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S Moriya

Publications and source records attributed to S Moriya.

At least 37 records · Page 2Linked to original sources
PubMed1999

Regulation of initiation of Bacillus subtilis chromosome replication.

Bacterial chromosome replication is tightly regulated at the initiation stage to coordinate with mass increase. Together with chromosome partition at cell division, this regulation mechanism ensures the proper number of chromosomes in daughter cells at any growth rate. Therefore, elucidation of this regulation mechanism is important for understanding the bacterial cell cycle. Despite much effort in Escherichia coli and Bacillus subtilis for many years, the mechanism remains to be completely elucidated. In E. coli, it is proposed that a critical amount of DnaA protein determines the time of initiation of replication in the cell cycle. Our study strongly suggested that this might not be the case in B. subtilis. Recently, remarkable progress has been made in bacterial cytology. The new techniques enable us to examine the subcellular location of proteins of interest and DNA regions of the chromosome (for example, the replication origin) and, therefore, to determine directly when in the cell division cycle and where within the cell initiation of chromosome replication takes place. Using the techniques, we detected the initiation complex by examining subcellular location of several Dna-initiation proteins in B. subtilis. Based on our new findings, we propose a novel model for regulation of the time of initiation of chromosome replication in the cell cycle.

Bacillus subtilis↗
PubMed1998

Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein.

Replication of the Escherichia coli chromosome is initiated at a unique site, oriC. Concurrent initiation occurs at all oriC sites present in a cell once, and only once, per cell cycle. A mechanism to ensure cyclic initiation events was found operating through the chromosomal site, datA, a 1-kb segment located at 94.7 min on the genetic map that titrates exceptionally large amounts of the bacterial initiator protein, DnaA. A strain lacking datA grew normally but exhibited an asynchronous initiation phenotype as a result of extra initiation events. This mutant phenotype was suppressed by DnaA-titrating plasmids. Furthermore, mutations in a 9-bp DnaA-binding sequence (the DnaA box) in datA were enough to induce the mutant phenotype. Thus, datA is a novel chromosomal element that appears to adjust a balance between free and bound DnaA for a single initiation event at a fixed time in the bacterial cell cycle. Titration of DnaA to newly duplicated datA during oriC sequestration, which is mediated by hemimethylated GATC sequences in oriC and the SeqA protein, would contribute to prevention of reinitiations when oriC is desequestered.

Bacterial Outer Membrane Proteins↗
PubMed1998

Phylogenetic position of symbiotic protist Dinenympha [correction of Dinemympha] exilis in the hindgut of the termite Reticulitermes speratus inferred from the protein phylogeny of elongation factor 1 alpha.

The phylogenetic position of the symbiotic oxymonad Dinenympha exilis, found in the hindgut of the lower termite Reticulitermes speratus, was determined by analysis of translation elongation factor 1 alpha (EF-1 alpha). cDNA corresponding to a major part of the amino acid coding region of EF-1 alpha mRNA was amplified by the reverse transcription polymerase chain reaction (RT-PCR) method from total mRNA of termite hindgut microorganisms without cultivation. The product was cloned into a plasmid vector, pGEM-T, and the clones were isolated and sequenced. One of the EF-1 alpha clones isolated was assigned to the protist D. exilis by whole-cell in-situ hybridization using a specific oligonucleotide probe with enzymatic signal amplification. The deduced amino acid sequence was aligned with those of other eukaryotic and archaeabacterial EF-1 alpha s, and the phylogenetic relationships among early branching eukaryotes were inferred by using the distance matrix method and the maximum parsimony method. The phylogenetic analysis indicated that the D. exilis offshoot occurred before mitochondria-containing organisms and D. exilis branched out after the diplomonads clade. These results indicate that the oxymonad D. exilis is one of the early branching organisms and suggest that the oxymonads form a lineage independent of other early branching organisms.

Amino Acid Sequence↗
PubMed1998

Molecular phylogenetic identification of the intestinal anaerobic microbial community in the hindgut of the termite, Reticulitermes speratus, without cultivation.

