Biomedical subjects

R Kiyama

Publications and source records attributed to R Kiyama.

At least 37 records · Page 2Linked to original sources

PubMed1996

In vitro transcription of a poly(dA) x poly(dT)-containing sequence is inhibited by interaction between the template and its transcripts.

Transcription of poly(dA) x poly(dT)-containing sequences was investigated in vitro using plasmids carrying a (dA)34 x (dT)34 tract in the coding region of the lacZ gene. The efficiency of transcription of the (dT)34 sequence on the transcribing strand by Escherichia coli RNA polymerase was substantially lower (approximately 60%) than that of the (dA)34 sequence or of the control lacZ gene. Analysis of the transcription process of the (dT)34 sequence by T3 RNA polymerase showed that the transcription was frequently arrested or terminated at the middle as well as immediately proximal of the (dA)34 x (dT)34 tract, and it occurred more prominently following accumulation of transcription products. This inhibition was strongly enhanced by the addition of the oligonucleotide (dT)34 or poly(U) to the reaction mixture, while (dA)34 and the duplex (dA)34 x (dT)34 suppressed the inhibition. A similar transcriptional inhibition was also observed in transcription mediated by T7 RNA polymerase and eukaryotic RNA polymerase II. We also demonstrated RNA x DNA complex formation of the (dA)34 x (dT)34 tract with poly(U), but not with poly(A). These findings strongly suggest that poly(dT)-containing template sequences interact and form a complex with its transcription products, possibly an RNA x DNA triplex, which blocks further transcription. This would explain the instability of the plasmids transcribing mRNAs with poly(U) but not poly(A) tracts and the underrepresentation of poly(U) but not poly(A) tracts in mRNAs.

DNA↗

PubMed1996

Conservation and periodicity of DNA bend sites in eukaryotic genomes.

DNA bend sites appear every 680 bp on average in the human epsilon- and beta-globin gene regions. Although most of their molecular nature has not been unraveled, a potential bend core sequence A2N8A2N8A2 (A/A/A) and its complementary T2N8T2N8T2 (T/T/T) appeared preferentially either in or very close to most of the bend sites, whereas other combinations of A2 and T2 dinucleotides, A/T/T + A/A/T, T/T/A + T/A/A and A/T/A + T/A/T, did not. The distances between any two of the core sequences in the entire beta-globin locus showed a strong bias to a length of 701-800 bp and multiples thereof, suggesting that there is periodicity throughout the locus. This bias was not found for other combinations of A2 and T2. Again, this periodicity was identified in many eukaryotic genes, whereas the tendency was absent in mRNAs and prokaryotic as well as viral genomes.

Animals↗

PubMed1996

Construction of highly extensive polymorphic DNA libraries by in-gel competitive reassociation procedure.

Differential genomic DNA libraries between two mouse strains and from two human individuals were constructed by means of the in-gel competitive reassociation (IGCR) procedure, a procedure developed for cloning altered anonymous restriction fragments. The libraries were highly enriched in RFLP fragments, approximately 60 and 40% for the mouse and human libraries, respectively, and, more importantly, maintained most of the original complexities of the RFLP fragments. Therefore, differential genomic DNA libraries constructed by the IGCR procedure, particularly for human genomic DNA, should offer highly extensive sources for polymorphic DNA sequences necessary for a variety of genome analyses, including studies on the origin and mechanism of biological diversity among the same species.

Animals↗

PubMed1996

An intrachromosomal repeating unit based on DNA bending.

DNA bending has been observed in conjunction with transcription, replication, and recombination. Furthermore, nucleosomes in eukaryotic cells are positioned through DNA bending, suggesting an active role for DNA bending in the chromosome organization. We reported previously that DNA bend sites appear every 680 bp in the human epsilon- and beta-globin gene regions. Here we showed that these sites are present at an interval of roughly 700 bp in the G gamma-A gamma-psi beta-globin gene region and that they divide the region into units. They were conserved in the promoter regions of nearly all beta-like globin genes and between human beta- and mouse beta maj-globin genes, although the periodicity of the sites was locally disturbed at the junctions of the duplicated G gamma- and A gamma-globin genes and in their second introns. This suggested that the periodicity is ranked lower in the hierarchy of genomic DNA organization than genome rearrangement and gene expression. A close inspection of one of the sites in the A gamma-globin gene region indicated that a 20-bp sequence containing periodic short (dA)n tracts was partly responsible for the bending. This sequence was shown to phase nucleosomes in this region by preferential binding to the core histones.

