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M Yanagida

Publications and source records attributed to M Yanagida.

At least 19 recordsLinked to original sources
PubMed2001

Fission yeast living mitosis visualized by GFP-tagged gene products.

The fission yeast Schizosaccharomyces pombe has been used as a model organism to study cell cycle control and dynamic chromosome behavior during anaphase segregation as genetic and cytological approaches are easily amenable. To understand the role of gene products involved in these cellular events, it is important to determine intracellular localization of each gene product during the cell cycle. In this article, visualization in living cells of several gene products involved in cell cycle control and sister chromatid separation is described. The genes tagged with jellyfish green fluorescent protein (GFP) include sad1(+) (encoding a spindle pole body (SPB) protein), atb2(+) (alpha-tubulin), mis6(+) (a kinetochore protein), eat1(+) (a novel actin-like protein localized in the nucleus) and cdc13(+) (a mitotic cyclin). In addition, LacI which is bound to a DNA segment containing LacO repeat sequences integrated near the centromere (cen1) is visualized. These are useful to monitor cell cycle events in living cells.

Cell Cycle↗
PubMed2000

Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast.

Mammalian kinetochores contain the centromere-specific histone H3 variant CENP-A, whose incorporation into limited chromosomal regions may be important for centromere function and chromosome segregation during mitosis. However, regulation of CENP-A localization and its role have not been clear. Here we report that the fission yeast homolog SpCENP-A is essential for establishing centromere chromatin associated with equal chromosome segregation. SpCENP-A binding to the nonrepetitious inner centromeres depended on Mis6, an essential centromere connector protein acting during G1-S phase of the cell cycle. Mis6 is likely required for recruiting SpCENP-A to form proper connection of sister centromeres.

Amino Acid Sequence↗
PubMed2000

Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast.

Sister kinetochores are bioriented toward the spindle poles in higher eukaryotic prometaphase before chromosome segregation. We show that, in budding yeast, the sister kinetochores are separated in the very early spindle, while the sister arms remain associated. Biorientation of the separated kinetochores is achieved already after replication. Mtw1p, a homolog of fission yeast Mis12 required for biorientation, locates at the centromeres in an Ndc10p-dependent manner. Mtw1p and the sequences 1.8 and 3.8 kb from CEN3 and CEN15, respectively, behave like the precociously separated kinetochores, whereas the sequences 23 and 35 kb distant from CEN3 and CEN5 previously used as the centromere markers behave like a part of the arm. Mtw1p and Ndc10p are identically located except for additional spindle localization of Ndc10p. A model explaining small centromeres and early spindle formation in budding yeast is proposed.

Antineoplastic Agents↗
PubMed2000

A 38 kb segment containing the cdc2 gene from the left arm of fission yeast chromosome II: sequence analysis and characterization of the genomic DNA and cDNAs encoded on the segment.

A genomic 38 kbp segment on the c1750 cosmid clone containing the cdc2 gene, located in the left arm of chromosome II from Schizosaccharomyces pombe, was sequenced. The segment was found to have five previously known genes, pht1, cdc2, his3, act1 and mei4. Among 11 coding sequences (CDSs) predicted by the gene finding software INTRON.PLOT., four CDSs, pi007, pi010, pi014 and pi016, had considerable similarity to 40S ribosomal protein, glycosyltransferase, cdc2-related protein kinase and alpha-1, 2-mannosyltransferase, respectively. Another unusually huge open reading frame (ORF) (pi011), consisting of 2233 amino acids, existed, having significant homology to alpha-amylase, granule-bound glycogen synthase and the Sz. pombe YS 1110 clone product at the N-terminal, middle and C-terminal regions, respectively. All the predicted 11 CDSs were experimentally analysed by RACE PCR. The sequencing of the RACE products revealed that there were two small overlaps at the 3' untranslated regions (UTRs) between pi004 and pi005 (17 bp) and between pi007 and pi008 (2 bp). The distances between 5' end of the 5'UTR and the putative translation initiation codon varied from 10 to 302 nucleotides (nt) among the nine CDSs successfully analysed by 5'-RACE. The expression level of each CDS on this clone was determined. Among the 16 genes on this clone, the previously determined genes, pht1, cdc2, his3 and act1, were found to be most highly expressed. Finally, cDNAs of all the newly identified genes were detected by RACE, proving the actual expression of these genes. The nucleotide sequence has been submitted to the EMBL database under Accession No. AB004534.

