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.     

Processing of RNA in Escherichia coli is limited in the absence of ribonuclease III,ribonuclease E and ribonuclease P

A strain of Escherichia coli lacking RNAase III and containing thermolabile RNAase E and RNAase P was labeled with ³²P_i at a non-permissive temperature. RNA molecules were separated by two-dimensional polyacrylamide gel electrophoresis. Most of the small RNA species were isolated and analyzed for the presence of 5′ nucleoside triphosphates. In 16 of the 22 species analyzed a significant number of the individual molecules contained 5′ di or triphosphates. We conclude, therefore, that very little endonucleolytic RNA processing occurs in the absence of the three RNA processing enzymes RNAase III, RNAase E and RNAase P.     

Chemoenzymic Synthesis of Pα-Methyl Deoxynucleoside Triphosphates

Abstract

5′-O-(methylphosphonyl)-N-(phenylacetyl)-2 ′-deoxycytidine, deoxyadenosine and deoxyguanosine were pyrophosphorylated and the resulting N-protected P α-methyl nucleoside triphosphates were deblocked by treatment with penicillin amidase at pH 7.8, 25°C to give P α-methyl nucleoside triphosphates.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignment of human guanylate kinase

Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain ¹H, ¹³C, and ¹⁵N nuclei as a first step towards the enzyme’s structural and mechanistic analysis with atomic resolution.     

An Efficient Protection-Free One-Pot Chemical Synthesis of Modified Nucleoside-5′-Triphosphates

A simple, reliable, and an efficient “one-pot, three step” chemical method for the synthesis of modified nucleoside triphosphates such as 5-methylcytidine-5′-triphosphate (5-MeCTP), pseudouridine-5′-triphosphate (pseudoUTP) and N¹-methylpseudouridine-5′-triphosphate (N¹-methylpseudoUTP) starting from the corresponding nucleoside is described. The overall reaction involves the monophosphorylation of nucleoside, followed by the reaction with pyrophosphate and subsequent hydrolysis of the cyclic intermediate to furnish the corresponding NTP in moderate yields with high purity (>99.5%).     

Continuous electron spin resonance detection of biochemical reactions of nucleoside triphosphates.

A technique of continuous recording of the kinetics of biochemical reactions of nucleoside triphosphates by means of an ESR (electron spin resonance) method is proposed. The technique is based on the differential ability of NTP (nucleoside triphosphates) and the products of their conversion to coordinate Mn²⁺ ions. Due to this fact the concentration of free (hydrated) Mn²⁺ ions changes in the course of the reaction and, consequently, the intensity of their ESR signal also changes. The proposed technique makes it possible to determine changes of concentration ?0.1% of the total free ion concentration. The technique was applied to observation of reactions catalyzed by RNA polymerase, alkaline phosphatase, and aminoacyl-tRNA synthetase.     

Rapid Synthesis of 2′ and 3′ Fluorinated Nucleotides and Their Use in 19F-NMR-Spectroscopy of Nucleotide-Binding Enzymes

Abstract

Improved methods have been developed for the syntheses of nucleoside analogs with fluorine atoms in the furanose moiety. 5′-O-mono and triphosphates of these analogs were used in stuctural studies on adenylate kinase using ¹⁹F-NMR.     

A simple method for synthesizing [γ-32P]nucleoside triphosphates using [32P]acetylphosphate and acetate kinase

A simple, rapid, and inexpensive method is described for the synthesis of γ-³²P-labeled ribo- or deoxyribonucleoside triphosphates. The procedure involves chemical synthesis of [³²P]acetylphosphate and subsequent phosphorylation of nucleoside diphosphates using acetate kinase (EC 2.7.2.1) and a final purification step. The entire procedure is performed 8 h or less.     

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.     

Deoxyribonucleotide Pools in Plant Tissue Cultures

Two dimensional thin-layer chromatography on anion-exchange cellulose enables the separation of the normally occurring ribo- and deoxyribonucleoside triphosphates. This technique was applied to perchloric acid extracts of callus tissue of sycamore and tobacco and of pine pollen grown in ³²P-orthophosphate labelled media to quantitate the nucleoside triphosphate pools under different growth conditions. The results showed that the ratio of the deoxyribonucleo-side triphosphates to their corresponding ribonucleoside triphosphates is low in plant cells, similar to the ratio previously found for animal cells. During the period of most rapid DNA synthesis in the callus tissue, the deoxyribonucleoside triphosphate pools reach their highest values. This effect is also demonstrated with cells of Escbericbia coli.     

