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Biomedical subjects

I Hirao

Publications and source records attributed to I Hirao.

49 records · Page 3Linked to original sources
PubMed1990

Unique hairpin structures occurring at the replication origin of phage G4 DNA.

Recently we reported that a DNA fragment, GCCAAAGC, forms an extraordinarily stable hairpin structure with two G-C pairs at the terminus and A-A-A stacked structure. The sequence is present at the replication origin of bacteriophage G4 ssDNA, and so on. Several kinds of possible hairpin structures, corresponding to the replication origin of phage G4, were synthesized and their secondary structures were examined. It was found that the fragments are able to form interconvertible hairpin structures depending on the length of the base-paired regions. The hairpin structure consisting of GCGAAAGC was not digested by the exonuclease activity of T4 DNA polymerase and it was stable enough to be only minimally bound by a single-stranded DNA binding protein.

Base Sequence↗
PubMed1990

Stabilization of mRNA in the cell-free translation system from Escherichia coli.

In the RNA directed cell-free protein synthesizing system from E. coli, there is a problem of contaminating 3'-exonucleases which attack the mRNAs. Thus, we tried the following two methods to stabilize mRNAs against nucleases: (A) To use mRNAs having hairpin structures at their termini and (B) To hybridize mRNAs with small DNA fragments to the 3'-termini of mRNAs. It was found that degradation of a mRNA was inhibited by the method B rather than the method A in the translation system.

Amino Acid Sequence↗
PubMed1989

Extraordinary stable structure of short single-stranded DNA fragments containing a specific base sequence: d(GCGAAAGC).

Short single-stranded DNA fragments carrying a GCGAAAGC sequence were found to move unexpectedly faster than other fragments of the same length in electrophoresis on a polyacrylamide gel containing a denaturing agent. The fragments were noted to have a stable structure even in 7M urea solution, but the stability cannot be explained simply on the basis of base pair formation alone. Physical characterization of the GCGAAAGC fragment indicated that it takes a hairpin-like structure in spite of the short chain length with only two G-C base pairs, comprised of GCG and AAAGC subsegments, each possessing a helical configuration independent of the others. Some biological implications of this unusual structure are discussed.

Base Composition↗
PubMed1989

Interconvertible hairpin structure found in the replication origin of phage G4 single-stranded DNA.

We found a synthetic GCGAAAGC fragment with a mobility greater than that of other oligodeoxyribonucleotides in gel electrophoresis to take on a stable hairpin structure possessing two terminal G-C base pairs. The GCGAAAGC sequence is also found in the replication origin of phage G4 single-stranded DNA, but the hairpin structure originally proposed differs from that of the GCGAAAGC fragment we have studied. Possibility of rearrangement of the secondary structure in the replication origin of phage G4 was examined in relation to its replication initiation mechanism.

Bacteriophages↗
PubMed1988

Single-stranded DNA fragments holding unusual conformation.

In the chemical synthesis of DNA, we found that the single-stranded DNA (ssDNA) fragments containing the sequence GCGAAAGC showed higher mobilities than the fragments without this sequence on a denaturing polyacrylamide gel electrophoresis. Physical structure of these DNA fragments was studied by enzyme digestion and optical analysis. The abnormal mobilities on electrophoresis seem to depend on an unusual conformation.

Base Sequence↗
PubMed1988

Synthetic oligodeoxyribonucleotides showing abnormal mobilities on polyacrylamide gel electrophoresis.

We found that synthetic DNA fragments containing a GCGAAAGC sequence showed higher mobilities than oligonucleotides without the sequence on denaturing polyacrylamide gel electrophoresis. For example, the fragment, GCGAAAGCT (9mer), showed higher mobility than the corresponding 8mer (CGAAAGCT). In addition, on Maxam-Gilbert sequencing, a 21mer containing the GCGAAAGC sequence showed an abnormal pattern, which were similar to those due to compression observed on sequencing of DNAs with high GC contents, as recently reported. It was suggested that this compression was due to the increased mobilities of the specific fragments with the GCGAAAGC sequence and that these fragments took on abnormal conformations.

Base Sequence↗
PubMed1986

Effects of modification of 4-thiouridine in E. coli tRNAfMet on methylation reaction with a thermostable Gm-methylase.

