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Methods in Enzymology 2007Naturally occurring modified residues derived from canonical RNA nucleotides are present in most cellular RNAs. Their detection in RNA represents a difficult task... (Review)
Review
Naturally occurring modified residues derived from canonical RNA nucleotides are present in most cellular RNAs. Their detection in RNA represents a difficult task because of their great diversity and their irregular distribution within RNA molecules. Over the decades, multiple experimental techniques were developed for the identification and localization of RNA modifications. Most of them are quite laborious and require purification of individual RNA to a homogeneous state. An alternative to these techniques is the use of reverse transcription (RT)-based approaches. In these approaches, purification of RNA to homogeneity is not necessary, because the selection of the analyzed RNA species is done by specific annealing of oligonucleotide DNA primers. However, results from primer extension analysis are difficult to interpret because of the unpredictable nature of RT pauses. They depend not only on the properties of nucleotides but also on the RNA primary and secondary structure. In addition, the degradation of cellular RNA during extraction, even at a very low level, may complicate the analysis of the data. RT-based techniques for the identification of modified residues were considerably improved by the development of selected chemical reagents specifically reacting with a given modified nucleotide. The RT profile obtained after such chemical modifications generally allows unambiguous identification of the chemical nature of the modified residues and their exact location in the RNA sequence. Here, we provide experimental protocols for selective chemical modification and identification of several modified residues: pseudouridine, inosine, 5-methylcytosine, 2'-O-methylations, 7-methylguanosine, and dihydrouridine. Advice for an optimized use of these methods and for correct interpretation of the data is also given. We also provide some helpful information on the ability of other naturally occurring modified nucleotides to generate RT pauses.
Topics: Animals; Humans; Purine Nucleotides; Pyrimidine Nucleotides; RNA; RNA Processing, Post-Transcriptional; Reverse Transcription
PubMed: 17673078
DOI: 10.1016/S0076-6879(07)25002-5 -
Journal of Inorganic Biochemistry Aug 2005The interactions between pyrimidine nucleotides: cytidine-5'-diphosphate (CDP) and cytidine-5'-triphosphate (CTP) and Cu(II) ions, spermine (Spm) and...
The interactions between pyrimidine nucleotides: cytidine-5'-diphosphate (CDP) and cytidine-5'-triphosphate (CTP) and Cu(II) ions, spermine (Spm) and 1,11-diamino-4,8-diazaundecane (3,3,3-tet) have been studied. The composition and stability constants of the complexes formed have been determined by means of the potentiometric method, while the centres of interactions in the ligands have been identified by the spectral methods (UV-Vis, Ultraviolet and Visible spectroscopy; EPR, electron spin resonance; NMR). In the systems without metal, formation of the molecular complexes nucleotide-polyamine with the interaction centres at the endocyclic nitrogen atom of purine ring N3, the oxygen atoms of the phosphate group from the nucleotide and protonated nitrogen atoms of the polyamine have been detected. Significant differences have been found in the metallation between the systems with Spm and with 3,3,3-tet. In the systems with spermine, mainly protonated species are formed with the phosphate group of the nucleotide and deprotonated nitrogen atoms of the polyamine making the coordination centres, while the donor nitrogen atom of the nucleotide N3 is involved in the intramolecular interligand interactions, additionally stabilising the complex. In the systems with 3,3,3-tet, the MLL' type species are formed in which the oxygen atoms of the phosphate group and nitrogen atoms of the polyamine are involved in metallation, whereas the N3 atom from the pyrimidine ring of the nucleotide is located outside the inner coordination sphere of copper ion. The main centre of Cu(II) interaction in the nucleotide, both in the system with Spm and 3,3,3-tet is the phosphate group of the nucleotide.