Animals↗

PubMed1995

Preference of the recombination sites involved in the formation of extrachromosomal copies of the human alphoid Sau3A repeat family.

The human alphoid Sau3A repetitive family DNA is one of the DNA species that are actively amplified to form extrachromosomal circular DNA in several cell lines. The circularization takes place between two of the five approximately 170 bp subunits with an average of 73.1% homology as well as between identical subunits. To investigate the nature of the recombination reaction, we cloned and analyzed the subunits containing recombination junctions. Analysis of a total of 68 junctions revealed that recombination had occurred preferentially at four positions 10-25 (A), 40-50 (B), 85-90 (C) and 135-160 (D) in the 170bp subunit structure. Two regions (B and C) were overlapped with the regions with higher homology between subunits, while other two regions (A and D) cannot be explained solely by the regional homology between the subunits. These regions were located at both junctions of the nucleosomal and the linker region, and overlapped with the binding motifs for alpha protein and CENP-B. Approximately 90% of the recombination occurred between the subunits located next but one (+/- 2 shift), although the frequency of recombination between the adjoining subunits (+/- 1 shift) was approximately 10%.

Base Sequence↗

PubMed1995

Construction of libraries for methylation sites by in-gel competitive reassociation (IGCR).

The in-gel competitive reassociation (IGCR) procedure was successfully applied to construct a comprehensive library enriched in DNA fragments containing C5mCGG sequences from mouse liver and brain genomic DNA. For IGCR, methylation-insensitive restriction enzyme (Msp I) digests were used as target DNA and methylation-sensitive restriction enzyme (Hpa II) digests as competitor DNA. Southern blot analysis indicated that 60 to 70% of the clones in the library were derived from the methylated sites and overall enrichment was 200- to 1000-fold. IGCR was further applied to construct a library for the sites differentially methylated between brain and liver DNA. In the library, approximately 20% of the Hpa II sites exhibited different degrees of methylation between these tissues.

Animals↗

PubMed1995

Novel angiotensin II receptor antagonists. Design, synthesis, and in vitro evaluation of dibenzo[a,d]cycloheptene and dibenzo[b,f]oxepin derivatives. Searching for bioisosteres of biphenylyltetrazole using a three-dimensional search technique.

Three-dimensional substructure searching (3D search), using the program MACCS-3D, was utilized for designing novel angiotensin II receptor antagonists which contain a bioisostere of the biphenylyltetrazole moiety of DuP 753. A 3D query was prepared from an overlay model of substructures of several potent AII antagonists. The search system retrieved 139 compounds from the database MDDR-3D, which consisted of 29,400 medicinal patent compounds. A tricyclic compound was selected from the retrieved compounds and then evolved by considering steric fitness to the overlay model and synthetic feasibility. Finally, various novel AII antagonists having dibenzo[a,d]cycloheptene or dibenzo[b,f]oxepin were designed and synthesized. The receptor binding activity (Ki) for several members of this series was in the 10(-10) M range, demonstrating the ability of 3D search technique to explore new lead structures.

Angiotensin II↗

PubMed1995

Conservation and periodicity of DNA bend sites in the human beta-globin gene locus.

A total of seven DNA bend sites were mapped in the 4.4-kilobase human beta-globin gene region by the circular permutation assay. The periodicity of these sites (except one) was about every 700 (average 685.5 +/- 267.7) base pairs. All of the sites contained the sequence feature of short poly(dA) tracts, which are typical of DNA bending. The relative positions of the sites to the cap site were identical to those in the epsilon-globin gene region, suggesting that the bend sites were conserved during molecular evolution of the two globin genes. To explain this periodicity and conservation of the sites within the evolutionary unstable noncoding regions, we focused upon the appearance of a potential bend core sequence, A2N8A2N8A2 (A/A/A), and its complement, T2N8T2N8T2 (T/T/T). These sequences appeared in or very close to most of the bend sites of the globin gene regions, whereas other A+T-rich sequences or candidates for DNA bending did not. The distances between any two of the core sequences in the entire beta-globin locus showed a strong bias to a length of about 700 base pairs and its multiples, suggesting that the periodicity exists throughout the locus. The data presented here strengthen the idea of sequence-directed nucleosome phasing.

