Nucleic acids symposium series最新文献

In Bacillus subtilis, selenocysteine tRNA has not been identified in a total genome sequence so far (1). To explore the system of selenocysteine incorporation in B. subtilis, we screened serine-acceptable tRNAs to find an unknown tRNA for selenocysteine by the combined method of specific biotinylation of aa-tRNA (2) and RT-PCR (3). cDNAs obtained from the serine-acceptable tRNA pool were amplified and cloned into plasmid to read its sequence. This procedure gave cDNA library corresponding known serine tRNAs, but no candidate for selenocysteine has been found. Thus, this result, together with the previous data (4), might reveal that there is no selenocysteine tRNA in B. subtilis and/or metabolism of selenium is considerably different from known one as seen in other bacteria.

To elucidate the contribution of LINE-like retrotransposon Zepps in formation and maintenance of chromosomal telomeres, newly formed mini-chromosomes in irradiated Chlorella vulgaris cells were isolated and structurally characterized. A mini-chromosome Y32 (approximately 400 kbp in size) was shown to have several copies of Zepp elements on both termini. On the right arm terminus, two copies of Zepps were found in a tandem array with poly(A) tracts facing towards the chromosome end. The poly(A) tail and a 3'-end of approximately 400 bp of the distal copy was replaced by telomeric repeats. On 5'-side of the proximal copy was another Zepp element in a reversed orientation. This newly formed telomeric structure is very similar to that found in the left arm terminus of chromosome I and support the model of Zepp-mediated maintenance of Chlorella telomeres.

In this study, the solid-phase synthesis of oligodeoxyribonucleotides having a guanosine pyrophosphate cap structure (Gpp-) was achieved by using a new guanosine monophosphate unit having the DMTr group capable of estimation of coupling efficiency of the pyrophosphate bond formation. Since 7-methylguanosine base was unstable under basic conditions, Gpp-capped DNA oligomers were synthesized by using a new linker having a silanediyl bond, which allowed to release the DNA chain from the solid support by treatment with fluoride anion under neutral conditions.

We have designed a new type of a DNA dendrimer which has rigid branched structure. The branching molecule was prepared from 1,3,5-tribromobenzene. The dendrimer unit, in which three oligonucleotide-chains, two molecules of T15 and one molecule of A15, linked to the branching molecule, was synthesized by an automated DNA synthesizer. The properties of the dendrimer unit and dendrimer formation by inter-molecular association of T15 and A15 chains of the dendrimer unit will be presented.

A facile stereospecific synthetic method for alpha-anomeric 2'-deoxypyrimidine nucleoside unit utilizing aminooxazoline derivative of ribofuranose was investigated. Thus, easily accessible riboaminooxazoline derivative prepared by ribose and cyanamid was allowed to react with ethyl alpha-bromoethylacrylate to give corresponding adduct. The adduct was cyclized by strong base such as potassium t-butokiside. The resulted 2,2'-cyclonucleoside was then treated with acetyl bromide followed by n-butyltin hydride to give alpha-anomeric 3',5'-di-O-acetylthymidine. 3',5'-Di-O-acety groups of the nucleoside were easily removed by the action of excess of triethyl amine in methanol. Essentially same procedure afforded corresponding 2'-deoxyuridine, which was further, converted to alpha-anomeric 2'-deoxycytidine.

Isopoly(S-carboxymethyl-L-cysteine) derivatives of nucleic acid bases were prepared as antisense compounds. These compounds in vitro have been found to form stable complex with oligo-DNA or RNA. This paper deals with effect of antisense compounds in vivo. The target in this paper is the sequence of the PSD-95 protein linked with NMDA receptor. Excess passing of calcium ions through the loss of the signal pathway without PSD-95 proteins caused by antisense compound. The cells detailing with L-cysteine derivatives showed the lowest percentage of 19.1%. The data were compared with that of phosphotioate antisense compound.

We examined the suppression of virus expression by cleavage of the HIV-1 RNA gene using a mammalian tRNA 3' processing endoribonuclease and an External Guide Sequence Oligozyme (EGS) in vivo. We constructed an EGS expression vector that used the tRNA(met) promoter as an expression cassette for EGS. The EGS expression vector was targeted to the upstream region of gag, region. The EGS expression vector was co-transfected into COS cells with the HIV-1 gene plasmid vector. As compared with the EGS non-expressing cells and the EGS expressing cells, the EGS expressing cells with the targeted gag start codon had a clearly decreased amount of the HIV-1 gag p24 protein. The EGS expressing cells with the targeted gag start codon showed effective suppression of HIV-1 gene expression. Thus, these studies describe novel gene targeting agents for the inhibition of gene expression and antiviral activity.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems designed on the basis of the "pseudoknot" model were synthesized and their tertiary interactions were analyzed by NMR spectroscopy. Rz-1 is a cis-acting ribozyme system (the cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. Rz-2 is a trans-acting ribozyme system (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-2 was partially labeled with stable isotopes in guanosine residues of enzyme 35mer. Rz-4 is a trans-acting ribozyme system (substrate: 8mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme 53mer) which was designed by Perrotta and Been. Rz-4 has the same sequence and an extra loop closing stem IV. From 2D-NOESY and 2D-HSQC (except for Rz-4) spectra, it was suggested each ribozyme forms "pseudoknot" type structure in solution. Additionally, it was found that G38 of Rz-1, G28 and G29 of Rz-2 and Rz-4 form base-pairs. These novel base-pairs are observed in the crystal structure of a modified genomic HDV RNA. From temperature change experiment of Rz-2, the imino proton signal of G28 disappeared at 50 degrees C earlier than the other corresponding signals. Upon MgCl2 titration of Rz-2, this signal showed the largest shift.

DNA enzymes are RNA-cleaving single stranded DNA molecules. The structure and the catalytic domain of a DNA enzyme were determined by Santro et al. in 1997. In this study, we have designed several types of DNA enzymes (PB2Dz) targeted to the PB2 mRNA translation initiation region of influenza A virus, and examined their cleavage kinetics, nuclease resistance, and a luciferase gene reporter assay. Using a synthetic substrate, these DNA enzymes were shown to have cleavage activity that is dependent on the length of the substrate recognition domain. To confer serum nuclease resistance to the DNA enzymes, we designed a new type of DNA enzyme that has the N3'-P5' phosphoramidate modification (PB2Dz-N) at each terminal. We examined the activity of this DNA enzyme in vivo. The DNA enzymes used in this study inhibited the expression of the PB2-luciferase gene in COS cells. These results suggest that DNA enzymes are potentially useful as gene inactivating agents of influenza A virus.

A novel fluorophore viz. 4-dansylamido-1,8-naphthalimido-N-pentanol has been designed, prepared and characterised. The comparative fluorescence has been studied in different solvents, solvent gradient, aqueous solutions of inorganic ions and buffers. This can be used for covalent tagging of oligonucleotides having potential application in Molecular Biology.