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T Nishimoto

Publications and source records attributed to T Nishimoto.

At least 73 records · Page 4Linked to original sources
PubMed1997

VP16 targets an amino-terminal domain of HCF involved in cell cycle progression.

The herpes simplex virus (HSV) regulatory protein VP16 activates HSV immediate-early gene transcription through formation of a multiprotein-DNA complex on viral promoters that includes the preexisting nuclear proteins HCF and Oct-1. The HCF protein is a complex of amino- and carboxy-terminal polypeptides derived from a large (approximately 2,000-amino-acid) precursor by proteolytic processing. Here we show that a 361-residue amino-terminal region of HCF is sufficient to bind VP16, stabilize VP16-induced complex assembly with Oct-1 and DNA, and activate transcription in vivo. This VP16 interaction region contains six kelch-like repeats, a degenerate repeat motif that is likely to fold as a distinctive beta-propeller structure. The third HCF kelch repeat includes a proline residue (P134) that is mutated to serine in hamster tsBN67 cells, resulting in a temperature-sensitive defect in cell proliferation. This missense mutation also prevents direct association between HCF and VP16, suggesting that VP16 mimics a cellular factor required for cell proliferation. Rescue of the tsBN67 cell proliferation defect by HCF, however, requires both the VP16 interaction domain and an adjacent basic region, indicating that HCF utilizes multiple regions to promote cell cycle progression.

Amino Acid Sequence↗
PubMed1997

Yrb2p, a Nup2p-related yeast protein, has a functional overlap with Rna1p, a yeast Ran-GTPase-activating protein.

The Ran-GTPase cycle is important for nucleus-cytosol exchange of macromolecules and other nuclear processes. We employed the two-hybrid method to identify proteins interacting with Ran and the Ran GTP/GDP exchange factor. Using PRP20, encoding the Ran GTP/GDP exchange factor, we identified YRB1, previously identified as a protein able to interact with human Ran GTP/GDP exchange factor RCC1 in the two-hybrid system. Using GSP1, encoding the yeast Ran, as bait, we isolated YRB2. YRB2 encodes a protein containing a Ran-binding motif similar to that found in Yrb1p and Nup2p. Yrb1p is located in the cytosol whereas Nup2p is nuclear. Similar to Yrb1p, Yrb2p bound to GTP-Gsp1p but not to GDP-Gsp1p and enhanced the GTPase-activating activity of Rna1p. However, unlike Yrb1p, Yrb2p did not inhibit the nucleotide-releasing activity of Prp20p. While overproduction of Yrb1p inhibited the growth of a mutant possessing a PRP20 mutation (srm1-1) and suppressed the rna1-1 mutation, overproduction of Yrb2p showed no effect on the growth of these mutants. Disruption of YRB2 made yeast cold sensitive and was synthetically lethal with rna1-1 but not with nup2delta. Nuclear protein import and the mRNA export were normal in strains possessing mutations of YRB2. We propose that Yrb2p is involved in the nuclear processes of the Ran-GTPase cycle which are not related to nucleus-cytosol exchange of macromolecules.

Base Sequence↗
PubMed1997

An altered nuclear migration into the daughter bud is induced by the cyclin A1-mediated Cdc28 kinase through an aberrant spindle movement in Saccharomyces cerevisiae.

A strain of Saccharomyces cerevisiae that contains an integrated copy of a Xenopus cyclin A1 gene under the control of the GAL1 promoter has been constructed. On inducing expression of cyclin A1, the nuclear migration that occurs prior to division becomes aberrant. Instead of migrating to the neck between the mother cell and daughter bud, the nucleus, the short mitotic spindle and its associated two spindle pole bodies entered the daughter bud. This phenotype was induced by expression of an indestructible cyclin mutant, but not by a mutated cyclin A1 unable to activate Cdc28 kinase. The nuclear abnormality induced by cyclin A1 was overcome by cdc28 mutations that abolish its ability to bind cyclin A1. Both yeast cyclin Clb3 and Xenopus mitotic cyclin B produced the same phenotype, whereas G1 cyclin Cln2 did not. The results suggest that the proper movement of the nucleus through the spindle function during mitosis requires the appropriate activity of Cdc28 kinase mediated by specific cyclins.