A termite maintains an anaerobic microbial community in its hindgut, which seems to be the minimum size of an anaerobic habitat. This microbial community consists of bacteria and various anaerobic flagellates, and it is established that termites are totally dependent on the microbes for the utilization of their food. The molecular phylogenetic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy that does not rely on cultivation of the resident microorganisms. Small subunit ribosomal RNA (ssrRNA) genes were directly amplified from the mixed-population DNA of the termite gut by polymerase chain reaction (PCR) and clonally isolated. Most sequenced clones were phylogenetically affiliated with the four major groups of the domain Bacteria: the Proteobacteria group, the Spirochete group, the Bacteroides group, and the Low G + C gram-positive bacteria. The 16S rRNA sequence data show that the majority of the intestinal microflora of the termite consists of new species that are yet to be cultured. The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite (R. speratus) was analyzed without cultivation. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order. The diversity of nitrogen-fixing organisms was also investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the mixed-population DNA of the termite gut and were clonally isolated. The phylogenetic analysis of the nifH amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. The molecular phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of R. speratus was also examined by the same strategy. The whole-cell hybridization experiments indicated that the sequence originated from a large hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that the hypermastigote and trichomonads shared a recent common ancestry.

Animals↗
PubMed1998

A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition.

We have analysed the function of a gene of Bacillus subtilis, the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23 degree C but not at 37 degree C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23 degree C and in the conditional null mutant grown in low concentrations of IPTG at 37 degree C. In addition, immunofluorescence microscopy showed that subcellular localization of the SpoOJ partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild-type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed.

Bacillus subtilis↗
PubMed1998

ClpC regulates the fate of a sporulation initiation sigma factor, sigmaH protein, in Bacillus subtilis at elevated temperatures.

Using a strain carrying a clpC-bgaB transcriptional fusion at the amyE locus, we found that the expression of a clpC operon was induced at the end of exponential growth in a sigmaB-independent manner and ceased around T3.5 in the wild type but not in a spo0H mutant. This suggests that some gene product(s) whose expression is dependent on sigmaH function is required for the turn-off of clpC transcription during an early stage of sporulation. A clpC deletion mutant showed a temperature-sensitive sporulation phenotype and exhibited an abnormally large accumulation of sigmaH in the cell at 45 degrees C after T2, at which time the sigmaH level in the wild type had begun to decrease. These results, together with the fact that spo0H transcription in the clpC deletion mutant was similar to that of the wild type, suggested that ClpC may be responsible for the degradation of sigmaH after the accomplishment of its role in sporulation. Moreover, as expected from these results, overproduction of Spo0A was also observed after the initiation of sporulation in the clpC deletion mutant at 45 degrees C.

Bacillus subtilis↗
PubMed1998

Comparative study of sialidase activity and G(M3) content in B16 melanoma variants with different metastatic potential.

We previously demonstrated that transfection of a sialidase cDNA into B16-BL6 cells, a highly metastatic and invasive cell line derived from the mouse B16 melanoma, resulted in a marked suppression of metastasis accompanied by decreased cellular content of the GM3 that is one of the target molecules of the sialidase expressed (Tokuyama et al., 1997 Int. J. Cancer, 73, 410-415). To obtain further insight into the involvement of sialidase in metastasis, we made a comparison of the levels of sialidase activity and GM3 content between B16 melanoma cell lines with low (B16-F1) and high (B16-F10 and -BL6) metastatic potential. The cells exhibited sialidase activity towards 4-methylumbelliferyl N-acetylneuraminic acid (4MU-Neu5Ac) and gangliosides at acidic pH in the particulate fractions, but not in the cytosol. The activity toward 4MU-NeuAc was significantly lower in highly metastatic cells. The activity toward gangliosides, on the other hand, varied independently of metastatic potential: B16-F10 cells with a high potential for experimental metastasis showed the lowest level and B16-BL6 cells having high invasiveness had rather a higher level of ganglioside sialidase along with a much greater GM3 synthase activity than the other two cell lines. Flow cytometric analysis with anti-GM3 antibody revealed that highly metastatic cell lines were higher in the binding affinity as compared to B16-F1 cells, B16-BL6 cells containing twice as much cellular GM3 as B16-F1 cells on thin-layer chromatography.

Animals↗
PubMed1997

Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA.

