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M Ohtsubo

Publications and source records attributed to M Ohtsubo.

At least 55 records · Page 3Linked to original sources
PubMed1993

Cloning, sequence and expression of the argG gene from Streptomyces lavendulae.

The argG gene, encoding argininosuccinate synthetase, was cloned from Streptomyces lavendulae KCCS0055 by colony hybridization using the argG-carrying 2.1-kb fragment of S. coelicolor DNA as a probe. The restriction map of the cloned DNA fragment was very similar to that of S. coelicolor. This DNA fragment could complement the argG mutation of both S. lividans 1326 I10 and Escherichia coli K-12 JE5694, suggesting that the fragment contained a promoter for both E. coli and S. lividans. The subcloning experiment using E. coli K-12 JE5694 as a host has indicated that the essential region for argG is contained in the 2.5-kb DNA fragment. The translational product was identified as a 56-kDa kDa protein in minicells and by conventional gel electrophoresis. Determination of the nucleotide (nt) sequence of the 2.5-kb DNA fragment revealed one open reading frame of 1449 bp. The amino acid (aa) sequence analysis showed that the N-terminus was Ser, and 9 aa from the N terminus were completely identical with those deduced from the nt sequence. Nuclease S1 mapping indicated that the transcription start point is located near the start codon.

Amino Acid Sequence↗
PubMed1992

[Computerized tomography in pulmonary infarction].

We performed a retrospective study of CT findings in 14 patients with 61 lesions of pulmonary infarction diagnosed clinically and radiographically. All cases except one had multiple pleural-based parenchymal lesions, and in five cases the lesions were bilateral. The site of pulmonary infarction was the right lower lobe in 24 lesions, left lower lobe in 19 lesions, right upper lobe and left upper lobe in seven lesions each, and right middle lobe in four lesions. Fifty-nine infarctions (excluding two segmental lesions) were divided into two groups according to the size of the lesions: a group of lesions 2 cm or more in size and a group less than 2 cm. Nineteen lesions in eight patients were in the larger group and 40 lesions in 12 patients in the smaller group. The typical CT findings of larger infarctions were pleural based parenchymal density with truncated apex, centrally directed linear shadow and inside low attenuation area indicating viable lung. Pleural thickening and effusion were frequently seen. No air bronchograms were seen in these subsegmental lesions. Smaller lesions were believed to indicate infarction of a single secondary pulmonary lobule, considering their size and shape. CT scan was able to detect these small lesions (single lobular infarctions) more frequently than chest radiography. One segmental lesion with air bronchogram was thought to represent atelectasis and the other one to represent pulmonary hemorrhage and edema. CT examination is useful for the detection and diagnosis of pulmonary infarction.

Adult↗
PubMed1991

Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non-permissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non-permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.

Animals↗
PubMed1991

Mutation of the hamster cell cycle gene RCC1 is complemented by the homologous genes of Drosophila and S.cerevisiae.

The RCC1 gene has been isolated from several vertebrates, including human, hamster and Xenopus. Genes similar to RCC1, namely BJ1 and SRM1/PRP20, have been isolated from the insect Drosophila and from the budding yeast Saccharomyces cerevisiae. A mutation of the RCC1 gene in the hamster BHK21 cell line, tsBN2, confers pleiotropic phenotypes, including G1 arrest and premature induction of mitosis in cells synchronized at the G1/S boundary. Similarly, mutations of the SRM1/PRP20 gene are pleiotropic; the srm1 mutant shows G1 arrest and suppression of the mating defect of mutants lacking pheromone receptors, and the prp20 mutant shows an alteration in mRNA metabolism. Here we show that both BJ1 and SRM1/PRP20 complement the temperature sensitive phenotype of the tsBN2 cells. Like RCC1 proteins of vertebrates, the protein products of the Drosophila and yeast RCC1 homologues were located in the nuclei of the mammalian cells. These results suggest that the BJ1 and SRM1/PRP20 genes are functionally equivalent to the vertebrate RCC1 genes, and that the RCC1 gene plays an important role in the regulation of gene expression in the eukaryotic cell cycle.

Amino Acid Sequence↗
PubMed1991

Complete nucleotide sequence of the human RCC1 gene involved in coupling between DNA replication and mitosis.

