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K Mizobuchi

Publications and source records attributed to K Mizobuchi.

At least 19 recordsLinked to original sources
PubMed1992

Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region.

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.

Bacterial Proteins↗
PubMed1992

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells.

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100 beta protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.

Adult↗
PubMed1992

15N and 13C labeling of Escherichia coli tRNAs toward the NMR analysis.

Escherichia coli tRNAs were labeled with stable isotope 15N in vivo. Three species of tRNA, tRNA(Glu), tRNA(Lys) and tRNA(Ile), were purified by an HPLC system and their NMR spectra were observed. In heteronuclear 1H-15N multiple or single quantum coherence (HMQC or HSQC) spectra, the crosspeaks corresponding to NH3 of U and NH1 of G can be distinguished clearly since their 15N chemical shifts are significantly different from each other. Thus, this combination of 15N-labeling and the proton detected heteronuclear experiments are useful for the signal assignment and the conformational analysis of tRNAs. Furthermore, C1'- selective 13C-labeling of nucleotides was examined in vivo in order to resolve the H1' signals of tRNAs. By using a newly constructed E. coli mutant strain, the isotopic enrichments of more than 90% at C1' and of less than 10% for other ribose carbons were achieved.

Carbon Isotopes↗
PubMed1991

An induced mRNA secondary structure enhances repZ translation in plasmid ColIb-P9.

Translation of the repZ gene encoding a DNA replication initiation protein of plasmid ColIb-P9 depends on not only the translation of a transcribed leader sequence (repY) but also the specific intergenic base pairing within RepZ mRNA between two short complementary sequences located in the repY and inc gene regions. In addition, repZ translation can be negatively regulated by Inc RNA, the product of the inc gene and a countertranscript to RepZ mRNA. Here we present evidence indicating that a stable secondary structure of RepZ mRNA, designated as structure III, sequesters one of the complementary sequences and the ribosome-binding site, thereby preventing repZ translation. When site-directed mutagenesis was used to destabilize structure III without changing the ribosome-binding site, a significant level of repZ expression was observed even in the absence of repY translation. Under these conditions, however, repZ expression could be substantially reduced by additional mutations that directly diminished the intergenic base pairing at the mRNA level between the two complementary sequences. These results indicate that repY translation is essential for the disruption of structure III by inducing the formation of a new secondary structure through the intergenic base pairing, and more importantly, that this new structure enhances repZ translation. We also found that the site of repY translation termination played a critical role in the intramolecular conformational alteration of structure III.

Bacteriocin Plasmids↗
PubMed1991

Positive and negative regulations of plasmid CoLIb-P9 repZ gene expression at the translational level.

Expression of the repZ gene involved in DNA replication of the ColIb-P9 plasmid depends on translation of a transcribed repZ leader sequence (repY) and is negatively regulated by Inc RNA, the product of the inc gene and a countertranscript to RepZ mRNA. To further understand the regulatory loop of repZ expression, we isolated and characterized replication-defective ColIb-P9 mutants that affected the level of repZ expression. Here we report that mutations occurring in two complementary sequences, one (5'GGCG3') in the inc region and one in the repY region, reduce the level of repZ expression without affecting transcription. The mutations in one complementary sequence were suppressed by compensatory base changes in the other sequence, restoring the ability of repZ expression. These results indicated that interaction by base pairing between the two complementary sequences of RepZ mRNA was essential for repZ translation. The two sequences, separated by 107 bases from each other, have a potential to form a novel pseudoknot in the RepZ mRNA leader. We also found that some mutations in the 5'GGCG3' sequence altered the specificity of Inc RNA, thereby reducing significantly its regulatory activity. Thus, this single specific sequence is involved in both positive and negative regulations for repZ expression. Possible regulatory mechanisms of repZ expression are discussed.

Amino Acid Sequence↗
PubMed1990

Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid.

The frequency of replication initiation of the ColIb-P9 plasmid depends on the level of repZ expression, which has been shown to be negatively regulated by inc RNA, the approximately 70-base-long product of the inc gene. To further understand the regulatory mechanism of repZ gene expression, we isolated mutants defective in ColIb-P9 replication using a lambda:ColIb-P9 hybrid phage. Among six mutants isolated, one amber mutant, rep57, failed to synthesize the RepZ protein. The mutation occurred in the repZ leader sequence that encodes a 29-amino-acid reading frame, designated as repY. We also isolated mutants that suppressed the rep57 phenotype. These mutations were single base insertions between the repY initiation codon and the rep57 mutation site and resulted not only in a frame shift of repY but also in the formation of repY-repZ fusions without changing the amino acid sequence of RepZ. Thus, repY is not directly involved in the replication reaction but rather functions as a positive regulator for repZ expression. We propose that repZ expression is coupled with repY translation, which acts to disrupt a secondary structure sequestering the repZ translation initiation signal. The positive and negative regulations of repZ expression were discussed. The other mutants were mapped in repZ, confirming that repZ is essential for ColIb-P9 replication.

