Nucleic Acids Symposium Series

http://nass.oxfordjournals.org

List of Papers (Total 1,527)

A novel convenient method for high bacteriophage titer assay

The recent various applications of phages (bacteriophages) including phage therapy have brought about a revival of phage investigation. The phage titer assay is indispensable for phage experiments. However, the conventional standard method is a plaque counting method which requires a little skill with tedious repeating operation. Furthermore, it is not directly applicable to high...

Expression behavior of high-pressure-compacted plasmid DNA in mammalian cell

We have been developing a novel compaction method of plasmid DNA using high pressure technology, and previously found that the size of the plasmid DNA was decreased with increasing the pressurizing-strength and time. In the present study, we investigated the tertiary structural change and the expression behavior of the pressure-compacted plasmid DNA in cell in vitro. When the...

Role of exposed aromatic residues in substrate-binding of CBM family 5 chitin-binding domain of alkaline chitinase

Chitinase J (ChiJ) from alkaliphilic Bacillus sp. strain J813 has a multidomain structure containing a catalytic domain (CatD), a fibronectin type III like domain (FnIIID) and a chitin-binding domain (ChBD). It has been shown that the ChBD binds to an insoluble chitin and enhances its degradation by the CatD. Further binding study of the ChBD was performed with a glutathione-S...

Incipient complex formation between AP endonucleases and DNA containing AP site: A vital role of the tryptophan residue

To elucidate whether the tryptophan residues in the vicinity of the catalytic site are involved in AP site recognition and are critical for AP endonuclease activity, the AP endonucleases of the four subtypes in the ExoIII AP endonuclease family were characterized and compared the positions of the tryptophan residues. The positions of the catalytic amino acid residues...

Physiological role of RsgA in ribosome biosynthesis

RsgA is a unique GTP hydrolytic protein, in which the GTPase activity is significantly enhanced by the small ribosomal subunit. Depletion of RsgA causes slow cell growth as well as defects in the subunit assembly of the ribosome and the 16S rRNA processing, suggesting its involvement in the maturation of the small subunit. Several antibiotics bound to the decoding center of the...

Trans-translation by tmRNA and SmpB

tmRNA has a dual function both as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch translation from a truncated or other problematic mRNA to the tmRNA's tag-encoding sequence. During trans-translation, tmRNA enters the ribosomal A-site without a codon-anticodon interaction, but with an SmpB which is a tmRNA binding protein. To further study the...

Production of yeast (m2G10) methyltransferase (Trm11 and Trm112 complex) in a wheat germ cell-free translation system

Transfer RNA (guanine-N2-)–methyltransferase [tRNA (m2G10) methyltransferase] catalyzes a methyl-transfer from S-adenosyl-l-methionine to N2-atom of guanine at position 10 (G10) in tRNA and generates N2-methylguanine at position 10 (m2G10). Yeast enzyme contains two protein subunits (Trm11 and Trm112). Trm11 protein is expected to be a catalytic subunit and Trm112 contains a Zinc...

Precise analysis of modification status at various stage of tRNA maturation in Saccharomyces cerevisiae

Transfer RNAs (tRNAs) are decorated with various post-transcriptional modifications which are enzymatically introduced at various stages of maturation. It is known that eukaryotic tRNAs are modified both in nucleus and cytoplasm. However, the order of tRNA modifications remains to be investigated. To unveil the precise timing of each modification associated with tRNA processing...

Screening of amber suppressor tRNAs suitable to introduce nonnatural amino acids into proteins by real-time monitoring of cell-free translation

Incorporation of nonnatural amino acids into proteins is a useful technique to analyze protein structure and function. We have reported that amber suppressor tRNAs suitable for efficient and specific incorporation of nonnatural amino acids into proteins can be obtained by screening a wide variety of naturally occurring tRNAs in an E. coli. cell-free translation system. The amber...

Preparation of an ochre suppressor tRNA recognizing exclusively UAA codon by using the molecular surgery technique

In order to create an ochre suppressor tRNA which exclusively recognizes UAA codon, we replaced the G34 at the first position of yeast tRNATyr[GΨA] anticodon with pseudouridine34 (Ψ34) by using the molecular surgery technique. This tRNATyr[ΨΨA] recognized only the UAA codon as expectedly, but tRNATyr[UΨA] made as a control also behaved similarly. This result may suggest that U34...

Association of nuclear-intermediate filament lamin B1 with necrotic- and apoptotic-morphologies in cell death Induced by 5-fluoro-2′-deoxyuridine

We report that anticancer 5-fluoro-2′-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression...

Molecular mechanisms of apoptosis induced by 3′-ethynylcytidine

1-(3-C-Ethynyl-β-d-ribo-pentofuranosyl)cytosine (3′-Ethynylcytidine; ECyd), a ribonucleoside analog, has a potent cytotoxic activity against cancer cells. We have investigated the cancer-cell death induced by ECyd, focusing on its molecular mechanisms. In ECyd-treated cells, RNase L is activated and involved in c-jun NH2-terminal kinase (JNK) phosphorylation, followed by...