Total genomic DNA of the human RCC1 gene was isolated from HeLa DNA and its complete nucleotide sequence (34,641 bp) was determined by the shotgun sequencing method. The exon-intron junctions were precisely assigned to this sequence by comparing the nucleotide sequence of RCC1 genomic DNA with that of its cDNA. The RCC1 gene was found to have 14 exons, 8 of which (starting from the seventh one) coded the seven repeated sequences of RCC1 protein. A single exon corresponded roughly to each repeat of the RCC1 protein except for the middle one, indicating that the RCC1 gene was generated through amplification of a primordial exon. Primer extension analysis revealed the presence of an internal promoter.

Base Sequence↗
PubMed1991

The yeast SRM1 protein and human RCC1 protein share analogous functions.

The Saccharomyces cerevisiae protein SRM1 and the mammalian protein RCC1 have amino acid sequence similarity throughout their lengths. SRM1 was defined by a recessive mutation in yeast that both activates the signal transduction pathway required for mating and leads to arrest in the G1 phase of the cell cycle. RCC1 was defined by a recessive mutation in hamster cells that causes premature chromosome condensation and other characteristics of entry into mitosis. Despite the seemingly different roles implied by these phenotypes, we suggest that RCC1 and SRM1 proteins have similar functions. In particular, we find that RCC1 can complement the temperature-sensitive growth phenotype of two independent srm1 mutations and also complements, at least partially, phenotypes associated with activation of the pheromone response pathway, such as transcription induction of FUS1. However, RCC1 fails to complement an srm1 null allele. Further characterization of the srm1 mutant phenotype reveals a defect in plasmid and chromosome stability, suggesting that the mutants have a defect in DNA replication, mitosis, or their coordination. Finally, like RCC1, SRM1 is a nuclear protein. Together, these data imply that SRM1 and RCC1 have a common role in their respective organisms.

Amino Acid Sequence↗
PubMed1990

Site-directed mutagenesis of Asp-376, the catalytic phosphorylation site, and Lys-507, the putative ATP-binding site, of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase.

Point mutations of Asp-376 of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase (the site of phosphorylation during the catalytic cycle) to Asn, Glu or Thr led to virtual abolishment of Na+/K(+)-ATPase activity and ouabain-binding capacity. Replacement of Lys-507 of the same subunit (the putative ATP-binding site) by Met resulted in decreases in Na+/K(+)-ATPase activity and ouabain-binding capacity. These results are in agreement with those reported for rabbit sarcoplasmic reticulum Ca2(+)-ATPase (Maruyama, K. and MacLennan, D.H. (1988) Proc. Natl. Acad. Sci. USA 85, 3314-3318).

Adenosine Triphosphate↗
PubMed1990

Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells.

When BHK21 cells synchronized in early S phase were exposed to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, mitosis specific events such as premature chromosome condensation, the production of MPM-2 antigens, dispersion of nuclear lamins and the appearance of mitotic asters were induced, and then disappeared upon further incubation. These mitosis specific events occurred even in the presence of cycloheximide. Within 1 h of exposure to OA, cdc2/histone H1 kinase activity rose 10-fold compared with untreated controls, but returned to the control level upon further incubation. Using antibodies against either p34cdc2 or cyclin B it was found that p34cdc2 complexed with cyclin B was dephosphorylated after OA treatment concomitant with the activation of cdc2 kinase, and that cyclin B was subsequently degraded concomitant with a decrease in cdc2 kinase activity, as in normal mitosis. In contrast, when cells in G1 phase were treated with OA no increase in cdc2 kinase activity was observed. Moreover when cells in pseudo-metaphase induced by nocodazole were treated with OA, cdc2 kinase was inactivated. These results suggest that OA sensitive protein phosphatases control both the activation and inactivation of the p34cdc2 kinase.

Animals↗
PubMed1990

Cloning of Xenopus RCC1 cDNA, a homolog of the human RCC1 gene: complementation of tsBN2 mutation and identification of the product.

We isolated a cDNA clone from Xenopus cDNA libraries for the homolog of the human RCC1 gene, which is considered to be a regulator for the onset of chromosome condensation. The cloned Xenopus RCC1 cDNA encoded a protein of 424 amino acids which had the same seven homologous repeated domains of about 60 amino acids as found in human RCC1 cDNA. The overall identity of amino acid sequence between the human and Xenopus RCC1 protein was 76%. Specifically, in the repeated domain the amino acid sequence was highly conserved between both species. The identity of amino acids in this region was 82%. In the N-terminal region, albeit the overall identity was low (35%), some positively charged amino acids were conserved. The transcript for Xenopus RCC1 gene with the length of 2.2-kb was detected in both Xenopus oocytes and cultured somatic cells, A6. The antibody prepared against Xenopus RCC1 protein produced in E. coli recognized 45 and 46 kDa proteins not only in Xenopus oocytes and A6 cells, but also in human and hamster cells. Xenopus RCC1 cDNA complemented the tsBN2 mutation, depending on the amount of its product in transformants. Thus, the RCC1 protein was suggested to regulate the onset of chromosome condensation in the eukaryote from amphibian to mammalian cells.

