Biomedical subjects

E Ohtsuka

Publications and source records attributed to E Ohtsuka.

At least 145 records · Page 8Linked to original sources

PubMed1992

Oligo(2'-O-methyl)ribonucleotides. Effective probes for duplex DNA.

To find novel probes for duplex DNA, we prepared four types of triplexes containing a homopurine-homopyrimidine 15-mer duplex DNA, and examined their thermal stabilities (Tm values). The single strand used for triplex formation were a DNA 15-mer having a defined C-T mixed sequence, and its sugar-modified analogs, namely 2'-fluoro DNA, RNA, and 2'-O-methyl RNA. The 2'-O-methyl RNA and the RNA-containing triplexes were similar in their enhanced stabilities at pH 6.1 and, amongst the four triplexes, the 2'-O-methyl was the most stable at pH 5.0. Furthermore, an experiment using a 34-mer duplex DNA suggested that the 2'-O-methyl RNA-triplex was destabilized, mostly as a result of the incorporation of a mismatched triplet, as compared to the DNA triplex counterpart. Thus, 2'-O-methyl RNA can serve as an effective probe for duplex DNA.

Base Sequence↗

PubMed1992

A hybrid ribonuclease H. A novel RNA cleaving enzyme with sequence-specific recognition.

A hybrid enzyme which site-specifically hydrolyzes RNA was created by covalently linking an oligodeoxyribonucleotide to Escherichia coli ribonuclease HI, an enzyme which specifically cleaves RNA moiety of DNA/RNA hybrids. A cysteine residue was substituted for Glu135 by site-directed mutagenesis in the mutant enzyme, in which all 3 free cysteine residues were replaced by alanine (Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990) Biochem. J. 271, 59-66), and coupled with a maleimide group, which is attached to the 5' terminus of the nonadeoxyribonucleotide (5'-GTCATCTCC-3') with a flexible tether. The resulting hybrid enzyme, d9-C135/RNase H, cleaved the phosphodiester bond between the fifth and sixth residues of the complementary nonaribonucleotide, without addition of the oligodeoxyribonucleotide. The nonaribonucleotide is cleaved by the wild-type or unmodified mutant enzyme only when the complementary oligodeoxyribonucleotide is present. When the kinetic parameters of the hybrid enzyme for the hydrolysis of the nonaribonucleotide were compared with those of the unmodified mutant enzyme for the hydrolysis of the nonanucleotide duplex, the hybrid enzyme exhibited a 7- and 4-fold decreases in the Km and kcat values, respectively, indicating that it performs multiple turnovers and has a sufficiently high hydrolytic activity. Hybrid ribonucleases H with various oligodeoxyribonucleotides in size and sequence, therefore, might be used as excellent tools for structural and functional studies of RNA.

Autoradiography↗

PubMed1992

X-ray structure of T4 endonuclease V: an excision repair enzyme specific for a pyrimidine dimer.

The x-ray structure of T4 endonuclease V, an enzyme responsible for the first step of a pyrimidine-dimer-specific excision-repair pathway, was determined at a 1.6-angstrom resolution. The enzyme consists of a single compact domain classified into an all-alpha structure. This single domain has two distinct catalytic activities; it functions as a pyrimidine dimer glycosylase and as an apurinic-apyrimidinic endonuclease. The amino-terminal segment penetrates between two major helices and prevents their direct contact. The refined structure suggests the residues involved in the substrate binding and the catalysis of the glycosylation reaction.

Amino Acid Sequence↗

PubMed1992

Induction of mutation of a synthetic c-Ha-ras gene containing hypoxanthine.

The second base of codon 61 of a synthetic c-Ha-ras gene was replaced with a hypoxanthine residue in a site-specific manner. Transfection of this gene into NIH3T3 cells by the calcium phosphate procedure resulted in increased focus formation. Total DNA was extracted from transformed cells, and the sequences of the inserted c-Ha-ras DNA were analyzed by the polymerase chain reaction-single-strand conformation polymorphism method. Mutations with A (or hypoxanthine) to G transition were detected exclusively. These results suggest that the synthetic c-Ha-ras gene can be used for investigations of mutagenesis caused by DNA lesions.

3T3 Cells↗

PubMed1992

Sequence-dependent cleavage of DNA by alkylation with antisense oligodeoxyribonucleotides containing a 2-(N-iodoacetylaminoethyl)thio-adenine.

Antisense oligodeoxyribonucleotides (15mers), containing a 2-(N-iodoacetylaminoethyl)thio-adenine, were synthesized and tested for their ability to cleave complementary DNAs (21mers). Cleavage of the target DNAs was done by alkylation followed by treatment with piperidine, and the positions of the alkylated sites were estimated by identification of the cleaved products. By using several combinations of the modified strands and their target DNAs, it was determined that alkylation occurred at adenine or guanine, depending on the torsion angle of the modified nucleoside.

Adenine↗

PubMed1992

Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with non-cognizable ribonucleotide, 2'AMP, and its comparison with a specific complex with 2'GMP.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.

Adenosine Monophosphate↗

PubMed1992

Recognition of bases in Escherichia coli tRNA(Gln) by glutaminyl-tRNA synthetase: a complete identity set.