Regulation of vascular endothelial growth factor gene under hypoxia by using artificial transcription factors

The vascular endothelial growth factor A (VEGF-A) gene is an attractive therapeutic target because both activation and repression of the gene are useful for treatment or cure of many diseases related to abnormal angiogenesis. To up- or downregulate the endogenous gene expression at will, we previously designed a 6-finger AZP to recognize a 19-bp target DNA in the VEGF-A gene, and...

Inhibition of influenza virus infection by targeting genome conserved region with non-natural nucleic acid

Two highly conserved 15 base sequences of influenza A virus genome were identified by CONSERV software, which can detect contiguous conserved sequences of biological sequences. Antiviral effect of phosphorothioate oligonucleotide that target these conserved sequences was evaluated by plaque formation assay and cell viability assay. Pre-treatment of cells with anti-PB2 (RNA...

Thymidylate kinase: The lost chemotherapeutic target

Here we highlight the unusual substrate specificity of Plasmodium falciparum thymidylate kinase (PfTMK) and the validity of the enzyme as a new drug target. Furthermore, we predict that the Anaplasma marginale enzyme has attractive domain constituents and may be functionally different from other TMPKs. We postulate that thymidylate kinases could have multiple attractive functions...

Construction of plants resistant to TYLCV by using artificial zinc-finger proteins

Previously, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to tomato yellow leaf curl virus (TYLCV) causing serious damage to an important agricultural crop, tomato. An...

Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease

To refine zinc-finger nuclease (ZFN) technology, we constructed a sandwiched ZFN, in which a DNA cleavage enzyme was sandwiched with two artificial zinc-finger proteins (AZPs). Because the sandwiched ZFN is designed to cleave the DNA between the two AZP-binding sites, the sandwiched ZFN is expected to bind preferentially to a DNA substrate rather than to cleavage products and...

Preparations of hammerhead ribozymes for investigations of their cleavable sequences

Recently, in hammerhead ribozymes, newly identified loop-loop interaction was found to be important for their activation. Therefore, we chemically synthesized a hammerhead ribozyme with this extra loop sequences and its mutant ribozymes, as well as their substrate RNA strands in order to clarify their cleavable sequences. After purification with an anion exchange column...

Characterization of RNA aptamers against SRP19 protein having sequences different from SRP RNA

SELEX is a conventional method to obtain high affinity nucleic acids to target molecules. In this study, high affinity RNA molecules against SRP19 protein were selected by using a randomized library. The primary and predicted secondary structures of the aptamers are different from those of S-domain RNA which is the natural target of SRP19 protein. Comparison of structural...

Structural aspects for the function of ATP-binding ribonucleopeptide receptors

We describe here analyses of the secondary structure of ATP-binding ribonucleopeptide (RNP) receptors. Mapping of the RNA structure of ATP-binding RNP receptors by using hydrolytic enzymes, chemical probing with dimethyl sulfate (DMS), and in-line probing indicated that ATP-binding RNP receptors take the loop structure at the nucleotide position of the “variable region”. In...

Covalently linked fluorescent ribonucreopeptide sensors

Fluorescent biosensors based on the biological macromolecule are convenient tools for investigating the event occurring in the living cell. As for one of the candidates of such biosensors, we have reported a fluorescent sensor by utilizing a ribonucleopeptide (RNP) framework. Fluorescent RNP sensors are obtained from the fluorescent RNP library constructed by the combination of...

Acridone-labeled DNA aptamer for the detection of biomolecules

An acridone-labeled DNA aptamer was synthesized to produce an aptamer probe. This aptamer probe could detect a specific molecule based on conformational changes upon binding of the specific molecules and the quenching of acridone emission.

Release of DNA binary complexes from the ternary complexes by carboxymethyl Poly(l-histidine)

The DNA ternary complexes with carboxymethyl poly(l-histidine) (CM-PLH) and poly(ethylenimine) (PEI) have released the DNA binary complexes with PEI by the protonation of CM-PLH at endosomal/lysosomal pH. The dissociation of the CM-PLH from the CM-PLH/PEI/DNA ternary complexes is proved by the fluorescence resonance energy transfer (FRET) analysis between the CM-PLH and PEI. The...

Influence of 3′-azido-2′,3′-dideoxyguanosine treatment on telomere length in human telomerase-immortalized human fibroblast cells

In order to study telomerase activation in normal cells, a telomerase-immortalized fibroblast cell line, hTERT-BJ1, treated with a telomerase inhibitor, 3′-azido-2′,3′-dideoxyguanosine (AZddG), is considered to be a good model. Long-term treatment with AZddG resulted in telomere shortening from 10-20 kbp to 5-6 kbp in cultured hTERT BJ1 cells. However, the telomere length then...

Identification of DNA binding specificity for TLS

TLS (Translocated in liposarcoma) has been characterized as a rearranged gene in chromosomal translocations specific of human myxoid liposarcoma. The various cellular functions of TLS participating either in transcription or splicing processes are thought the involvement of an interaction of TLS with DNA and/or RNA. To investigate insight into DNA-TLS interaction, we performed...