Base Sequence↗
PubMed1990

Organization of the replication control region of plasmid ColIb-P9.

We identified a 1,845-base-pair sequence that contains essential information for the autonomous replication and regulation of the 93-kilobase-pair IncI alpha group ColIb-P9 plasmid. Biochemical and genetic analyses revealed that this sequence specifies at least two structural genes, designated repZ and inc. The repZ gene encodes a protein with a molecular weight of 39,000, which probably functions as an initiator for the ColIb-P9 replicon. The inc gene that phenotypically governs the incompatibility encodes an RNA with a size of about 70 bases. This small RNA acts in trans to repress the expression of repZ, thereby functioning to maintain a constant copy number of the ColIb-P9 replicon in host cells.

Amino Acid Sequence↗
PubMed1990

Posttranscriptional control of plasmid ColIb-P9 repZ gene expression by a small RNA.

The replication frequency of plasmid ColIb-P9 depends on the level of repZ gene expression, which is negatively regulated by the action of the inc gene (C. Hama, T. Takizawa, H. Moriwaki, Y. Urasaki, and K. Mizobuchi, J. Bacteriol. 172:1983-1991, 1990). To further understand the mechanism of this regulation, we analyzed transcripts of the ColIb-P9 replication control region. Four RNA species, designated RNAI to RNAIV, were observed in plasmid pCH11, which contained the whole inc gene region and the 5' portion of the repZ gene. RNAII, RNAIII, and RNAIV, with sizes of approximately 200, 500, and 1,500 bases, respectively, were identified as rightward transcripts that shared common transcription initiation sites; RNAIV was determined to be equivalent to a part of repZ mRNA, which was observed in pCH10, a plasmid that contained sufficient information for replication and control of ColIb-P9. Conversely, RNAI, with a size of about 70 bases, was transcribed leftward and was identified as the product of the inc gene and hence equivalent to inc RNA detected by in vitro RNA synthesis. This small RNA was found to be complementary to a part of repZ mRNA. These results and quantitative analyses of the transcripts in Inc- mutants indicate that the inc RNA negatively regulates repZ expression mainly at the posttranscriptional level through the possible formation of an inc RNA-repZ mRNA hybrid in the host cells.

Bacterial Proteins↗
PubMed1989

The organization of the purL gene encoding 5'-phosphoribosylformylglycinamide amidotransferase of Escherichia coli.

Escherichia coli 5'-phosphoribosylformylglycinamide (FGAR) amidotransferase (EC 6.3.5.3) encoded by the purL gene catalyzes the conversion of FGAR to formylglycinamidine in the presence of glutamine and ATP for the de novo purine nucleotide biosynthesis. On the basis of the nucleotide sequence of purL, the enzyme was dissected along the polypeptide chain into at least three discrete regions, designated as domains I, II, and III, by genetic complementation tests. Domain III (255 amino acids), which resides in the C-terminal region, is similar in amido acid sequence to several glutamine amidotransferases and exerts the transfer of the amide nitrogen of glutamine. Domain I (791 amino acids) resides in the N-terminal region and contains a potential ATP binding motif. Domain II (249 amino acids) locates between domains I and III and is composed of an alternating structure of at least eight predicted beta-strand and alpha-helix elements, as has been observed in the family of triosephosphate isomerases. The functions of domains I and II have been discussed in relation to the transfer of the carbonyl oxygen of FGAR into the gamma-phosphorus moiety of ATP. These results support a model that the E. coli purL gene is a fused gene of at least three different gene families. The highly repetitive sequences of the E. coli genome appeared to play an important role in the process of the gene fusion.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence analysis of genes purH and purD involved in the de novo purine nucleotide biosynthesis of Escherichia coli.