Base Sequence↗
PubMed2000

Matrix assisted laser desorption/ionization-time of flight-mass spectrometry analysis of proteins detected by anti-phosphotyrosine antibody on two-dimensional-gels of fibrolast cell lysates after tumor necrosis factor-alpha stimulation.

We describe efficient methods for using functional proteomics analysis to study signal transduction pathways in murine fibroblast L929 cells following stimulation with tumor necrosis factor (TNF)-alpha. After stimulation with TNF-alpha, cellular proteins of L929 cells were extracted with a lysis buffer containing 0.3% sodium dodecyl sulfate (SDS) for 10-30 min time intervals, and were separated by two-dimensional (2-D) electrophoresis followed by immunoblot analysis with anti-phosphotyrosine antibody and alkaline phosphatase-anti IgG antibody conjugate. To improve detection sensitivity by immunoblot analysis we used a chemifluorescent substrate for alkaline phosphatase. One hundred protein spots were detected in the TNF-alpha stimulated L929 cell extract by immunoblot analysis. The use of chemifluorescence allowed us to quantitate immunoblotted spots with fluoroscanner so that we were able to detect time-dependent changes of a number of immunoblotted spots. Protein spots on a silver-stained 2-D gel corresponding to those detected by immunoblot analysis were subjected to in-gel trypsin digestion- matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry analysis, respectively. Twenty-one proteins detected by immunoblot analysis were identified by MS-Fit database search analysis. Among them, the proteins that show time-dependent changes in staining intensity include vimentin, tubulin beta-chain, eukaryotic translation initiation factor 1A, chromatin assembly factor 1 (P48 subunit), probable protein disulfide isomerase P5, and several other proteins. Vimentin and tubulin beta-chain have been reported to be phosphorylated at tyrosine residues and involved in the signal transduction pathway induced by TNF-alpha. However, the other proteins have no previously known function in the signal transduction pathway. Thus, the methods used in this study seem to be suitable for the identification of time-dependent changes in many proteins that are involved in signal transduction. Usefulness of the method for comprehensive analysis of the proteins involved in signal transduction pathway and the limitations of the method are discussed.

Acrylic Resins↗
PubMed2000

Kinetic analysis of the interaction between recombinant human Fc(epsilon)RIalpha and serum IgEs from allergic patients.

Binding of IgE to the high-affinity IgE receptor (Fc(epsilon)RI) is the essential event for allergic reaction. Although there are many reports on binding kinetics between myeloma IgE and Fc(epsilon)RI, little is known about the kinetics between heterogeneous polyclonal IgE in the serum and Fc(epsilon)RIalpha. To elucidate the binding characteristics of heterogeneous serum IgE, we measured kinetic parameters of binding between IgE from allergic patients and a recombinant ectodomain of the human Fc(epsilon)RIalpha subunit by real-time interaction analysis based on surface plasmon resonance. Purified IgE monomer from the plasma of allergic patients displayed kinetics for the interaction with Fc(epsilon)RIalpha similar to those of myeloma IgE. In the case of crude IgE samples from allergic patients, one of seven specimens showed significantly higher affinity than highly purified IgE, suggesting that it is possible for IgEs in this specimen to form complexes of higher molecular weight.

Dermatitis, Atopic↗
PubMed2000

Cell cycle mechanisms of sister chromatid separation; roles of Cut1/separin and Cut2/securin.