Base Sequence↗

PubMed1995

Kinetic studies on the interaction of nonlabeled antagonists with the angiotensin II receptor.

Angiotensin AT1 receptor antagonists are divided into two types, surmountable and insurmountable, based on the way they inhibit angiotensin II-induced vasoconstriction. To elucidate what causes the difference, we studied how antagonists associate with and dissociate from AT1 receptor sites in rat liver membranes. Three antagonists, 6-propyl-7-oxo-4[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-4,7- dihydropyrazolo[1,5-a]pyrimidine-3-carboxylic acid (SRL1080227), 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylic acid (CV-11974) and 2-butyl-3-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo- [4,5-b]pyridine (FK739), showed competitive antagonism when they were added simultaneously with [125I]angiotensin II, but CV11974 and SRL1080227 showed apparently noncompetitive antagonism when membranes were preincubated with each antagonist. The longer the preincubation time with CV11974 or SRL1080227 was, the more effectively the antagonist inhibited [125I]angiotensin II binding, while the inhibition by FK739 did not change with the preincubation time. To estimate their dissociation rate from the receptor binding site, we studied [125I]angiotensin II binding to membranes which had been preincubated with each antagonist and washed twice. Membranes pretreated with FK739 completely recovered the ability to bind [125I]angiotensin II with a period of 60 min, while membranes preincubated with CV11974 did not read this level of recovery. [125I]angiotensin II binding to membranes preincubated with SRL1080227 increased gradually, but did not reach the control level during the experiment. The kinetic properties of SRL1080227, CV11974 and FK739 were consistent with their characteristic modes in inhibiting angiotensin II-induced contraction of isolated rabbit aorta and decreasing blood pressure of spontaneously hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin Receptor Antagonists↗

PubMed1995

Analysis of recombination junctions in extrachromosomal circular DNA obtained by in-gel competitive reassociation.

Essentially all eukaryotic cells contain circular extrachromosomal DNA as a result of excision from the chromosomes. To obtain insight into the nature of recombination associated with the occurrence of such DNA species and its biological significance, we analyzed a library enriched in recombination junctions which was constructed by a novel DNA subtraction technique; in-gel competitive reassociation (IGCR). Furthermore, we also introduced inverse PCR to characterize chromosomal DNA fragments containing the recombination junctions. At least 45% of the clones in the library constructed by the IGCR procedure comprised DNA with recombination junctions. Nucleotide sequence analysis of the recombination junctions indicated that three of four extrachromosomal DNAs thus analyzed were produced through recombination between sequences with a 3-5 bp homology in the chromosomes. One extrachromosomal DNA was apparently generated through non-homologous recombination, possibly by end-to-end joining. These results have demonstrated the usefulness of IGCR in concentrating recombination junctions, which provide the most direct evidence for the mechanism of the recombinational events involved, from highly complex genomes.

Base Sequence↗

PubMed1995

Construction of a human genomic library of clones containing poly(dG-dA).poly(dT-dC) tracts by Mg(2+)-dependent triplex affinity capture. DNA polymorphism associated with the tracts.

Microsatellite DNA is a useful tool for detecting DNA polymorphisms among species or individuals, especially those among closely related individuals. We constructed a library of clones that contained poly(dG-dA).poly(dT-dC) tracts from human genomic DNA by Mg(2+)-dependent triplex DNA formation. Examination of triplex DNA formation in the presence of various metal ions Mg2+, Mn2+, or Zn2+ revealed that the procedure worked best in the presence of Mg2+. Affinity enrichment was performed with AluI-digested chromosomal DNA mixed with biotinylated (dG-dA)17 in the presence of Mg2+. A library constructed after three cycles of affinity enrichment showed that over 80% of the clones contained at least one poly(dG-dA).poly(dT-dC) tract. Most of them contained a perfect (dG-dA)n repeat 30-84 base pairs in length, while some contained variants such as (dC-dT)10-(dC)-(dC-dT)9. Using the clones from the library as a probe, we detected DNA polymorphisms associated with the repeat length of the tracts in the Japanese population. We also detected a microsatellite instability among the tracts in a cancer tissue sample.

