleotides: evidence for metal chelation (N-7-M-PO3) and the effect of macrochelate formation on the nucleotide sugar conformation.

The interaction of the La (III) and Tb (III) ions with adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP), and 2'-deoxyguanosine-5'-monophosphate (5'-dGMP) anions with metal/nucleotide ratios of 1 and 2 has been studied in aqueous solution in acidic and neutral pHs. The solid complexes were isolated and characterized by Fourier transform ir and 1H-nmr spectroscopy. The lanthanide (III)-nucleotide complexes are polymeric in nature both in the solid and aqueous solutions. In the metal-nucleotide complexes isolated from acidic solution, the nucleotide binding is via the phosphate group (inner sphere) and an indirect metal-N-7 interaction (outer-sphere) with the adenine N-1 site protonated. In the complexes obtained from neutral solution, metal chelation through the N-7 and the PO3(2-) group is prevailing. In aqueous solution, an equilibrium between the inner and outer sphere metal-nucleotide interaction has been observed. The ribose moiety shows C2'-endo/anti pucker in the free AMP anion and in the lanthanide (III)-AMP complexes, whereas the GMP anion with C2'-endo/anti sugar conformation exhibits a mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers in the lanthanide (III)-GMP salts. The deoxyribose has O4'-endo/anti sugar pucker in the free dGMP anion and a C3'-endo/anti, in the lanthanide (III)-dGMP complexes.

Adenosine Monophosphate↗
PubMed1995

The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase.

The fluorescent properties and their sensitivity to the surrounding environment of the nucleotide analog 2-aminopurine (2-AP) have been well documented. In this paper we describe the use of 2-AP as a direct spectroscopic probe of the mechanism of nucleotide incorporation by Escherichia coli Pol I Klenow fragment (KF) and bacteriophage T4 DNA polymerase. The nucleotidyl transfer reaction may be monitored in real time by following the fluorescence of 2-AP, allowing the detection of transient intermediates along the reaction pathway that are inaccessible through traditional radioactive assays. Previous studies with Klenow fragment [Kuchta, R. D., Mizrahi, V., Benkovic, P. A., Johnson, K. A., & Benkovic, S. J. (1987) Biochemistry 26, 8410-8417] have revealed the presence of a nonchemical step prior to chemistry and have identified this conformational change as the rate-limiting step of correct nucleotide incorporation. During correct incorporation, phosphodiester bond formation occurs at a rate greater than the conformational change and has not been measured. However, during misinsertion, the rate of the chemical step becomes partially rate limiting and it becomes possible to detect both steps. We have successfully decoupled the chemical and conformational change steps for nucleotide insertion by KF using the misincorporation reaction, and we present direct spectroscopic evidence for an activated KF'-DNA-dNTP species following the conformational change step which features hydrogen bonding between the incoming and template bases. In addition, we have utilized these same experiments to demonstrate the existence of a similar nonchemical step in the mechanism of dNTP incorporation by bacteriophage T4 DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Aminopurine↗
PubMed2001

Rapid characterization of DNA oligomers and genotyping of single nucleotide polymorphism using nucleotide-specific mass tags.

Using currently available MS-based methods, accurate mass measurements are essential for the characterization of DNA oligomers. However, there is a lack of specificity in mass peaks when the characterization of individual DNA species in a mass spectrum is dependent solely upon the mass-to-charge ratio (m/z). Here, we utilize nucleotide-specific tagging with stable isotopes to provide internal signatures that quantitatively display the nucleotide content of oligomer peaks in MS spectra. The characteristic mass-split patterns induced by the partially (13)C/(15)N-enriched dNTPs in DNA oligomers indicate the number of labeled precursors and in turn the base substitution in each mass peak, and provide for efficient SNP detection. Signals in mass spectra not only reflect the masses of particular DNA oligomers, but also their specific composition of particular nucleotides. The measurements of mass tags are relative in the mass-split pattern and, hence, the accuracy of the determination of nucleotide substitution is indirectly increased. For high sample throughput, (13)C/(15)N-labeled sequences of interest have been generated, excised in solution and purified for MS analysis in a single-tube format. This method can substantially improve the specificity, accuracy and efficiency of mass spectrometry in the characterization of DNA oligomers and genetic variations.