A cytosolic sialidase cDNA was transfected into a highly metastatic and invasive cell line, B16-BL6, derived from the murine B16 melanoma. Stable transfection of a cytosolic sialidase expression vector yielded 4 transfectants with high content of the exogenous sialidase protein as well as enzyme activity. These transfectants exhibited markedly decreased experimental pulmonary metastasis, invasiveness in collagen gels and cell motility on colloidal gold-coated glass plates but no change in cell attachment to fibronectin, collagen type VI or laminin. To cast light on the underlying mechanisms, cellular constituents of the transfectants were analyzed. Sialidase over-expression did not lead to any significant changes in cell surface carbohydrates or intracellular glycoproteins, as revealed by lectin flow cytometry and lectin blotting, respectively. Thin layer chromatography of intracellular glycolipids, however, revealed decreased ganglioside GM3 and increased lactosylceramide as major changes.

Animals↗
PubMed1997

Mechanism of anucleate cell production in the oriC-deleted mutants of Bacillus subtilis.

We constructed oriC-deleted mutants of Bacillus subtilis by integrating the minimal replication region of plasmid pLS32 into the proA (115 degrees), spoIIIJ (360 degrees) and thrS (256 degrees) loci of the chromosome, respectively. All three mutants produced anucleate cells and the DNA/protein ratio was lower than that of the wild-type strain when grown in nutrient broth. However, when grown in minimal-glucose medium, the frequency of anucleate cells was reduced in all of them and the DNA/protein ratio was restored to normal. Especially, the oriC-deleted mutant in which the plasmid was integrated near oriC produced almost no anucleate cell. These results indicate that initiation frequency of chromosome replication from the integrated plasmid origin were reduced disproportionately to cell mass increase in rich medium, which in turn disrupted coordination between DNA replication cycle and cell division cycle. The locations of the plasmid origin relative to the natural oriC locus affected the production of anucleate cell remarkably, suggesting that partition mechanism of chromosome was also impaired by the translocation of its replication origin.

Bacillus subtilis↗
PubMed1997

Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli.

BACKGROUND: The timing of the cell division in Escherichia coli is highly regulated, but its mechanism has not been identified. Previously we have found that the cfcA1 mutation uncouples DNA replication and cell division and elevates the frequency of cell division. We further analysed the structure and the role of the cfc genes of cfcA11, a derivative of cfcA1, and another cfc mutant, cfcB1. RESULTS: The cfc mutants divided prior to the ordinary stage of cell division and produced many small cells with nucleoid. However, the cells grew exponentially, and the length of a cell cycle and the initiation mass for chromosome replication were not altered by the cfc mutations. These properties resulted from a reduction of the period between the nucleoid division and the cell division in a cell cycle, and a compensatory lengthening of the period between the cell division and the initiation of the next round of DNA replication. CfcA11 has a mutation in glySa which encodes the alpha-subunit of glycyl-tRNA synthetase, and cfcB1 has an IS2 insertion in apaH which encodes Ap4A hydrolase. The cfc properties of both cfc mutants were suppressed by a multicopy plasmid carrying apaH+, and the intracellular level of Ap4A in cfcA1 was 15-fold higher, and cfcB1 was 100-fold higher than their parent. Experiments using wild-type cells showed that a high level of Ap4A caused early cell division, and that a low level of Ap4A caused delayed cell division. CONCLUSION: Ap4A is a signal for the induction of cell division. High levels of Ap4A are responsible for the initiation of cell division. The glyS mutation permits an efficient synthesis of Ap4A.

Cell Cycle↗
PubMed1997

Suppression of initiation defects of chromosome replication in Bacillus subtilis dnaA and oriC-deleted mutants by integration of a plasmid replicon into the chromosomes.

We constructed Bacillus subtilis strains in which chromosome replication initiates from the minimal replicon of a plasmid isolated from Bacillus natto, independently of oriC. Integration of the replicon in either orientation at the proA locus (115 degrees on the genetic map) suppressed the temperature-sensitive phenotype caused by a mutation in dnaA, a gene required for initiation of replication from oriC. In addition, in a strain with the plasmid replicon integrated into the chromosome, we were able to delete sequences required for oriC function. These strains were viable but had a slower growth rate than the oriC+ strains. Marker frequency analysis revealed that both pyrD and metD, genes close to proA, showed the highest values among the markers (genes) measured, and those of other markers decreased symmetrically with distance from the site of the integration (proA). These results indicated that the integrated plasmid replicon operated as a new and sole origin of chromosome replication in these strains and that the mode of replication was bidirectional. Interestingly, these mutants produced anucleate cells at a high frequency (about 40% in exponential culture), and the distribution of chromosomes in the cells was irregular. A change in the site and mechanism (from oriC to a plasmid system) of initiation appears to have resulted in a drastic alteration in coordination between chromosome replication and chromosome partition or cell division.