Base Sequence↗

PubMed1995

Protection of DNA sequences by triplex-bridge formation.

We have demonstrated that the DNA sequence between two triplex-forming polypurine.polypyrimidine (Pu.Py) tracts was protected from DNA modifying enzymes upon formation of triplex DNA structures with an oligodeoxyribonucleotide in which two triplex-forming Pu or Py tracts were placed at the termini (triplex-bridge formation). In model experiments, when two triplex structures were formed between double-stranded DNA with the sequence (AG)17-(N)18-(T)34, and an oligodeoxyribonucleotide, (T)34-(N)18-(GA)17, not only the Pu.Py tracts but also the 18 bp non-Pu.Py sequence in the duplex DNA between the tracts was protected from restriction enzymes, HpaII methylase and DNase I. This protection occurred only when both of the Pu.Py tracts were involved as triplexes. The length of the tracts could be as short as 21 bp, while the difference in length between the non-Pu.Py sequences on the duplex and the oligodeoxyribonucleotide should be within 10 nucleotides. The efficiency of protection was enhanced in the presence of a cationic detergent, cetyltrimethylammonium bromide, during triplex formation. Protection was also observed with another type of the triplex bridge formed between (G)34 and (T)34 tracts with an oligodeoxyribonucleotide, (T)34-(N)20-(G)34. These findings suggest that the protection of specific DNA sequences from enzymes by triplex-bridge formation can be applied to any DNA sequence by placing it between two triplex-forming sequences.

Base Sequence↗

PubMed1995

A differential cloning procedure for rearranged or altered genomic DNA based on in-gel competitive reassociation.

We have developed a substantially improved differential cloning procedure designed for cloning anonymous altered restriction DNA fragments from higher organisms. The improvements include (i) in-gel dissociation and reassociation of biotinylated restriction digests of target DNA fragments, (ii) replacement of agarose gel by a synthetic gel material for electrophoresis, (iii) use of a reassociation enhancing reagent (CTAB) for in-gel reassociation, and (iv) introduction of PCR. After several cycles of IGCR, we attained considerable enrichment of altered or rearranged DNA fragments which were originally present at one copy or less per complex eukaryotic genome. Examples of enrichment include those of an exogenously added DNA fragment, a chromosomal DNA sequence that has undergone a deletion, and DNA fragments containing a recombination junction.

Animals↗

PubMed1995

Synthesis and evaluation of novel nonpeptide angiotensin II receptor antagonists: imidazo[4,5-c]pyridine derivatives with an aromatic substituent.

Starting from recently reported nonpeptidic angiotensin II (AII) receptor antagonists, we have designed and prepared a new series of 6-arylimidazo[4,5-c]pyridine derivatives. Variation of phenyl groups at the 4-, 6- or 7-position of imidazo[4,5-c]pyridine showed that substitution at the 6-position resulted in receptor-binding activity almost as potent as that of DuP 753. This led to synthesis and evaluation of an extensive series of 6-aryl-imidazo[4,5-c]pyridine derivatives. Some of them were 4-fold more potent in vitro than DuP 753, but only showed weak antihypertensive activity in vivo when given orally to rats.

Angiotensin Receptor Antagonists↗

PubMed1995

Synthesis and evaluation of novel pyrazolo[1,5-alpha]pyrimidine derivatives as nonpeptide angiotensin II receptor antagonists.

