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R Ohki

Publications and source records attributed to R Ohki.

31 records · Page 2Linked to original sources
PubMed1997

The structure and organization of the human NPAT gene.

Ataxia telangiectasia (AT) is an autosomal recessive gene disorder, and ATM, a housekeeping gene, has been identified as the gene responsible for AT. Recently we found that another housekeeping gene, NPAT, is located upstream of ATM on human chromosome 11. The two housekeeping genes are transcribed in opposite directions and share a 0.5-kb 5' flanking sequence. The structure and organization of NPAT were determined by direct sequencing of cosmid clones carrying the gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon/intron boundaries and all of the exons. The gene spans at least 44 kb and consists of 18 exons and 17 introns. It has been suggested that AT heterozygotes have an increased risk of developing cancer, especially breast cancer in women. Frequently, loss of heterozygosity at loci on 11q22-q24 has been observed in DNA isolated from tumors of the breast, uterine cervix, and colon, perhaps suggesting the location of a tumor suppressor gene in 11q22-q24. For investigation of the role of NPAT in AT and these tumors with allelic loss of 11q22-q24, appropriate primer sequences and PCR conditions for amplification of all the NPAT exons from genomic DNA were determined. We previously reported that no recombinations are found among Atm, Npat, and Acat1 (acetoacetyl-CoA thiolase) loci as determined by fine genetic linkage mapping of the mouse AT region. The results of the LA-PCR analysis using NPAT- and ACAT-specific primers and human genomic DNA allowed us to map ACAT 12 kb centromeric to NPAT.

Animals↗
PubMed1997

bmr3, a third multidrug transporter gene of Bacillus subtilis.

A third multidrug transporter gene named bmr3 was cloned from Bacillus subtilis. Although Bmr3 shows relatively low homology to Bmr and Blt, the substrate specificities of these three transporters overlap. Northern hybridization analysis showed that expression of the bmr3 gene was dependent on the growth phase.

Amino Acid Sequence↗
PubMed1996

Cloning and sequencing of the gene encoding the soluble fumarate reductase from Saccharomyces cerevisiae.

A gene of the soluble fumarate reductase (FRDS) that binds FAD non-covalently was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotides designed from partial amino acid sequences of highly purified enzyme. The nucleotide sequence of a 0.99-kb amplified product was found to be nearly identical to a partial sequence of an open reading frame (ORF) previously reported (EMBL database accession number S-30830). According to the sequence in the EMBL database, we cloned 1.7-kb fragment containing entire sequence of this ORF by PCR and found that this fragment contained a perfect match to the 0.99-kb sequence amplified with the degenerate primers. From these results, we concluded that this ORF is the FRDS gene. The amino acid sequences of the regions involved in the non-covalent binding of FAD and the active site, which are conserved among the flavoprotein subunits of membrane-bound fumarate reductase and succinate dehydrogenase, were found in FRDS. However, unlike the membrane-bound enzymes, FRDS did not contain the histidine residue that covalently binds the isoalloxazine ring of FAD at or near the corresponding position. FRDS showed high homology to the product of S. cerevisiae OSM1 gene which was reported to be required for growth in hypertonic media.

Amino Acid Sequence↗
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

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

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↗
PubMed1992

Escherichia coli dnaJ deletion mutation results in loss of stability of a positive regulator, CRP.

The dnaJ deletion mutant K7052(lambda dnaK) has a temperature-sensitive defect in the synthesis of beta-galactosidase. We confirmed this operon-specific and temperature-sensitive defect in cell-free extracts prepared from the mutant cells and found that the missing factor was CRP. In the mutant, the cellular concentration of CRP was too low to allow the expression of the lac operon at a nonpermissive temperature. Introduction of a CRP over-producing plasmid into the dnaJ deletion mutant suppressed the defect of beta-galactosidase synthesis. The lower content of CRP in the mutant was found to result from extreme instability of the protein. These results strongly suggested that the heat shock protein dnaJ is involved in the stabilization (or degradation) of CRP.

Autoradiography↗
PubMed1991

[Effects on the retina and choroid of transpupillary diode laser photocoagulation].

A transpupillary retinal photocoagulator utilizing GaAlAs diode laser which emits an 810 nm beam has been constructed. It has a continuous-wave mode with a maximum output of 400 mW at the cornea. Animal experiments with monkeys were carried out successfully at various intensities to produce controlled coagulations. Observation by ophthalmoscopy, light and electron microscopes at different time intervals proved diode laser photocoagulation produced more intensive effects in choroid than krypton red laser (647 nm). However, due to the greater absorption of the choroid, there is a tendency to over-coagulation unless conditions are carefully set. No serious side effects in the retina and choroid were observed during the experiments.

Animals↗
PubMed1989

A complete deletion mutant of the Escherichia coli dnaKdnaJ operon.

Southern hydridization analyses of genomic DNAs from various dnaJ mutants of Escherichia coli showed that mutant K7052, which has well characterized dnaK706 and dnaJ705 double mutantions, is a deletion mutant. The deletion is about 8.0 kb long and encompasses the whole of the dnaKdnaJ operon.

Cloning, Molecular↗
PubMed1987

The Escherichia coli dnaJ mutation affects biosynthesis of specific proteins, including those of the lac operon.

Temperature-sensitive dnaJ mutants of Escherichia coli showed a thermosensitive defect in the synthesis of beta-galactosidase. Synthesis of the lac mRNA was greatly reduced at the restrictive temperature. The mutants were also conditionally defective in the synthesis of a subset of membrane proteins such as succinate dehydrogenase, whereas the synthesis of anthranilate synthetase, encoded by trpED, as well as that of most cellular proteins, was unaffected at the restrictive temperature. The defect was specific for the dnaJ mutants among several dna mutants which are known to be involved in the initiation of DNA synthesis: dnaK, dnaA, and dnaB mutants synthesized each of these proteins normally even at the restrictive temperature. At the restrictive temperature, growth of the dnaJ mutants was arrested at a specific stage of the cell cycle.

Anthranilate Synthase↗
PubMed1986

Krypton and argon laser photocoagulation effects in subretinal hemorrhage.

Previous studies suggested that krypton laser photocoagulation was more effective in the treatment of macular diseases than argon laser. Furthermore, it could perform photocoagulation more effectively in some lesions with subretinal hemorrhage, because the krypton laser beam was poorly absorbed by hemoglobin. In the present experiment, hemorrhagic retinal detachment was produced in monkey eyes with Q-switched Nd-YAG laser, and 4 weeks later photocoagulation was performed with krypton and argon lasers to compare the differences in the effects of these two lasers. When the subretinal hemorrhage and a heavy coagulation effect was produced in the detached retina, but no coagulation effects were observed in the choroid. Krypton laser beam could go through the hemorrhage and certain coagulation effects were observed in the choroid and the detached retina. It is suggested that krypton laser photocoagulation is more effective in the lesions behind subretinal hemorrhages than photocoagulation with argon laser.

Animals↗