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Y Kohwi

Publications and source records attributed to Y Kohwi.

27 records · Page 2Linked to original sources
PubMed1990

Hierarchical binding of DNA fragments derived from scaffold-attached regions: correlation of properties in vitro and function in vivo.

On its upstream side, the human interferon-beta gene is flanked by a 7-kb SAR (scaffold-attached region) DNA element. The core of this element is determined and subjected to in vitro reassociations with isolated scaffolds. Binding properties of SAR fragments with decreasing length are quantified and related to consensus sequences like the topoisomerase II box and an ATATTT motif. Characteristics as the stoichiometry, affinity, and cooperativity of the binding process are shown to depend on the length of SAR DNA and suggest a model involving a multiple-site attachment to protein scaffolds. We propose a rational approach for predicting the SAR mediated transcriptional enhancements in vivo from their binding properties in a standardized in vitro assay. The efficiency of this approach is demonstrated for a marker (huIFN-beta) and a selector gene (neor).

Animals↗
PubMed1989

Cationic metal-specific structures adopted by the poly(dG) region and the direct repeats in the chicken adult beta A globin gene promoter.

Naturally occurring contiguous deoxyguanine residues and their surrounding sequences in the chicken adult beta A globin gene promoter were analyzed for their inherent potential to adopt non-B DNA structures in supercoiled plasmid DNA. In particular, cationic effects on structure were studied by treating the supercoiled plasmid DNA harboring the chicken adult beta A globin 5' flanking sequence with an unpaired DNA base-specific probe, chloroacetaldehyde in the presence of either Mg++, Cu++, Zn++, Ca++ or Co++ ions. The chloroacetaldehyde-reactive bases were mapped at a single base resolution by a chemical cleavage method that specifically cleaves DNA at the chloroacetaldehyde modified sites. These experiments revealed that while Mg++ and Ca++ ions induce a dG.dG.dC triple helix structure at the contiguous dG residues, Zn++, Cu++ and Co++ ions induce yet another structure at the direct repeats immediately 5' of the dG residues. When Mg++ and Zn++ ions are both present, Zn++ inhibits the dG.dG.dC triplex at the contiguous dG residues and induces a particular non-B DNA structure at the adjacent direct repeats. The specific induction of non-B DNA structures by metal ions at the two adjacent sequences within the promoter region may be of biological significance.

Animals↗
PubMed1989

Non-B DNA structure: preferential target for the chemical carcinogen glycidaldehyde.

The effect of DNA conformation on the reaction specificities of the chemical carcinogen glycidaldehyde (GDA) was examined. Supercoiled plasma DNA harboring a poly(dG)-poly(dC) tract, which folds sharply into halves from the center of the tract to form a tetra-stranded structure containing either a dG.dG.dC triplex structure in the presence of Mg2+ or a dC+.dC.dC triplex structure in the absence of Mg2+ was chosen as the reaction substrate. The reactive sites alkylated by GDA were determined at a single base resolution after these sites were specifically cleaved with a combination of either the hydrazine and piperidine or formic acid and piperidine reactions. The results show that at pH 5-7, GDA reacts preferentially with DNA bases that are involved in the altered DNA conformations. Interestingly, in addition to the known reaction of GDA with guanine residues, it was also found to be highly reactive with specific cytosine residues that reside in the altered DNA conformations. These GDA-reactive cytosine residues were unpaired as judged by their reactivity with the unpaired DNA base specific probe, chloroacetaldehyde. Therefore, it appears that DNA conformation plays a major role in determining the reaction specificities of GDA.

Aldehydes↗
PubMed1988

Magnesium ion-dependent triple-helix structure formed by homopurine-homopyrimidine sequences in supercoiled plasmid DNA.

DNA can be chemically cleaved at the site of chloroacetaldehyde-modified residues by the chemicals used for Maxam-Gilbert sequencing reactions. Use of this technique facilitates fine structural analysis of unpaired DNA bases in DNA with non-B-DNA structure. This method was used to study the non-B-DNA structure adopted by the poly-(dG).poly(dC) sequence under torsional stress at various ionic conditions. In the presence of 2 mM Mg2+, the 5' half of the deoxycytosine tract is very reactive to chloroacetaldehyde, while the 3' half is virtually unreactive. In the poly(dG) tract, chloroacetaldehyde reaction is restricted to the center guanine residues. In the absence of Mg2+, however, it is the 5' half of the deoxyguanine tract that is reactive to chloroacetaldehyde, while the 3' half is unreactive. And chloroacetaldehyde reaction is restricted to the center cytosine residues in the poly(dC) stretch. These results strongly suggest that the poly(dG).poly(dC) sequence is folded into halves from the center of the sequence to form a tetra-stranded-like structure. Such a structure contains either a triplex consisting of poly(dG).poly(dG).poly(dC) strands in the presence of Mg2+ or a triplex consisting of poly(dC).poly(dG).poly(dC) strands in the absence of Mg2+. The fourth strand, not involved in triplex formation, is closely associated with the triplex and is positioned in such a way that DNA bases are exposed and freely accessible to the chloroacetaldehyde reaction.

