Nucleic acids research. Supplement (2001)最新文献

N3-Phenacyl-N1-substituted uracils 3a-q were synthesized by introduction of substituents at the N1-position of N3-phenacyluracil 2, and their hypnotic and sedative activities were evaluated. Pharmacological activities of these N3-phenacyl-N1-substituted uracils were examined using hypnotic activity and synergistic effects with pentobarbital or diazepam for the hypnotic and sedative activities.

Various nonnatural amino acids has been incorporated into proteins by using four-base codons in an E. coli in vitro translation system. Here, design and synthesis of novel fluorescently labeled nonnatural amino acids and their incorporation into proteins were investigated. Transfer RNAs that contained a CCCG anticodon and were aminoacylated with BODIPY FL-labeled amino acids were prepared by a chemical aminoacylation method, and added to an in vitro translation system in the presence of a streptavidin mRNA containing a CGGG codon. SDS-PAGE and Western blot analysis of the synthesized proteins indicate that BODIPY FL-labeled aminophenylalanine derivatives are efficiently incorporated into proteins through the four-base codon decoding.

MicroRNAs (miRNAs) are phylogenetically widespread small RNAs in animals and plants. These small RNAs can regulate a gene expression at a translational level and play roles during the development of C. elegans, D. melanogaster and plants. Although more than two hundred miRNAs have been found in mammals, the target mRNAs of miRNAs is unknown. Recently, we identified Hes1, bHLH transcriptional repressor, as a target of miR-23 in NT2 cells. In this study, we further investigate that the expression of Hes1 is regulated by miR-23 during the retinoic acid (RA)-induced neural differentiation of NT2 cells. Reduction in the level of miR-23 by siRNAs resulted in the accumulation of Hes1 in differentiated NT2 cells. Moreover, a reduction in the level of miR-23 by siRNA-miR-23 affected the RA-induced neural differentiation of NT2 cells. Thus, our results indicate that miR-23 has a critical role in the RA-induced neuronal differentiation of NT2 cells.

To investigate the recognition mechanism of tRNA(Pro) by prolyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1, various tRNA(Pro) transcripts were prepared by in vitro transcription system. These transcripts were aminoacylated with proline by overexpressed A. pernix prolyl-tRNA synthetase. From prolylation experiments, recognition elements of A. pernix tRNA(Pro) were determined to be G35 and G36 of anticodon, discriminator base A73, and G1-C72 base pair at acceptor stem end.

Transcription factor AP-2 seems to play an important role in the malignancy of melanoma. In this study, we constructed ribozyme expression vectors to suppress the expression of AP-2 (AP-2-ribozymes) and then examined gene expression in human A375P melanoma cells that stably expressed ribozymes targeted to the AP-2 transcript. A comparison of the gene-expression profiles of A375P cells that expressed AP-2-ribozymes and those transfected with the empty vector revealed changes in levels of expression of several genes. Here we described that the combination of gene suppression by ribozymes and the analysis of gene expression using a macro-array provides a good approach for elucidating signal transduction pathways. These results provide further insight into the role of AP-2 in human melanoma cells.

The combination of two stabilizing strategies to increase stability of nucleic acid assembly is not always resulted in synergistic effect. In the present study, to explore the rational way to select the combination for synergistically stabilizing strategies, we examined the kinetic effects of different triplex-stabilizing strategies, poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO). The former increased the association rate constant, whereas the latter decreased the dissociation rate constant in triplex equilibrium. Each strategy increased the binding constant of the triplex formation by nearly two orders of magnitude. The combination of both stabilizing strategies, which was the triplex formation with PN TFO in the presence of the copolymer, increased the binding constant by nearly four orders of magnitude, implying successful synergy of their activities. The kinetically orchestrated effects in which the copolymer and the PN modification contribute to distinct ingredients in triplex equilibrium achieved the observed synergistic stabilization. We conclude that kinetic analyses of stabilizing effects enable us to select a rational combination of stabilizing strategies.

5-Formyluracil is a major oxidative thymine lesion with mutagenic and cytotoxic properties. In this study, we have partially purified and characterized a mammalian 5-formyluracil-DNA glycosylase (FDG) from rat liver. FDG was a monofunctional DNA glycosylase and removed 5-formyluracil, uracil, 5-hydroxyuracil, 5-hydroxylmethyluracil in single-stranded and double-stranded DNA. Several lines of evidence indicate that FDG is a rat SMUG1 homologue. Human SMUG1 also exhibited similar enzymatic properties.

CSNA is a computer system which classifies a set of RNA structures based on their structural characters; hydrogen bond and base-base stacking. CSNA has been applied to the RNA structure determination by NMR and it was found that CSNA could provide well converged groups as the lowest energy structures. Here, we further applied CSNA to the structure determination of a 31mer RNA forming a psuedoknot structure. It was demonstrated that CSNA is a useful tool for the RNA structure determination by NMR.

Ribonucleopeptide receptors for ATP have been designed by using a structure-based design and in vitro selection method. The ATP binding ribonucleopeptide receptors revealed submillimolar affinity to ATP and discriminate ATP against other ribonucleotides. In this research, we have developed a simple strategy to convert the ATP-binding ribonucleopeptide receptor into a ribonucleopeptide sensor by introducing a fluorophore in the peptide subunit. Fluorophore labeled ribonucleopeptide complex showed a large change in the fluorescence intensity upon addition of ATP.