Animals↗
PubMed1997

Synthesis by an alpha-glucosidase of glycosyl-trehaloses with an isomaltosyl residue.

Glycosyl-trehaloses with an isomaltosyl residue were synthesized by alpha-glucosidase from Aspergillus niger by using maltotetraose as a glucosyl donor and trehalose as the acceptor. The one trisaccharide and two tetrasaccharides formed were isolated by successive column chromatography. The results of an enzymatic digestion, methylation analysis, and 13C-NMR studies indicated that these oligosaccharides were alpha-isomaltosyl alpha-glucoside, alpha-isomaltotriosyl alpha-glucoside and alpha-isomaltoside. These oligosaccharides were not fermented to an acid by Streptococcus mutans, and they effectively inhibited water-insoluble glucan synthesis from sucrose by glucosyltransferase. In an in vitro utilization test with human intestinal bacteria, these oligosaccharides were predominantly utilized by Bifidobacteria.

Bacteroides↗
PubMed1996

Genetic interaction of DED1 encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35 degrees C, but not at 37 degrees C. A revertant of srm1-1 ded1-21 that became able to grow at 37 degrees C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2.

Cell Cycle Proteins↗
PubMed1996

Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1.

Using the two-hybrid method, we isolated a Saccharomyces cerevisiae cDNA encoding a protein homologous to Schizosaccharomyces pombe protein Dis3sp, using as bait, human GTPase Ran. The DIS3 gene is essential for viability and complements S.pombe mutant dis3-54 which is defective in mitosis. Although Dis3sc has no homology to RanBP1, it bound directly to Ran and the S.cerevisiae Ran homologue Cnr1, but not to the S.cerevisiae RCC1 homologue Srm1. Upon binding to Ran with a 1:1 molar ratio, Dis3sc enhanced a nucleotide-releasing activity of RCC1 on Ran. In the presence of Dis3sc, the K(m) of RCC1 on Ran decreased by half, while the kcat was unchanged. In vivo, Dis3sp was present as oligomers of M(r) 670-200 kDa as previously reported, and the 200 kDa oligomer of Dis3sp was found to include Spi1 and Pim1, the S.pombe homologues of Ran and RCC1, respectively. Although the biological function of the heterotrimeric oligomer consisting of Dis3, Spi1 and Pim1 is unknown, our results indicate that Dis3 is a component of the RCC1-Ran pathway.

Amino Acid Sequence↗
PubMed1996

Premature chromatin condensation induced by loss of RCC1 is inhibited by GTP- and GTPgammaS-Ran, but not GDP-Ran.

RCC1 is a guanine nucleotide exchanging factor acting on nuclear G protein Ran. Premature chromatin condensation occurs in the temperature-sensitive rcc1- mutant of the BHK21 cell line, tsBN2, at the restrictive temperature. This observation can be explained if the premature activation of MPF is normally inhibited by GTP-Ran. In the absence of RCC1, GDP-Ran predominates, resulting in MPF activation. However, experiments with Ran mutants to determine whether GTP- or GDP-Ran prevents activation of MPF have yielded conflicting results. In order to clarify this point, we have microinjected nucleotide-bound Ran, instead of mutated Ran, into the nuclei of tsBN2 cells treated to reduce RCC1-mediated guanine nucleotide exchange. GTP-Ran, GTPgammaS-Ran, and GDP-Ran all inhibited chromatin condensation. However, the inhibition of chromatin condensation by GDP-Ran could be completely abolished by co-injection with GDP, but not GTP. Thus, we conclude that GTP-Ran blocks the activation of MPF and that hydrolysis of GTP is not required to prevent MPF activation.

Animals↗
PubMed1996

Cloning and sequencing of trehalose synthase gene from Pimelobacter sp. R48.