Aldehyde Oxidoreductases↗
PubMed1996

Structure of the dnaA region of the endosymbiont, Buchnera aphidicola, of aphid Schizaphis graminum.

Buchnera aphidicola is an intracellular prokaryote (endosymbiont) that lives in the body cavity of the aphid. Phylogenetic studies indicated that it is closely related to Escherichia coli and members of Enterobacteria. The gene order of the region containing the dnaA gene is well conserved in many bacteria. Seven genes of the endosymbiont of the aphid Schizaphis graminum, gyrB, dnaN, dnaA, rpmH, rnpA, yidD, and 60K. were found to be homologous in sequence and relative location to those of E. coli. We have further sequenced the region downstream of the 60K gene to elucidate the boundary of the conserved region, and found that one more gene, thdF, is conserved. The comparison of gene organizations of the dnaA region of the related bacteria supported the close phylogenetic relationship of B. aphidicola to E. coli. In addition, we have identified groES and groEL genes next to the thdF gene. GroEL protein was reported to be expressed at an elevated level in the endosymbionts of aphids, and is considered to play an important role in their association with the aphid host. Comparison of the structure of the groE operon with that of the endosymbiont of the aphid Acyrthosiphon pisum revealed the conservation of a sequence resembling the E. coli consensus heat shock promoter, and this sequence may be responsible for the high expression of the groEL gene in aphid endosymbionts.

Animals↗
PubMed1996

Mapping of the replication origin of the Bacillus subtilis chromosome by the two-dimensional gel method.

Analysis of replication intermediates produced by in vitro replication of the Bacillus subtilis oriC plasmid revealed that replication initiated at an untranslatable DnaA box region downstream of the dnaA gene. In order to show that replication of the B. subtilis chromosome also starts at the same region in vivo, we have analyzed replication intermediates generated in vivo by the two-dimensional gel method. A bubble arc was detected when the downstream region was used as a probe. In contrast, only a simple Y arc was found when the upstream DnaA box region required for autonomous replication of the oriC plasmid was used as a probe. Furthermore, the bubble arc ranged from unit to almost double the size of a fragment in which the downstream region was located near the middle. These results indicate that replication of the B. subtilis chromosome initiates at the downstream DnaA box region of the dnaA gene and proceeds bidirectionally.

Bacillus subtilis↗
PubMed1996

EGF or PDGF receptors activate atypical PKClambda through phosphatidylinositol 3-kinase.

Overexpression of a TPA-insensitive PKC member, an atypical protein kinase C (aPKClambda), results in an enhancement of the transcriptional activation of TPA response element (TRE) in cells stimulated with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). EGF or PDGF also caused a transient increase in the in vivo phosphorylation level and a change in the intracellular localization of aPKClambda from the nucleus to the cytosol, indicating the activation of aPKClambda in response to this growth factor stimulation. These immediate signal-dependent changes in aKPClambda were observed for a PDGF receptor add-back mutant (Y40/51) that possesses only two of the five major autophosphorylation sites and binds PI3-kinase, and were inhibited by wortmannin, an inhibitor of PI3-kinase. Furthermore, an N-terminal fragment of the catalytic subunit of PI3-kinase, p110alpha, inhibited aPKClambda-dependent activation of TRE in Y40/51 cells stimulated with PDGF. Overexpression of p110alpha resulted in an enhancement of TRE expression in response to PDGF and the regulatory domain of aPKClambda inhibited this TRE activation in Y40/51 cells. These results provide the first in vivo evidence supporting the presence of a novel signalling pathway from receptor tyrosine kinases to aPKClambda through PI3-kinase.

Amino Acid Sequence↗
PubMed1996

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.

Cell Compartmentation↗
PubMed1996

Dominant negative effect of the truncated p110 subunit of phosphatidylinositol-3 kinase.

Effects of p110, the catalytic subunit of PI-3 kinase, on induction of TPA response element-driven promoter by EGF was examined. The induction was enhanced by co-expression of the wild type of p110. The truncated p110 mutants containing the binding site for p85 but missing the catalytic activity repressed the induction. A mutant with no binding activity to p85 did not show this effect. These results suggest that PI-3 kinase is involved in signal transduction of EGF and that the truncated p110s capable of binding to p85 serves as a dominant negative reagent for PI-3 kinase.

Animals↗