Base Composition↗
PubMed2006

Tapping the nucleotide pool of the host: novel nucleotide carrier proteins of Protochlamydia amoebophila.

Protochlamydia amoebophila UWE25 is related to the Chlamydiaceae comprising major pathogens of humans, but thrives as obligate intracellular symbiont in the protozoan host Acanthamoeba sp. The genome of P. amoebophila encodes five paralogous carrier proteins belonging to the nucleotide transporter (NTT) family. Here we report on three P. amoebophila NTT isoforms, PamNTT2, PamNTT3 and PamNTT5, which possess several conserved amino acid residues known to be critical for nucleotide transport. We demonstrated that these carrier proteins are able to transport nucleotides, although substrate specificities and mode of transport differ in an unexpected manner and are unique among known NTTs. PamNTT2 is a counter exchange transporter exhibiting submillimolar apparent affinities for all four RNA nucleotides, PamNTT3 catalyses an unidirectional proton-coupled transport confined to UTP, whereas PamNTT5 mediates a proton-energized GTP and ATP import. All NTT genes of P. amoebophila are transcribed during intracellular multiplication in acanthamoebae. The biochemical characterization of all five NTT proteins from P. amoebophila in this and previous studies uncovered that these metabolically impaired bacteria are intimately connected with their host cell's metabolism in a surprisingly complex manner.

Adenosine Triphosphate↗
PubMed1973

Nucleotide sequence analysis with polynucleotide kinase and nucleotide "mapping" methods. 5'-Terminal sequences of deoxyribonucleic acid from bacteriophages lambda and 424.

The polynucleotide kinase reaction was used in analyses of complex mixtures of oligodeoxynucleotides which were fractionated by various two-dimensional nucleotide ;mapping' procedures. Parallel ionophoretic analyses on DEAE-cellulose paper, pH2, and AE-cellulose paper, pH3.5, of venom phosphodiesterase partial digests of 5'-terminally labelled oligonucleotides enabled the sequence of the nucleotides to be deduced uniquely. A ;diagonal ionophoresis' method has been used with mixtures of nucleotides. Application of these methods to 5'-terminally labelled DNA from bacteriophage lambda gave the terminal sequences pA-G-G-T-C-G and pG-G-G-C-G. Identical 5'-terminal sequences were found with DNA from bacteriophage 424.

Adenine Nucleotides↗
PubMed2007

The first pure LambdaHT rotamer of a complex with a cis-[metal(nucleotide)2] unit: a cis-[Pt(amine)2(nucleotide)2] LambdaHT rotamer with unique molecular structural features.

cis-[PtA2(nucleotide)2] complexes (A2 stands for two amines or a diamine) have been extensively investigated as model compounds for key cisplatin-DNA adducts. All cis-[metal(nucleotide/nucleoside)2] complexes with guanine and related purines characterized in the solid state thus far have the DeltaHT conformation (head-to-tail orientation of the two bases and right-handed chirality). In sharp contrast, the LambdaHT conformation (left-handed chirality) dominates in acidic and neutral aqueous solutions of cis-[PtA2(5'-GMP)2] complexes. Molecular models and solution experiments indicate that the LambdaHT conformer is stabilized by 5'-phosphate/N1H hydrogen-bond interactions between cis nucleotides with the normal anti conformation. However, this evidence, while compelling, is indirect. At last, conditions have been defined to allow crystallization of this elusive conformer. The structure obtained reveals three unique features not present in all other cis-[PtA2(nucleotide)2] solid-state structures: a LambdaHT conformation, very strong hydrogen-bond interactions between the phosphate and N1H of cis nucleotides, and a very small dihedral angle between the planes of the two guanines lying nearly perpendicular to the coordination plane. These new results indicate that, because there are no local base-base repulsions precluding the LambdaHT conformer, global forces rather than local interactions account for the predominance of the DeltaHT conformer over the LambdaHT conformer in the solid state and in both inter- and intrastrand HT crosslinks of oligonucleotides and DNA.

Cisplatin↗
PubMed1983

A method for measuring free-nucleotide pool sizes and incorporation of labeled precursors: its application in studying myocardial pyrimidine nucleotides.