5'-Phosphoribosylglycinamide synthetase (EC 6.3.4.13) and 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase (EC 2.1.2.3) are enzymes involved in the de novo purine nucleotide synthesis and are encoded by purD and purH genes of Escherichia coli, respectively. A 3535-nucleotide sequence containing the purHD locus and the upstream region of the rrnE gene was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected Mr of 57,329 and 46,140, respectively. The plasmids carrying ORF-1 complemented not only the mutant cells defective in purH of E. coli but also the cells of Salmonella typhimurium lacking the activity of IMP cyclohydrolase (EC 3.5.4.10) which catalyzes the conversion of 5'-phosphoribosyl 5-formylaminoimidazole-4-carboxamide to IMP. The E. coli purH gene, therefore, specifies bifunctional 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase-IMP cyclohydrolase. The plasmids carrying ORF-2 were able to complement the mutant cells defective in purD. Both purH and purD genes constitute a single operon and are coregulated in expression by purines as other purine genes are. A highly conserved 16-nucleotide sequence termed the PUR box (Watanabe, W., Sampei, G., Aiba, A., and Mizobuchi, K. (1989) J. Bacteriol. 171, 198-204; Tiedeman, A.A., Keyhani, J., Kamholz, J., Daum, H. A., III, Gots, J.S., and Smith, J.M. (1989) J. Bacteriol. 171, 205-212) was found in the control region of the purHD operon and compared with the sequences of the control regions of other purine operons.

Acyltransferases↗
PubMed1989

Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5'-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis.

It has been shown that the Escherichia coli purE locus specifying 5'-phosphoribosyl-5-amino-4-imidazole carboxylase in de novo purine nucleotide synthesis is divided into two cistrons. We cloned and determined a 2,449-nucleotide sequence including the purE locus. This sequence contains two overlapped open reading frames, ORF-18 and ORF-39, encoding proteins with molecular weights of 18,000 and 39,000, respectively. The purE mutations of CSH57A and DCSP22 were complemented by plasmids carrying ORF-18, while that of NK6051 was complemented by plasmids carrying ORF-39. Thus, the purE locus consists of two distinct genes, designated purE and purK for ORF-18 and ORF-39, respectively. These genes constitute a single operon. A highly conserved 16-nucleotide sequence, termed the PUR box, was found in the upstream region of purE by comparing the sequences of the purF and purMN operons. We also found three entire and one partial repetitive extragenic palindromic (REP) sequences in the downstream region of purK. Roles of the PUR box and REP sequences are discussed in relation to the genesis of the purEK operon.

Amino Acid Sequence↗
PubMed1989

[Primary intracranial T cell type malignant lymphoma: a case report].

Primary intracranial T cell type malignant lymphoma is extremely rare. Only 8 cases are reported in the literature so far. In this paper, a case with this type of malignant lymphoma is reported. A 41-year-old man was admitted because of abnormal behavior. Enhanced CT scan demonstrated high density mass and perifocal low-density area in the right frontal lobe, the right basal ganglia and the periventricular region. Specimen biopsied from right frontal lobe was submitted for histological examination. An immunohistochemical technique using monoclonal surface markers confirmed reactivity for LCA, MT-1, OKT-4 and OKT-8, while there was no reactivity for MB-1 and OKB-2. Pathological diagnosis was diffuse medium T cell type malignant lymphoma. Postoperatively, after 60 Gy irradiation, the tumor disappeared. There is no difference on CT findings between B cell type and T cell type malignant lymphoma.

Adult↗
PubMed1989

[A case of vacuolated meningioma].

A rare case of falx meningioma with extensive vacuolization was reported. A 57-year-old woman was admitted to Okayama University Hospital because of numbness in her left lower limb. A plain CT scan showed a hypodense mass in the right frontal region with marked enhancement after contrast medium injection. MRI on T1 weighted images showed low intense mass with the so-called "peritumoral band" of low intensity at the periphery of the mass. Angiogram showed a mass effect and a tumor stain fed by the right pericallosal artery and left middle meningeal artery. The mass was excised en bloc. Microscopic examination demonstrated tumor cells with varied sized vacuoles, which were mostly fat-negative, except for small islands with typical meningothelial cells in the periphery of the tumor. Ultrastructurally, there were two kinds of vacuole in the tumor cells. The smaller, round vacuoles were in the cytoplasm, while the larger ones were in the extracellular space. The cell processes had developed junctional complex, and numerous cytoplasmic filaments were evident. With these findings, this mass was diagnosed as vacuolated meningioma. It is important especially in frozen section not to misdiagnose the tumor as liposarcoma, chordoma, metastatic brain tumor and so on.