A novel series of 6-alkyl-7-oxo-4,7-dihydropyrazolo[1,5-alpha]pyrimidine-3-carboxyli c acid derivatives was prepared as angiotensin II (AII) receptor antagonists. When evaluated in an in vitro binding assay using COS cells transfected with a cDNA encoding a human AT1 angiotensin II receptor, the compounds in this series showed Ki values in the range of 0.4-4.0 nM. In anesthetized spontaneously hypertensive rats (SHRs), administration of the 6-propyl derivative 4d (1 mg/kg, i.v.) reduced the mean blood pressure (MBP) by a maximum of more than 30 mmHg from the normal value.

Angiotensin II↗

PubMed1994

Instability of plasmid DNA maintenance caused by transcription of poly(dT)-containing sequences in Escherichia coli.

A series of pUC19-derived plasmids which contain a (dA)34 x (dT)34 tract was constructed to examine the effect of this sequence on plasmid maintenance in Escherichia coli. When this sequence was placed downstream from the lacZ promoter (lacZp), plasmids transcribing the (dT)34 strand were rapidly lost from the cells irrespective of the orientation relative to the replication origin, while plasmids transcribing the (dA)34 strand were stably maintained. A plasmid with a deletion within the lacZp region, preventing transcription from the (dT)34 strand, exhibited no such instability. The apparent transcription-dependent plasmid instability was not observed for the TcR (tetracycline-resistance gene) promoter which has weaker activity than lacZp. Similar strand-specific instability was observed for another microsatellite sequence, (dG)34 x (dC)34. These findings suggest that accumulation of transcription products of the poly(dT) sequences could block DNA replication.

Escherichia coli↗

PubMed1994

Periodicity of DNA bend sites in human epsilon-globin gene region. Possibility of sequence-directed nucleosome phasing.

Analysis by the circular permutation assay of the human epsilon-globin gene region revealed that the DNA bend sites were located every 682.5 +/- 132.0 base pairs on average, separating the region into domains. Among 10 major and 1 minor bend sites mapped in the region, the transcription initiation and termination sites of the epsilon-globin gene were located close to the bend sites, and the first and the second exons of the epsilon-globin gene were separated from the third exon by another site. The bend sites were also located anterior to the two Alu family sequences. Short poly(dA).poly(dT) tracts typical for DNA bending were not always present in the sites. Fine mapping of a bend site having no poly(dA).poly(dT) tracts with concatenated oligonucleotides and analysis by S1 nuclease nicking assay indicated that the unusual structure, a base slippage or a partial triplex DNA structure, formed by a polypurine.polypyrimidine sequence in the region is the basis of bending. The bend sites were mapped in the promoter region (within approximately 300 base pairs from the cap site) of the human beta-globin and in c-myc and erythropoietin receptor genes, as well as in the mouse beta maj-globin gene. The conservation and the periodicity of the bend sites in the noncoding region suggest the active role of the sites that is a signal for nucleosome phasing.

Animals↗

PubMed1994

Enrichment of human DNAs that flank poly(dA).poly(dT) tracts by triplex DNA formation.

Human DNA fragments which contain poly(dA).poly(dT) tracts and their immediate flanking regions were enriched by means of triplex DNA formation. Human DNA fragments were mixed with biotinylated (dT)34 in the presence of Mg2+ and the triplex DNA [(dT)34.poly(dA).poly(dT)] was adsorbed onto streptavidin-coated magnetic beads and the DNA fragments which formed triplexes were eluted from the beads with a buffer containing EDTA. A control experiment using a plasmid with a poly(dA).poly(dT) tract indicated that DNA fragments with the tract could be enriched over 60-fold after one cycle of the treatment. After PCR amplification, the sample was subjected to the next cycle of the affinity enrichment. After four cycles, we obtained a human genomic DNA library of clones with inserts ranging from 500 to 1000 bp among which 86% had at least one poly(dA).poly(dT) tract. No less than 60% of the clones were close or distant members of the Alu family. Sequence determination of 25 clones revealed that the length of the poly(dA).poly(dT) tracts was 14 to 37 (average 28) bp and that they were located close to either end of the fragments. While 15 clones were identified as Alu family homologues and one as a human L1 family member, nine clones were of unknown origin. None of these nine clones were a highly repeated sequence nor a part of transcriptionally active regions.

Bacterial Proteins↗