The gene encoding trehalose synthase (catalyzing the conversion of maltose into alpha, alpha-trehalose by intramolecular transglucosylation) was cloned from Pimelobacter sp. R48. Sequence analysis revealed a 1719-bp synthase gene and a 573-residue amino-acid sequence. The 220 N-terminal residues were homologous to those of maltases from Saccharomyces carlsbergensis and Aedes aegypti.

Amino Acid Sequence↗
PubMed1996

D-type cyclin expression is decreased and p21 and p27 CDK inhibitor expression is increased when tsBN462 CCG1/TAFII250 mutant cells arrest in G1 at the restrictive temperature.

BACKGROUND: The tsBN462 temperature-sensitive mutant hamster cell line exhibits cell cycle arrest and apoptosis at the restrictive temperature of 39.5 degrees C, due to a point mutation in the CCG1/TAFII250 gene, which encodes a component of the general transcription factor TFIID. RESULTS: We now report that CCG1/TAFII250 persisted as a complex with TBP and associated proteins (TAFs) in tsBN462 cells at the restrictive temperature. FACScan analysis revealed that the tsBN462 mutation resulted in a failure to progress out of G0 into G1. Using two-dimensional gel electrophoresis we observed a decrease in the synthesis of several proteins, starting in the middle of the G1 phase, becoming very pronounced during late G1. The expression of the immediate early genes c-fos, c-jun and c-myc was normally induced by serum treatment of quiescent cells at the restrictive temperature, whereas expression of cyclins A, D1 and D3 was reduced. Expression of the cyclin-dependent kinase (CDK) inhibitor proteins p21 and p27 was enhanced. Consistent with the decreased cyclin D and increased p21/p27 expression, we found that phosphorylation of Rb was decreased at 39.5 degrees C. Cyclin A-, E- and Cdk2-associated histone H1 kinase activity was reduced concomitantly with the increase in p21 protein. CONCLUSION: Decreased cyclin/Cdk kinase activity and decreased Rb phosphorylation are possible causes of G1 cell cycle arrest in tsBN462 cells at the restrictive temperature.

Animals↗
PubMed1996

A single point mutation of hamster aminoacyl-tRNA synthetase causes apoptosis by deprivation of cognate amino acid residue.

BACKGROUND: We have isolated a series of temperature-sensitive mutants for cell-proliferation from the BHK21 cell line derived from the golden hamster (Nishimoto & Basilico 1978; Nishimoto et al. 1982). Using these mutants as a recipient of DNA-mediated gene transfer, we have been cloning human genes which complement these ts mutants. RESULTS: Cultures of tsBN269 cells, a temperature-sensitive mutant of the BHK21 cell line, underwent apoptosis at 39.5 degrees C, a nonpermissive temperature. The gene complementing the tsBN269 cells was cloned and found to encode lysyl-tRNA synthetase. Indeed, tsBN269 cells were found to have a single cytosine to a thymine point mutation at the first nucleotide of codon 542 in hamster lysyl-tRNA synthetases. Due to this mutation, the activity of lysyl-tRNA synthetase was reduced--even at 33.5 degrees C, a permissive temperature. Consistent with these findings, while supplementation with lysine permitted tsBN269 cells to grow at a nonpermissive temperature, the deprivation of lysine caused apoptosis in tsBN269 cells, even at 33.5 degrees C. Cycloheximide inhibited the apoptosis caused by lysine starvation at 33.5 degrees C, but not at 39.5 degrees C. We also found that another hamster temperature-sensitive mutant, tsBN250, which is defective in histidyl-tRNA synthetase, entered apoptosis with the deprivation of histidine. CONCLUSION: Our data suggested that the defect in aminoacyl-tRNA synthetase turned on the cascade of apoptosis that was already present in the cells.

Amino Acid Sequence↗
PubMed1996

A hamster temperature-sensitive G1 mutant, tsBN250 has a single point mutation in histidyl-tRNA synthetase that inhibits an accumulation of cyclin D1.