Cerebral Angiography↗
PubMed1988

Elastosis in meningioma. A case report and electron microscopic study.

A 55-year-old man underwent left frontoparietal craniotomy for total removal of a parasagittal meningioma. The tumor showed a predominant pattern of meningotheliomatous meningioma and a partial pattern of angiomatous, transitional and fibrous meningioma. A striking feature was the appearance of unusually numerous elastinophilic material in the intercellular stroma. We observed a process in which the progress of elastosis changed the appearance of meningotheliomatous meningioma to a hypocellular, and subsequently acellular fibroelastotic node. Three years after the first craniotomy, meningioma recurred at the same site. The recurrent tumor also showed the histological pattern of meningotheliomatous meningioma with stromal elastosis, as previously observed. This is the first report of a case of meningioma with stromal elastosis. The present case represents the first unequivocal demonstration of elastogenesis in meningioma.

Elastic Tissue↗
PubMed1988

Novel segregation patterns of infecting-mutant genotypes in plate complementation tests among amber mutants of bacteriophage BF23.

Amber mutants of bacteriophage BF23 were classified into two functional groups, types I and II, by the yields of the infecting-mutant genotypes in plate complementation tests. Type I mutants produced their genotypes at levels more than 20% of the total progeny phages, and type II mutants did so at levels of less than 5%. Comparison of the results of plate complementation tests with those of extract complementation tests revealed that all the type I mutants were defective in the tail formation, while most type II mutants were defective in the formation of either mature heads (type IIa) or both mature heads and tails (type IIb). Since in extract complementation tests the activated phages are always of genotypes corresponding to mutations defective in only the tail formation, the plate complementation test is comparable with the extract complementation test when judged on the basis of the yield of the mutant genotypes. Of 29 complementation groups, 8 type I, 14 type IIa, and 5 type IIb mutants were identified. Previously, amber mutations of BF23 were mapped on four genetic segments. These segments were ordered in one linkage map by crosses between deletion and amber mutants.

Coliphages↗
PubMed1988

Relationships among genes and gene products of bacteriophage BF23.

Twenty-five gene products of bacteriophage BF23 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functions were studied in relation to type I and II genes classified by means of genetic complementation tests. All the type I mutants were defective in the synthesis of a tail protein, L3. In addition, 4 type I gene products, L5 (gp21), L7 (gp20), L8 (gp29), and L9 (gp25), were identified as constituents of tails (gp21 denotes that a protein is a product of gene 21). Three type IIb mutants in genes 10, 14, and 19 diminished substantially the production of late proteins, including tail and head proteins, and the two other type IIb mutants in genes 1 and 2 were defective in the synthesis of both early and late proteins. Of 14 type IIa mutants, at least 6 were defective in phage DNA synthesis and 2 were defective in the synthesis of head proteins. The defect in the head donor activities of type IIa mutants in extract complementation tests was due to the failure of the formation of mature heads containing DNA. The above results support directly the results of the genetic characterization of BF23 genes.

Autoradiography↗
PubMed1988

Regulation of the temporal synthesis of proteins in bacteriophage BF23-infected cells.

Regulation of temporal synthesis of pre-early, early, and late proteins in bacteriophage BF23-infected cells has been studied by using five amber mutants defective in genes 1, 2, 10, 14, and 19. The synthesis of pre-early proteins is negatively regulated by the actions of gene 1, a pre-early gene. The switch from pre-early to early protein synthesis is mainly regulated by the second-step DNA transfer reaction, which is controlled by at least genes 1 and 2. Early proteins can be kinetically and genetically divided into two regulatory classes, designated Ea and Eb. The shutoff of Eb-early protein synthesis is associated with the turn-on of late protein synthesis. This step is controlled by genes 10, 14, and 19. Gene 10 also regulates negatively the synthesis of Ea-early proteins, indicating that this gene has a dual function in the regulation of early protein synthesis. The temporal synthesis of phage-encoded proteins is regulated mainly at the transcriptional level. Evidence is presented indicating that the host RNA polymerase is modified by the interaction with the gene products of genes 2, 10, and 14 (gp2, gp10, and gp14, respectively). gp2 interacts with the enzyme in the earlier stage of infection but is replaced by gp10 in the later stage. This exchange reaction depends on the presence of gp14 and gp19 and is related to the switch from Eb to late protein synthesis. Thus, the regulation of BF23 gene expression occurs in a coordinated manner throughout the development of this phage.

Autoradiography↗