Topics: Amines; Cations, Divalent; Copper; Cytidine Diphosphate; Cytidine Triphosphate; Molecular Structure; Spectrum Analysis
PubMed: 15993946
DOI: 10.1016/j.jinorgbio.2005.05.005 -
Chemistry & Biodiversity Sep 2007The pyrimidine bases of RNA are uracil (U) and cytosine (C), while thymine (T) and C are used for DNA. The C(5) position of C and U is unsubstituted, whereas the C(5) of... (Comparative Study)
Comparative Study
The pyrimidine bases of RNA are uracil (U) and cytosine (C), while thymine (T) and C are used for DNA. The C(5) position of C and U is unsubstituted, whereas the C(5) of T is substituted with a Me group. Miller et al. hypothesized that various C(5)-substituted uracil derivatives were formed during chemical evolution, and that C(5)-substituted U derivatives may have played important roles in the transition from an 'RNA world' to a 'DNA-RNA-protein world'. Hyperthermophilic bacteria and archaea are considered to be primitive organisms that are evolutionarily close to the universal ancestor of all life on earth. Thus, we examined the substrate specificity of several C(5)-substituted or C(5)-unsubstituted dUTP and dCTP analogs for several DNA polymerases from hyperthermophilic bacteria, hyperthermophilic archaea, and viruses during PCR or primer extension reaction. The substrate specificity of the C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides varied greatly depending on the type of DNA polymerase. The significance of this difference in substrate specificity in terms of the origin and evolution of the DNA replication system is discussed briefly.
Topics: Archaea; Archaeal Proteins; Bacteria; Bacterial Proteins; Bacteriophages; DNA; DNA Replication; DNA-Directed DNA Polymerase; Evolution, Molecular; Hot Temperature; Pyrimidine Nucleotides; RNA; Substrate Specificity; Viral Nonstructural Proteins
PubMed: 17886855
DOI: 10.1002/cbdv.200790165 -
Journal of Medicinal Chemistry Feb 2008Orotidine monophosphate decarboxylase (ODCase) generally accepts pyrimidine-based mononucleotides as ligands, but other nucleotides are also known to bind to this...
Orotidine monophosphate decarboxylase (ODCase) generally accepts pyrimidine-based mononucleotides as ligands, but other nucleotides are also known to bind to this enzyme. We investigated the kinetic properties of eight common and endogenous nucleotides with ODCases from three species: Methanobacterium thermoautotrophicum, Plasmodium falciparum, and Homo sapiens. UMP and XMP exhibited higher affinities as compared to the other nucleotides tested. The product of ODCase catalyzed decarboxylation, UMP, displayed inhibition constants (K(i)) of 330 microM against the Mt enzyme and of 210 and 220 microM against the Pf and Hs ODCases, respectively. The K(i) values for XMP were 130 microM and 43 microM, respectively, for Mt and Pf ODCases. Interestingly, XMP's affinity for human ODCase (K(i) = 0.71 microM) is comparable and even slightly better than that of the substrate OMP. Binding of various nucleotides and their structural features in the context of ODCase inhibition and inhibitor design are discussed.
Topics: Animals; Crystallography, X-Ray; Humans; Kinetics; Ligands; Methanobacterium; Models, Molecular; Orotidine-5'-Phosphate Decarboxylase; Plasmodium falciparum; Protein Binding; Protein Conformation; Purine Nucleotides; Pyrimidine Nucleotides; Species Specificity
PubMed: 18181562
DOI: 10.1021/jm700968x -
Basic Life Sciences 1975
Review
Topics: Animals; Bromouracil; DNA; DNA Repair; DNA Replication; Endonucleases; Molecular Weight; Protein Binding; Pyrimidine Nucleotides; Radiation Effects; Recombination, Genetic; Ultraviolet Rays
PubMed: 1103875
DOI: 10.1007/978-1-4684-2898-8_27 -
The Biochemical Journal Aug 2009
Topics: Animals; Antimetabolites; Cytidine; Cytidine Triphosphate; Deoxyadenine Nucleotides; Deoxycytidine; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyuridine; Heart; Humans; Mitochondria; Mitochondrial Diseases; Models, Biological; Phosphorylation; Pyrimidine Nucleotides; Rats; Thymine Nucleotides; Uracil; Uridine; Uridine Monophosphate; Uridine Triphosphate; Zidovudine
PubMed: 19698084
DOI: 10.1042/BJ20091194 -
Basic Life Sciences 1975
Review
Topics: Base Sequence; DNA; DNA Repair; Nucleic Acid Conformation; Pyrimidine Nucleotides; Radiation Effects; Ultraviolet Rays
PubMed: 1103835
DOI: 10.1007/978-1-4684-2895-7_1 -
Annals of the New York Academy of... Aug 1975
Topics: Animals; Carcinoma, Ehrlich Tumor; Cytosine Nucleotides; DNA; DNA, Neoplasm; Deoxyuridine; Kinetics; Mice; Polyribonucleotide Nucleotidyltransferase; Pyrimidine Nucleotides; Spectrophotometry, Ultraviolet; Sulfhydryl Compounds; Thymidine; Uracil Nucleotides
PubMed: 1059371
DOI: 10.1111/j.1749-6632.1975.tb29255.x -
Biochemical and Biophysical Research... Apr 2000Oligonucleotides can be used as sequence-specific DNA ligands by forming a local triple helix. In order to form more stable triple-helical structures or prevent their...