RNA SYNTHESIS BY EXOGENOUS RNA POLYMERASE ON CYTOLOGICAL PREPARATIONS OF CHROMOSOMES

Cytological preparations of Drosophila polytene chromosomes serve as templates for RNA synthesis carried out by exogenous RNA polymerase (Escherichia coli). Incorporation of labeled ribonucleoside triphosphates into RNA may be observed directly by autoradiography. Because of the effects of rifampicin, actinomycin D, ribonuclease, high salt, and the requirement for all four nucleoside triphosphates, we conclude that the labeling observed over chromosomes is due to DNA-dependent RNA polymerase activity. Using this method, one can observe RNA synthesis in vitro on specific chromosome regions due to the activity of exogenous RNA polymerase. We find that much of the RNA synthesis in this system occurs on DNA sequences which appear to be in a nondenatured state.     

Gene expression in isolated plastids from fruits of capsicum annuum

Plastids were obtained from the ripening fruits of Capsicum annuum, and incubated in vitro in the presence of [³⁵S]methionine (Met). There was polypeptide synthesis at all stages of pepper tissue studied in both chloroplasts and chromoplasts, dependent on the addition of nucleoside triphosphates and phsophoenolpyruvate and inhibited by D-threo-chloramphenicol.     

Endocytosis of surface bound dopamine β-hydroxylase and plasma membrane following catecholamine secretion by bovine adrenal chromaffin cells

Cultured chromaffin cells were stimulated with either Ba²⁺ or nicotine to secrete catecholamines. This resulted in the appearance of the chromaffin granule membrane protein, dopamine β-hydroxylase (DBH), on the cell surface. The DBH exposed on the cell surface was labeled using fluorescently tagged anti-DBH Fab fragments and the cell surface was simultaneously labeled with fluorescently tagged concanavalin A. Immediately after labeling, both fluorescent markers were localized on or near the cell surface; anti-DBH fluorescence was distributed as patches, but Con A fluorescence was uniformly distributed. Approximately 30 min after labeling, anti-DBH fluorescence appeared to be almost completely internalized without apparent redistribution on the surface whereas much of the Con A fluorescence remained on the cell surface.The rate of DBH endocytosis was quantified using ¹²⁵I labeled anti-IgG to measure surface bound anti-DBH. Following stimulation of catecholamine secretion, DBH and DBH/anti-DBH complexes both disappeared from the cell surface at similar rates. The half-life on the cell surface was approximately 7 min. These results demonstrate that DBH was rapidly and selectively retrieved from the cell surface, probably from the site of exocytosis.     

Isolation and kinetic properties of 5′-nucleotidase from guinea-pig skeletal muscle

5′-Nucleotidase (EC 3.1.3.5) has been solubilized and purified 1200-fold from guinea-pig skeletal muscle, to a specific activity of 40 U/mg protein. The purified enzyme yields a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Guinea-pig skeletal muscle 5′-nucleotidase is extremely sensitive to inhibition by nucleoside di- and triphosphates. The inhibition is of the competitive type, and can be reversed only by strong excess of Mg²⁺. Nucleoside diphosphates are more powerful inhibitors than nucleoside triphosphates. The K_i values for ADP and ATP are 0.036 and 0.28 μM, respectively. The purified enzyme does not require exogenous cations for maximal activity and is inhibited by EDTA. This inhibition is reversed by divalent cations. This indicates that the enzyme contains a tightly bound metal cation.     

A batch procedure for the assay of cyclic nucleotide phosphodiesterase.

A procedure for the assay of cyclic nucleotide phosphodiesterase is described in which labeled cyclic nucleotide is separated from labeled nucleoside by the batchwise addition of ethanolic slurries of Dowex 2 fluoride. Under the conditions described there is no detectable adsorption of nucleoside by the anion exchanger, which removes more than 95% of the tritium in boiled samples of [8-³H]cAMP or [8-³H]cGMP. Linear time courses and enzyme vs activity relationships are described for 10^?3 and 10^?7m cAMP and 10^?4m cGMP. The method is limited by interference by neutral salts and by the enzymatic conversion of adenosine into inosine.     