A procedure for a rapid and accurate determination of nucleotide pool sizes in heart muscle is described. The method involves an enzymatic cleavage of all nucleotides by phosphodiesterase to nucleoside 5'-monophosphates and an HPLC separation (Partisil 10 SAX) by isocratic or two-step elution. This method permits reproducible measurements of the pools of pyrimidine nucleotides which are particularly small in cardiac tissue. Moreover, this technique may be conveniently applied in studies on the incorporation of labeled precursors into free nucleotides. Experimental evidence is presented showing the accuracy of the method.

Adenosine Monophosphate↗
PubMed1991

Interaction of adenine nucleotides with the adenine nucleotide translocase regulates the developmental changes in proton conductance of the inner mitochondrial membrane.

2-h-old neonatal liver mitochondria, when depleted of adenine nucleotides, showed an 'ohmic' current-voltage relationship and a higher passive proton permeability of the membrane, resembling fetal mitochondrial behaviors for the proton conductance. Incubation of fetal mitochondria with ATP, GDP or carboxyatractyloside promoted a significant reduction in the passive proton permeability of the membrane and the appearance of the characteristic biphasic behavior for the proton conductance. It is concluded that the postnatal increase in intramitochondrial adenine nucleotide concentration promotes, by the interaction of the nucleotides with the adenine nucleotide translocase, the reduction in the passive proton permeability of the mitochondrial membrane, allowing efficient energy conservation in the neonatal liver.

Adenine Nucleotides↗
PubMed1991

Improved ion-pair high-performance liquid chromatographic method for the quantification of a wide variety of nucleotides and sugar-nucleotides in animal cells.

An improved method including extraction procedures is presented for the analysis of nucleotides in suspension-cultivated animal cells. Quantification was performed by ion-pair high-performance liquid chromatography after perchloric acid extraction. It was found that the amount of perchloric acid taken for extraction influenced the yield and that cell washing procedures caused deterioration of the analysis results for triphosphates. More than thirty nucleotides and sugar-nucleotides were separated within 25 min using a Supelcosil reversed-phase column (3 microns) with tetrabutylammonium hydrogensulphate as pairing agent and methanol-pH gradient elution. Cultivated hybridoma cells showed variations in intracellular nucleotide concentrations as well as relative amounts during different growth phases, which could reflect the physiological state of a cell culture.

Adenosine Triphosphate↗
PubMed2001

The structure and nucleotide occupancy of bovine mitochondrial F(1)-ATPase are not influenced by crystallisation at high concentrations of nucleotide.

Analysis of tryptophan mutants of F(1)-ATPase from Escherichia coli [Löbau et al. (1997) FEBS Lett. 404, 15-18] suggested that nucleotide concentrations used to grow crystals for the determination of the structure of bovine F(1)-ATPase [Abrahams et al. (1994) Nature 370, 621-628] would be sufficient to occupy only two catalytic sites, and that higher concentrations of nucleotide would result in all three sites being occupied. We have determined the structure of bovine F(1)-ATPase at 2.9 A resolution with crystals grown in the presence of 5 mM AMPPNP and 5 microM ADP. Similar to previous structures of bovine F(1)-ATPase determined with crystals grown in the presence of lower nucleotide concentrations, only two beta-subunits have bound nucleotide and the third subunit remains empty.

Adenosine Diphosphate↗
PubMed1989

Isocratic reversed-phase high-performance liquid chromatography of ribonucleotides, deoxynucleotides, cyclic nucleotides and deoxycyclic nucleotides.

An isocratic reversed-phase high-performance liquid chromatographic system is described for the separation of a relatively large number (greater than 20) of ribonucleotides, deoxynucleotides, cyclic nucleotides and deoxycyclic nucleotides. A 25 cm x 5 mm ODS-Hypersil (5 microns particle size) column was used with methanol-triethylammonium phosphate buffer as eluent. The effects of methanol content, pH, and ionic strength of the buffer on retention and resolution of the nucleotides have been studied. The applicability of the system was demonstrated by the analysis of nucleotides in cells and tissue extracts.

Cells, Cultured↗