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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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.
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...
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...
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...