Erratum

RNA polynucleotide kinase has been shown to transfer [γ³²P] from ATP to 5-OH termini of endogenous nuclear RNA. The products of this reaction have been isolated in RNA larger than 125 after in vitro incubation of mouse L cell nuclei. About 20%–30% of these 5′-OH kinase products are polyadenylated. A sizeable fraction of the [γ³²P] label from ATP is also found in internal phosphodiester bonds after 30-minute nuclear incubation in vitro. The possibility of substantial [³²P] recycling via the α position of nucleoside triphosphate was ruled out because: (1) 2mM nucleoside triphosphates in the incubation medium, (2) limited nearestneighbor distribution 3′ and 5′ to the phosphodiester bond compared with that from [α³²P] UTP, (3) different nearest-neighbor distribution for RNA molecules > 12S and 12-3S, (4) relative insensitivity of the [γ³²P] incorporation to α-amanitin as compared with total RNA synthesis, (5) internal [³²P] appearance in RNA > 12S in less than five minutes of incubation, and (6) < 0.03% to 0.6% of the total [³²P] in the α position of nucleoside triphosphates after 30 minutes of incubation. The [γ³²P] incorporation was dependent on high ATP concentration and was insensitive to competition by inorganic phosphate. These results are consistent with the levels of 5′ RNA polynucleotide kinase activity in L cell nuclei and suggest the presence of an RNA ligase that can utilize the termini generated by the 5′-OH RNA kinase in a ligation reaction.     

Changes in ribo- and deoxyribonucleoside triphosphate pools within the cell cycle of a synchronized moused fibroblast cell line

Intracellular pool levels of ribo- and deoxyribonucleoside triphosphates were monitored throughout the cell cycle of C3H10T1/2 mouse embryo fibrolast cells synchronized by isoleucine deprivation. Absolute pool sizes of ribonucleoside triphosphates were approximately 30 fold greater than those of the corresponding deoxyribonucleoside triphosphates. Of the ribonucleoside triphosphates, pool sizes of ATP exhibited the greatest change, increasing from a low of 32.7 nmol/10⁷ cells during G₁ to a high of 81.6 nmol/10⁷ cells 2 h prior to mid S-phase. Levels of ATP subsequently declined to 40.2 nmol/10⁷ cells during late S-phase, followed by a second peak of 65.8 nmol/10⁷ with the onset of cell division. No significant changes in the pool sizes of UTP and GTP were found throughout the cell cycle. Of the deoxyribonucleoside triphosphates, pool sizes of pyrimidine deoxyribonucleoside triphosphates were approx. 5–10 fold greater than those of purine deoxyribonucleoside triphosphates. Low levels of deoxyribonucleoside triphosphates during G₁ (0.3–1.3 pmol/10⁷ cells) increased coordinately with the initiation of DNA synthesis to an initial peak during mid S-phase (0.5–6.4 pmol/10⁷ cells). Decling levels of deoxyribonucleoside triphosphates during late S-phase were followed by a subsequent larger second peak (1.7–10.7 pmol/10⁷ cells) during G₂-M.     

Lake Michigan sponge phosphatic metabolite variations with habitat: a P nuclear magnetic resonance study

The ³¹P NMR phosphatic profile of the sponge Eunapius fragilis differs considerably from profiles obtained from vertebrate tissues: phosphonic acids, glycan phosphates, and phosphoarginine are present in the sponge profile. Ethanolamine phosphate, a family of phosphodiesters, dinucleotides, and a family of pyrimidine-nucleotide-activated co-factors are elevated, while nucleoside monophosphates, inorganic orthophosphate, and nucleoside triphosphates are diminished with respect to vertebrate tissues. The profile contains 26 different phosphatic metabolite resonance bands, and all but one of these (phosphonates) can be used to differentiate among three southern Lake Michigan sponge habitats examined: a coastal lagoon; the shipwreck of the Material Service Barge in the open waters of Lake Michigan; and an industrial section of the Calumet River. The quantitative phosphatic metabolite concentration data, in conjunction with numerical indexes derived therefrom, demonstrate that, in analogy to sponge phospholipid profiling, ³¹P NMR phosphatic metabolites can be utilized to distinguish among habitats using a single species as a bioindicator. For the sponge, energy reserves are concentrated in nucleotide co-factors rather than nucleoside triphosphates. High concentrations of ethanolamine and choline phosphate intermediates are present suggesting that metabolic pathways leading to phospholipid biosynthesis are primed for rapid membrane biosynthesis leading to subsequent sponge growth.