BACKGROUND: We have previously isolated a series of temperature-sensitive mutants for cell-proliferation from the BHK21 cell line, derived from the golden hamster. These mutants proliferate at 33.5 degrees C, the permissive temperature, but not at 39.5 degrees C the restrictive temperature. Using DNA-mediated gene transfer, human genes complementing these ts mutants were cloned. RESULTS: At 39.5 degrees C the tsBN250 cell line, a temperature-sensitive mutant of the BHK21 cell line, had a defect in the G1 phase, but not in the S phase. The human gene complementing tsBN250 cells was found to encode histidyl-tRNA synthetase. Indeed, the tsBN250 cell line had a single base change--guanine to adenine at the second position of the 362nd codon of hamster histidyl-tRNA-synthetase, converting arginine to histidine. Following release from serum starvation, cyclin E, but not cyclin D1, was accumulated, while, at 39.5 degrees C, the mRNA of cyclin D1 was normally expressed in tsBN250 cells. A similar inhibition of cyclin D1 accumulation was observed in another ts mutant, tsBN269, which has a single point mutation in lysyl-tRNA synthetase. Overexpression of cyclin D1 enabled tsBN250 cells to enter the S phase. CONCLUSION: tsBN250 cells have a single point mutation in histidyl tRNA synthetase that causes a loss of histidyl-tRNA synthetase activity which in turn reduces the content of cyclin D1, but not of cyclin E, thereby resulting in G1 arrest.

Amino Acid Sequence↗
PubMed1996

Conserved histidine residues of RCC1 are essential for nucleotide exchange on Ran.

Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive rcc1- mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased K(m), while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of K(m) similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.

Alanine↗
PubMed1996

RCC1 in the Ran pathway.

RCC1 is a chromosomal protein that functions as a GEF of the nuclear G protein Ran, which GTPase activity is enhanced by RNA1 located in the cytoplasm. RCC1 has no preference for GTP or GDP-bound Ran, so that GTP-Ran formation in vivo is regulated by relative concentrations of GTP/GDP and regulatory proteins interacting with RCC1, Ran, and RNA1. Proteins possessing the special Ran-binding motif have been found to be conserved in species ranging from yeasts to mammalians. The finding of RanBP2/NUP358 clearly indicates the involvement of the Ran pathway in the nuclear pore transport function, in agreement with the finding that both rcc1- and rna1- show defects in this process. However, loss of RCC1 induces premature initiation of mitosis, resulting in G1 arrest with the micronuclei possessing mitotic condensed chromosomes. How both the cell cycle and nucleocytoplasmic transport are regulated by the RCC1-Ran pathway is a major question.

Amino Acid Sequence↗
PubMed1996

Replication of herpes simplex virus type 1 DNA is inhibited in a temperature-sensitive mutant of BHK-21 cells lacking RCC1 (regulator of chromosome condensation) and virus DNA remains linear.

tsBN2, a temperature-sensitive (ts) growth mutant of the hamster cell line BHK-21, has a point mutation in the RCC1 (regulator of chromosome condensation) gene, and prematurely enters mitosis at 39.5 degrees C, a nonpermissive temperature. In this mutant at 39.5 degrees C infectious progeny of herpes simplex virus type 1 (HSV-1) was not produced and replication of HSV-1 DNA was inhibited. HSV-1 DNA from virus particles is normally circularized upon infection, and circularized HSV-1 DNA molecules can serve as template for DNA replication. In tsBN2 at 39.5 degrees C, HSV-1 DNA appeared to remain linear after infection, suggesting the obstruction of HSV-1 DNA circularization, which could account for failure of HSV-1 DNA replication. In transient replication assays performed in tsBN2 at 39.5 degrees C, through superinfection with HSV-1 helper virus, there was no evidence of replication of circular DNA of the hybrid plasmid containing the HSV-1 replication origin. Production of mRNAs of HSV-1 early genes required for HSV-1 DNA replication was decreased in tsBN2 at 39.5 degrees C. Therefore, RCC1 was assumed to be involved in the formation of an HSV-1 DNA configuration suitable for replication (that is circularization) and the supply of proteins required for replication of the circularized HSV-1 DNAs.