Oligonucleotides can be used as sequence-specific DNA ligands by forming a local triple helix. In order to form more stable triple-helical structures or prevent their degradation in cells, oligonucleotide analogues that are modified at either the backbone or base level are routinely used. Morpholino oligonucleotides appeared recently as a promising modification for antisense applications. We report here a study that indicates the possibility of a triple helix formation with a morpholino pyrimidine TFO and its comparison with a phosphodiester and a phosphoramidate oligonucleotide. At a neutral pH and in the presence of a high magnesium ion concentration (10 mM), the phosphoramidate oligomer forms the most stable triple helix, whereas in the absence of magnesium ion but at a physiological monovalent cation concentration (0.14 M) only morpholino oligonucleotides form a stable triplex. To our knowledge, this is the first report of a stable triple helix in the pyrimidine motif formed by a noncharged oligonucleotide third strand (the morpholino oligonucleotide) and a DNA duplex. We show here that the structure formed with the morpholino oligomer is a bona fide triple helix and it is destabilized by high concentrations of potassium ions or divalent cations (Mg(2+)).
Topics: Base Sequence; DNA; DNA Primers; Electrophoresis; Kinetics; Magnesium; Morpholines; Pyrimidine Nucleotides; Spectrophotometry, Ultraviolet; Thermodynamics; Thionucleotides
PubMed: 10753631
DOI: 10.1006/bbrc.2000.2438 -
The Journal of Biological Chemistry Dec 1980We have measured the pyrimidine nucleotide contents of the culture fluid, acid-soluble fraction, and acid-insoluble fraction of cultures of hamster embryo fibroblasts...
We have measured the pyrimidine nucleotide contents of the culture fluid, acid-soluble fraction, and acid-insoluble fraction of cultures of hamster embryo fibroblasts (third subculture) through the final two divisions of growth in culture. The cells show a growth delay between the penultimate and ultimate division periods and a concomitant biochemical synchrony of pyrimidine metabolism. The cells exhibit normal excretion of pyrimidine nucleotides beginning with the ultimate division cycle. This excretion results from the net breakdown of ribonucleic acid and a cell-regulated maximum for pyrimidine mononucleoside polyphosphate content. This upper limit for the pyrimidine nucleoside polyphosphate content is not a steady state phenomenon but rather an absence of both synthesis and utilization. The hamster embryo fibroblast exhibits a directed flow of salvage uridine for ribonucleic acid synthesis. We show that de novo synthetic uridine 5'-monophosphate also can be used for ribonucleic acid synthesis without prior entry into the cytoplasmic uridine nucleoside polyphosphate pool. During attachment and first division salvage uridine does enter the cytoplasmic nucleotide pool. The properties of the cytidine pools differ from the uridine pools in specific activity and levels of cytidine, due to turnover of the terminal C-C-A of cytoplasmic transfer ribonucleic acid and the delay in conversion of of nonradioactive de novo synthetic uridine 5'-monophosphate to cytidine 5'-triphosphate. The partial synchrony in these cultures has been used as a temporal marker of the observed events.
Topics: Animals; Cell Cycle; Cells, Cultured; Cricetinae; Embryo, Mammalian; Female; Fibroblasts; Interphase; Kinetics; Pregnancy; Pyrimidine Nucleotides
PubMed: 7440538
DOI: No ID Found