The fidelity of protein biosynthesis rests largely on the correct aminoacylation of transfer RNAs by their cognate aminoacyl-tRNA synthetases. Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system for studying the basis of this highly specific recognition process. Correct aminoacylation depends on the set of nucleotides (identity elements) in tRNA(Gln) responsible for correct interaction with GlnRS. Specific contacts between tRNA(Gln) and GlnRS include the 2-amino group of guanosines. Therefore, we made a set of tRNA(Gln) variants in which specific guanosines were replaced by inosine using recombinant RNA technology. This resulted in a set of tRNAs that varied by single deletions of the amino group from guanine residues, thus allowing us to test the functional importance of these contacts. In addition, a number of mutants were made by transcription of mutated tRNA genes with base changes at position 10, 16 or 25. In vitro aminoacylation of these mutants showed decreases in the specificity constant (kcat/KM) of up to 300-fold, with kcat being the parameter most affected. These experiments reveal G10 as a new element of glutamine identity. In addition, the interaction of G2, G3 and G10 with GlnRS via the 2-amino group is significant for tRNA discrimination. Based on these results, and on earlier data, we propose a complete set of bases as identity elements for tRNA(Gln).

Amino Acid Sequence↗

PubMed1992

Ubiquitous presence in mammalian cells of enzymatic activity specifically cleaving 8-hydroxyguanine-containing DNA.

Here we report the finding of enzymatic activity that specifically cleaves DNA containing 8-hydroxyguanine (oh8Gua) residues in various mammalian cells. To detect this activity, we used a synthetic double-stranded DNA containing a single oh8Gua at a defined position as the substrate, and analyzed the products of enzymatic digestion by polyacrylamide gel electrophoresis. Two cleavage sites near the oh8Gua residue were detected with partially purified fractions from cow brain and rat liver, and also with preparations from all mammalian tissues examined. These results suggest that enzymatic activity for the removal of oh8Gua from DNA is widely distributed in mammalian cells.

Animals↗

PubMed1992

Cross-ligation and exchange reaction of RNA catalyzed by hairpin ribozymes.

The catalytic domain in the minus strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) assumes a hairpin-like secondary structure. This ribozyme catalyzes a cross-ligation reaction between substrate RNAs of different lengths. We constructed ribozymes to probe the activities of ligation and RNA fragment exchange.

Base Sequence↗

PubMed1992

[Upper gastrointestinal endoscopic findings and gastric mucosal blood flow in patients with rheumatoid arthritis].

In order to clarify the mechanism of peptic ulceration complicating rheumatoid arthritis (RA), the endoscopic examination of the stomach and duodenum, and the measurement of gastric mucosal blood flow (GMBF, ml/min/100 g) were performed in 49 patients with RA, in relation to the clinical variables (age, sex, disease duration, anatomical stage, functional class and disease activity) and drugs administered. Most of peptic ulcer (88%, 22/25) was found in the gastric mucosa in RA patients, of which antral ulcer occupied 82% (18/22). GMBF in the antrum was significantly lower in RA patients with antral ulcer than in those with normal gastric findings, while GMBF in the gastric body was rather higher in either patients with antral or other gastric ulcer, especially in patients with the angle or body ulcer. The prevalence of antral ulcer tended to be higher in females than in males, and GMBF in the antrum was significantly lower in the former than in the latter (42.7 +/- 11.0 vs 55.6 +/- 12.6). There was no relationship between the prevalence of antral ulcer or GMBF and the activity of RA. All the patients except 4 had received non-steroidal anti-inflammatory drugs (NSAIDs), and of these 45 patients, 24 had also steroids. The prevalence of antral ulcer was higher in patients taking NSAIDs and steroids more than 5mg prednisolone concomitantly than in those taking NSAIDs only, although there was no difference in GMBF in the antrum between them. The prevalence of antral ulcer in patients taking NSAIDs, according to the kind of NSAIDs taken, was the highest in phenylacetic acids group. The difference was significant between propionic acids group and phenylacetic acids group. GMBF in the antrum of phenylacetic acids group was the lowest among the three groups (34.7 +/- 7.1 in phenylacetic acids, 46.6 +/- 11.2 in oxicams and 47.7 +/- 12.6 in propionic acids). Those results indicate that long-term administration of NSAIDs may selectively inhibit GMBF in the antrum, leading to impairment of the protective mechanism in the gastric wall and subsequent occurrence of antral ulcer.

Aged↗

PubMed1991

Mutagenesis and self-ligation of the self-cleavage domain of the satellite RNA minus strand of tobacco ringspot virus and its binding to polyamines.

Several mutants for the minus strands of the self-cleaving domain of the satellite RNA of tobacco ringspot virus have been synthesized by joining chemically synthesized oligoribonucleotides with RNA ligase. Kinetic properties of the enzyme strands (50 nucleotides) against substrates (15-mer and 18-mer) were investigated. Structural properties of the unpaired part in the cleavage region were estimated from mutagenesis. The catalytic domain alone was proved to be responsible for the rejoining reaction of cleaved substrates. It was also found that the ribozyme could be divided into two strands without loss of activity. Effects of concentration of magnesium ion and polyamines on the cleavage reaction for the two-stranded ribozyme are also reported.