Acetaldehyde↗
PubMed1988

An ultimate chemical carcinogen, N-acetoxy-2-acetylaminofluorene, detects non-B DNA structures that are reactive with chloroacetaldehyde in supercoiled plasmid DNA.

A model ultimate carcinogen, N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF), reacts with specific DNA sites in supercoiled plasmid DNA that assume non-B DNA structures. The reaction was studied using supercoiled plasmid DNA harboring either inverted repeats or poly(dG)--poly(dC) sequences, the sequences which are known to adopt non-B DNA structure when under torsional stress. The sites of modification were determined by first digesting the chemically treated DNA with a restriction enzyme, and then by digesting the site of modification with S1 nuclease. Southern blot analysis of resulting DNA fragments revealed that N-acetoxy-AAF detects non-B DNA structures in common with another chemical carcinogen, chloroacetaldehyde, which reacts specifically with unpaired adenine and cytosine residues. These results suggest that specific DNA sites with unpaired DNA bases in supercoiled plasmid DNA, and possibly similar structures in chromatin, are hot-spots for certain chemical carcinogen attack.

2-Acetylaminofluorene↗
PubMed1987

Unusual conformational effect exerted by Z-DNA upon its neighboring sequences.

Supercoiled plasmid DNA harboring an insert of (dG-dC)16, a sequence known to form Z-DNA upon negative supercoiling, was reacted with chloroacetaldehyde. Chloroacetaldehyde, like bromoacetaldehyde, was found to be a specific probe for detecting unpaired DNA bases in supercoiled plasmid DNA. Under torsional stress (at bacterial superhelical density), chloroacetaldehyde reacted at multiple discrete regions within the neighboring sequences of the (dG-dC)16 insert. When the plasmid population was considered as a whole, the distribution of the chemically reactive bases exhibited a pattern of inversion symmetry with the center of inversion in the middle of the (dG-dC)16 insert. However, when a single supercoiled plasmid molecule was considered, chloroacetaldehyde reacted with only one of the neighboring sequences, either 5' or 3' of the (dG-dC)16 insert, but not with both. The possibility that the supercoiled plasmid DNA is in equilibrium with these two structural forms is discussed.

Acetaldehyde↗
PubMed1985

Poly(dG)-poly(dC) sequences, under torsional stress, induce an altered DNA conformation upon neighboring DNA sequences.

Supercoiled plasmid DNAs (at bacterial superhelical density) harboring the homopurine-homopyrimidine sequence, poly(dG)-poly(dC), were reacted with bromoacetaldehyde (BAA), a reagent that reacts with unpaired DNA bases. Not only did the poly(dG)-poly(dC) sequence react with BAA but, surprisingly, neighboring sequences located 3' to the contiguous G sequences also reacted. The altered conformation in the poly(dG)-poly(dC) sequence and in the neighboring sequence occurred in the same supercoiled plasmid DNA molecule. Furthermore, the occurrence of an "unpaired" conformation in the neighboring sequence is strictly due to a positional effect, since it is observed when the poly(dG)-poly(dC) segment is adjacent to a variety of neighboring sequences.

Acetaldehyde↗
PubMed1984

Amphipathic lipid-bound protein antigens in mouse bladder carcinomas detected by a monoclonal antibody.

We have found organic solvent soluble proteins that contain tightly bound fatty acids. They were demonstrated in mouse bladder carcinomas by a monoclonal rat antibody, 33E7C. The antigens recognized by antibody 33E7C copurified with lipids when extracted from bladder carcinomas with a mixture of chloroform-methanol. The antigens remained in the chloroform phase after solvent partition with water. Chromatographic analysis using an alpha-hydroxy-propylated Sephadex G-50 (LH60) column led to the isolation of a group of proteins binding to antibody 33E7C. Four proteins (10K, 18K, 25K, and 40K daltons) were found on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after radioimmunoprecipitation with antibody 33E7C. After preparative SDS-PAGE, the purified antigens were soluble in both water and a mixture of chloroform-methanol (2:1) but were freed from phospholipids and glycolipids. The antigens have been characterized by the presence of tightly bound fatty acids with the following findings: (1) They were metabolically colabeled by incubating bladder carcinoma cells with [9,10-3H]palmitic acid and [14C]leucine, followed by radioimmunoprecipitation assay and by SDS-PAGE. (2) After hydrolysis of proteins in NaOH-methanol, [3H]palmitic acid methyl ester was separated from the 40K and 18K proteins by thin-layer chromatography.

Animals↗