Amino Acid Sequence↗
PubMed1990

Premature chromosome condensation is induced by a point mutation in the hamster RCC1 gene.

At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts+ phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, serine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.

Amino Acid Sequence↗
PubMed1989

Expression of active alpha-3 subunit of rat brain Na+,K+-ATPase from the messenger RNA injected into Xenopus oocytes.

Cloned cDNA encoding the so-far uncharacterized alpha-3 subunit of rat brain Na+,K+-ATPase (Hara et al. (1987) J. Biochem. 102, 43-58, Shull et al. (1986) Biochemistry 25, 8125-8132) was incorporated into a vector carrying the SP6 promoter. The mRNA produced in vitro was injected into Xenopus oocytes with the mRNA encoding the Na+,K+-ATPase beta subunit of Torpedo electroplax. Increased Na+,K+-ATPase activity in the oocyte membrane was observed. This newly expressed activity was inhibited by ouabain (Ki = 1.5 x 10(-7) M), suggesting that the alpha-3 subunit of rat brain Na+,K+-ATPase is a highly ouabain-sensitive catalytic subunit.

Animals↗
PubMed1989

The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA.

The RCC1 gene, a regulator for the onset of chromosome condensation was found to encode a protein with a molecular mass of 45 kD, determined using the antibody against the synthetic peptides prepared according to the amino acid sequence of the putative RCC1 protein. The p45 located in the nuclei was released from the isolated nuclei, either by DNase I digestion or by treatment with 0.3 M NaCl. Consistently, p45 bound to the DNA-cellulose column was eluted with 0.3 M NaCl. After sequential treatment with DNase I and 2 M NaCl, almost all of the RCC1 protein were released from the nuclei. Thus, RCC1 protein locates on the chromatin and is not a component of the nuclear matrix. In mitotic cells, p45 is dispersed into the cytoplasm. Presumably, RCC1 protein plays a role in regulating the onset of chromosome condensation, at the level of transcription or of mRNA maturation.

Amino Acid Sequence↗
PubMed1989

Reconstituted F1-ATPase complexes containing one impaired beta subunit are ATPase-active.

Homogeneous populations of hybrid alpha 3 beta 3 gamma complexes of the thermostable F1-ATPase containing one, two, or three copies of the mutationally impaired beta subunits were produced using the solid phase reconstitution method. Two kinds of mutated beta subunits were used for the reconstitution, one of which lacked the ability to bind any adenine nucleotides. The complexes containing one impaired beta and two wild-type beta subunits retained a significant amount of ATPase activity with cooperative kinetics, whereas those containing two or three impaired beta subunits showed very little ATPase activity. These results imply that the catalysis of steady-state ATP hydrolysis can proceed even if one of the three beta subunits in F1-ATPase is not functional.

Adenosine Diphosphate↗
PubMed1989

[The epidemiological study of hepatolithiasis in the Kamigoto district, Nagasaki where hepatolithiasis is high incidence].

Regional distribution, age, sex, type of disease and history of surgery of the biliary tract were examined in 134 patients with hepatolithiasis in the Kamigoto district. The ratio of patient to population was 1:126 in the region showing the highest incidence. When classified by age at establishment of the diagnosis, patient in their sixties comprised the largest portion in both sexes. The ratio of men to women was 1:1.2. When classified in accordance with the draft by Research Group for the Study of Hepatolithiasis, 103 cases (76.9%) were of the intrahepatic type, and 31 cases (23.1%) were of the intra-extrahepatic type. The stones were found in the right lobe in 58 patients (43.3%), the left lobe in 59 patients (44.0%) and the both lobes in 17 patients (12.7%). Forty-four patients (32.8%) had a history of surgery of the biliary tract. Ultrasonographic examination of the abdomen in junior high school students revealed hepatolithiasis in one. As a result, the intrahepatic type and the right lobe type are high frequency in this district, and epidemiological survey of the family history and the food habit in this district is necessary.

Adolescent↗