Gm-methylase isolated from an extreme thermophile, Thermus thermophilus HB 27 methylates the 2'-OH of the ribose of G18 in the consensus GG sequence in the D loop, by recognizing the D loop-and-stem structure as a minimal substrate. Modification of s4U8 of E. coli tRNAfMet with S-benzylthioisothiourea resulted in a considerable decrease in methylation activity of Gm-methylase. The effect was cancelled by reduction with beta-mercaptoethanol. However, aminoacylation activity and methylation activity of m1A-methylase were scarcely influenced by the modification. These results suggest the involvement of s4U8 residue of tRNA in the recognition of Gm-methylase.

Escherichia coli↗
PubMed1985

Solid-phase synthesis of oligoribonucleotides.

Selective deprotection of the 5'-O-dimethoxytrityl group of oligoribonucleotides required for 5'-deprotection reaction during synthesis of an oligoribonucleotide was achieved by the treatment with 1% dichloroacetic acid in dichloromethane at room temperature, without removal of the 2'-O-tetrahydropyranyl group. Phosphorylation of protected ribonucleosides and coupling reaction to the 5' end of oligoribonucleotides attached to polystyrene solid support were carried out by the use of bifunctional reagent 2-chlorophenyl-O-O-bis(1-benzotriazolyl) phosphate. In this way, trinucleotides; TpTpT, dUpdUpT, and UpUpT, were synthesized.

Chromatography, High Pressure Liquid↗
PubMed1983

Difference of the 2',5'-oligoadenylate and the 3',5'-oligoadenylate in the formation of base pairings.

The 2',5'-oligoadenylates (tetramer, pentamer, and hexamer) were synthesized and their structural properties were compared with the corresponding 3',5'-oligoadenylates. Stacking effect observed with the 2',5'-oligoadenylates was weaker than that observed with the corresponding 3',5'-oligo-adenylates. With two polyuridylate (3',5') strands, a 2',5'-hexaadenylate formed a triple strand only in the presence of Mg++ion at room temperature, while a 3',5'-hexaadenylate did even without Mg++ion.

Adenine Nucleotides↗
PubMed1983

Chemical synthesis of 2',5'-oligoribonucleotides.

Partial phenylcarbamoylation of ribonucleosides by metal carboxylates - phenyl isocyanate system was found to be induced at 2'-position with high regioselectivity in good yields, similar to that by amines - phenyl isocyanate system; these results prompted us to perform synthesis of oligoribonucleotides bearing 2'-5' phosphodiester linkage starting either from 5'-0-acetyl-2'-0-phenylcarbamoyl or from 2', 5'-bis(phenylcarbamo)yl derivatives through the sequence of reactions, i.e. tetrahydropyranylation at 3'-position, decarbamoylation and deacylation with sodium methoxide in methanol both at 2'- and 5'-positions, dimethoxytritylation at 5'-position, and phosphorylation at 2'-position with 5-chloro-8-quinolylphosphate/8-quinolinesulfonyl chloride, followed by the usual coupling procedures by Takaku et al).

Chemical Phenomena↗
PubMed1980

Dibutyltin oxide--phenyl isocyanate system for regioselective phenylcarbamoylation of the hydroxy-groups of ribonucleosides.

For partial phenylcarbamoylation of the hydroxy-groups of ribonucleosides, dibutyltin oxide--phenyl isocyanate system was found to be surperior to the bis(tributyltin) oxide--phenyl isocyanate system from the standpoint of reaction procedures including isolation of the products; the reaction was proved to occur with similar regioselectivity and to give the corresponding 5'-, 3'-, and 2'-O-phenylcarbamoyl derivatives in good yields, respectively, due to the conditions used.

Cyanates↗
PubMedDate not supplied

The limits of specificity: an experimental analysis with RNA aptamers to MS2 coat protein variants.

It has been hypothesized that selections for aptamers with high affinity for a given target molecule will of necessity identify aptamers that have high specificity for that target. We have attempted to assess this hypothesis by selecting aptamers that can bind to MS2 coat protein or to single- or double-substitution variants of the coat protein. Some aptamers selected to bind MS2 coat protein or its variants were mildly specific for their cognate targets, discriminating by two- to fourfold against closely related proteins. Specificity determinants on both the coat proteins and the aptamers could be identified. However, many aptamers could readily bind to each of the different coat proteins. The identification of such aptamer 'generalists' belies the proposed relationship between the affinities and specificities of selected RNA ligands. These results imply that, while aptamers may not finely discriminate between closely related targets, neither will their binding be negated by mutations in targets. Aptamer pharmaceuticals may therefore better resist the evolution of resistance.

Allolevivirus↗