Animals↗
PubMed1996

Inhibition of generation of authentic genomic termini of herpes simplex virus type 1 DNA in temperature-sensitive mutant BHK-21 cells with a mutated CCG1/TAF(II)250 gene.

A temperature-sensitive (ts) mutant from the BHK-21 hamster cell line, tsBN462, has a defect in progression of the G1 phase at the nonpermissive temperature of 39.5 degrees C. The ts mutation in tsBN462 is located in the CCG1 gene, encoding the general transcription factor TAF(II)250. In tsBN462 at 39.5 degrees C, infectious progeny of herpes simplex virus type 1 (HSV-1) was not produced and generation of authentic genomic termini of HSV-1 was inhibited. HSV-1 concatemers containing L components in two possible orientations were produced in tsBN462 at 39.5 degrees C; hence, the generation of authentic genomic termini seemed to be dispensable for inversion of the L component. As production of mRNAs of HSV-1 genes of three kinetic classes in the tsBN462 at 39.5 degrees C was comparable to findings under permissive conditions, the sequential and regulated manner in which HSV-1 gene expression is processed is likely to be maintained in the nonpermissive condition.

Animals↗
PubMed1996

Putative GTPase Gtr1p genetically interacts with the RanGTPase cycle in Saccharomyces cerevisiae.

In order to identify a protein interacting with RCC1, a guanine nucleotide-exchange factor for the nuclear GTPase Ran, we isolated a series of cold-sensitive suppressors of mtr1-2, a temperature-sensitive mutant of the Saccharomyces cerevisiae RCC1 homologue. One of the isolated suppressor mutants was mutated in the putative GTPase Gtr1p, being designated as gtr1-11. It also suppressed other alleles of mtr1-2, srm1-1 and prp20-1 in contrast to overexpression of the S. cerevisiae Ran/TC4 homologue Gsp1p, previously reported to suppress prp20-1, but not mtr1-2 or srm1-1. Furthermore, gtr1-11 suppressed the rna1-1, temperature-sensitive mutant of the Gsp1p GTPase-activating protein, but not the srp1-31, temperature-sensitive mutant of the S. cerevisiae importin alpha homologue. mtr1-2, srm1-1 and prp20-1 were also suppressed by overexpression of the mutated Gtr1p, Gtr1-11p. In summary, Gtr1p that was localized in the cytoplasm by immunofluoresence staining was suggested to function as a negative regulator for the Ran/TC4 GTPase cycle.

Base Sequence↗
PubMed1996

Purification and properties of a novel enzyme, trehalose synthase, from Pimelobacter sp. R48.

A novel enzyme, trehalose synthase, was purified from a cell-free extract of Pimelobacter sp. R48 to an electrophoretically homogeneous state by successive chromatographies on DEAE-Toyopearl 650, Butyl-Toyopearl 650, and Mono Q HR5/5 columns. The molecular weight of the enzyme was estimated to be 62,000 by SDS-polyacrylamide gel electrophoresis, and the enzyme had a pI of 4.6 by gel isoelectrofocusing. The enzyme catalyzed the conversion of maltose into trehalose by intramolecular transglucosylation. The enzyme also converted into maltose but was inactive on other saccharides. The N-terminal amino acid of the enzyme was serine. The optimum pH and temperature were pH7.5 and 20 degrees C, respectively. The enzyme was stable in the range of pH 6.0-9.0 and up to 30 degrees C for 60 min. The rate of conversion of maltose into trehalose was independent of the maltose concentration. The maximum yield of trehalose from maltose were 81.8%, 80.9%, and 76.7% at 5, 15, and 25 degrees C, respectively. The activity was inhibited by Cu2+, Hg2+, Ni2+, Zn2+, and Tris.

Amino Acid Sequence↗