Base Sequence↗

PubMed1991

How does RNase H recognize a DNA.RNA hybrid?

The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA.RNA hybrid Escherichia coli RNase H complex. Site-directed mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out. A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively. In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme. The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model. Moreover, the specific cleavage of the RNA strand in DNA.RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes. A mechanism of enzymatic action is proposed.

Amino Acid Sequence↗

PubMed1991

NMR studies for identification of dI:dG mismatch base-pairing structure in DNA.

One- and two-dimensional NMR experiments have been undertaken to investigate deoxyinosine:deoxyguanosine (dI:dG) base pairing in a self-complementary dodecadeoxyribonucleotide, d(C1-G2-C3-I4-A5-A6-T7-T8-G9-G10-G11-G12) (designated IG-12), duplex. The NMR data indicate formation of a dI(syn):dG(anti) base pair in a B-DNA helix. This unusual base pairing results in altered NOE patterns between the base protons (H8 and H2) of the I4 residue and the sugar protons of its own and the 5'-flanking C3 residues. The dI(syn):dG(anti) base pair is accommodated in the B-DNA duplex with only a subtle distortion of the local conformation. Identification of the dI:dG base pairing in this study confirms that a hypoxanthine base can form hydrogen-bonded base pairs with all of the four normal bases, C, A, T, and G, in DNA.

Base Composition↗

PubMed1991

Non-cognizable ribonucleotide, 2'AMP, binds to a mutant ribonuclease T1 (Y45W) at a new base-binding site but not at the guanine-recognition site.

Complex of a mutant ribonuclease T1 (Y45W) with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined by X-ray diffraction at 1.7 A resolution. The 2'AMP molecule locates at a new base-binding site which is remote from the guanine-recognition site, where 2'GMP was found to be bound. The nucleotide adopts the anti conformation of the glycosidic bond and C3'-exo sugar pucker. There exists a single hydrogen bond between the adenine base and the enzyme, and, therefore, the site found is apparently a non-specific binding site. The results indicate that the binding of 2'AMP to the guanine-recognition site is weaker than that to the new binding site.

Adenosine Monophosphate↗

PubMed1991

An endonuclease activity in human polymorphonuclear neutrophils that removes 8-hydroxyguanine residues from DNA+.

An endonuclease that specifically removes 8-hydroxyguanine (oh8Gua) from DNA has been isolated from Escherichia coli. As the amount of oh8Gua produced in DNA of X-ray-irradiated mice is known to decrease with time after irradiation, an attempt was made to find a similar activity in human polymorphonuclear neutrophils (PMNs) using a synthetic dsDNA containing oh8Gua as a substrate. The PMN enzyme was isolated free of other DNases, and found to cleave the substrate DNA simultaneously at 2 sites, the phosphodiester bonds 5' and 3' to oh8Gua, producing free hydroxyl and phosphate groups, respectively. The enzyme showed almost no activity on DNAs containing other kinds of modified base tested or mismatched DNA. Thus human cells also contain an endonuclease that specifically removes oh8Gua residues from DNA.

Base Sequence↗

PubMed1991

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.

Base Sequence↗

PubMed1991

Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding.

Mg2+ is important for the RNase activity of the hammerhead ribozyme. To investigate the binding properties of Mg2+ to the hammerhead ribozyme, cleavage rates and CD spectra for substrates containing inosine or guanosine at the cleavage site were measured. The 2-amino group of this guanosine interfered with the rate of the cleavage reaction and did not affect the amount of Mg2+ bound to the hammerhead RNA. The kinetics and CD spectra for chemically synthesized oligoribonucleotides with a Sp or Rp phosphorothioate diester bond at the cleavage site indicated that 1 mol of Mg2+ binds to the pro-R oxygen of phosphate. The binding constant for Mg2+ was about 10(4) M-1, which represents outer-sphere complexation. The hammerhead ribozyme catalyzes the cleavage reaction via an in-line pathway. This mechanism has been proved for RNA cleavage by RNase A by using a modified oligonucleotide that has an Sp phosphorothionate bond at the cleavage site. From these results, we present the reaction pathway and a model for Mg2+ binding to the hammerhead ribozyme.

Amines↗

PubMed1991

NMR studies of a DNA containing 8-hydroxydeoxyguanosine.

The effects of hydroxylation at the C8 of a deoxyguanosine residue in DNA were studied by NMR analysis of a self-complementary dodecanucleotide, d(C1-G2-C3-oh8G4-A5-A6-T7-T8-C9-G10-C11-G12), which has an 8-hydroxy-2'-deoxyguanosine (oh8dG) residue at the 4th position. NMR data indicate that the 8-hydroxyguanine (oh8G) base takes a 6,8-diketo tautomeric form and is base-paired to C with Watson-Crick type hydrogen bonds in a B-form structure. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(CGCGAATTCGCG) duplex. The structural changes caused by 8-hydroxylation of the deoxyguanosine, if any, are localized near the modification site.

8-Hydroxy-2'-Deoxyguanosine↗