The correct transmission of chromosomes from mother to daughter cells is fundamental for genetic inheritance. Separation and segregation of sister chromatids in growing cells occurs in the cell cycle stage called 'anaphase'. The basic process of sister chromatid separation is similar in all eukaryotes: many gene products required are conserved. In this review, the roles of two proteins essential for the onset of anaphase in fission yeast, Cut2/securin and Cut1/separin, are discussed with regard to cell cycle regulation, and compared with the postulated roles of homologous proteins in other organisms. Securin, like mitotic cyclins, is the target of the anaphase promoting complex (APC)/cyclosome and is polyubiquitinated before destruction in a manner dependent upon the destruction sequence. The anaphase never occurs properly in the absence of securin destruction. In human cells, securin is an oncogene. Separin is a large protein (MW approximately 180 kDa), the C-terminus of which is conserved, and is thought to be inhibited by association with securin at the nonconserved N-terminus. In the budding yeast, Esp1/separin is thought to be a component of proteolysis against Scc1, an essential subunit of cohesin which is thought to link duplicated sister chromatids up to the anaphase. Whether fission yeast Cut1/separin is also implicated in proteolysis of cohesin is discussed.

Anaphase↗
PubMed2000

Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase.

Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. The eso1(+) gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G(1)-S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase eta of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase eta domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.

Acetyltransferases↗
PubMed1999

Control of metaphase-anaphase progression by proteolysis: cyclosome function regulated by the protein kinase A pathway, ubiquitination and localization.

Ubiquitin-mediated proteolysis is fundamental to cell cycle progression. In the fission yeast Schizosaccharomyces pombe, a mitotic cyclin (Cdc13), a key cell cycle regulator, is degraded for exiting mitosis, while Cut2 has to be destroyed for the onset of sister chromatid separation in anaphase. Ubiquitination of these proteins requires the special destruction box (DB) sequences locating in their N-termini and the large, 20S complex called the anaphase-promoting complex or cyclosome. Here we show that cyclosome function during metaphase-anaphase progression is regulated by the protein kinase A (PKA) inactivation pathway, ubiquitination of the cyclosome subunit, and cellular localization of the target substrates. Evidence is provided that the cyclosome plays pleiotropic roles in the cell cycle: mutations in the subunit genes show a common anaphase defect, but subunit-specific phenotypes such as in G1/S or G2/M transition, septation and cytokinesis, stress response and heavy metal sensitivity, are additionally produced, suggesting that different subunits take distinct parts of complex cyclosome functions. Inactivation of PKA is important for the activation of the cyclosome for promoting anaphase, perhaps through dephosphorylation of the subunits such as Cut9 (Apc6). Cut4 (Apc1), the largest subunit, plays an essential role in the assembly and functional regulation of the cyclosome in response to cell cycle arrest and stresses. Cut4 is highly modified, probably by ubiquitination, when it is not assembled into the 20S cyclosome. Sds23 is implicated in DB-mediated ubiquitination possibly through regulating de-ubiquitination, while Cut8 is necessary for efficient proteolysis of Cdc13 and Cut2 coupled with cytokinesis. Unexpectedly, the timing of proteolysis is dependent on cellular localization of the substrate. Cdc13 enriched along the spindle disappears first, followed by decay of the nuclear signal, whereas Cut2 in the nucleus disappears first, followed by decline in the spindle signal during metaphase-anaphase progression.

Anaphase↗
PubMed1999

Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4.

The condensin complex in frog extracts, containing two SMC (structural maintenance of chromosomes) and three non-SMC subunits, promotes mitotic chromosome condensation, and its supercoiling activity increases during mitosis by Cdc2 phosphorylation. Here, we report that fission yeast has the same five-member condensin complex, each of which is essential for mitotic condensation. The condensin complex was purified and the subunits were identified by microsequencing. Cnd1, Cnd2, and Cnd3, three non-SMC subunits showing a high degree of sequence conservation to frog subunits, are essential for viability, and their gene disruption leads to a phenotype indistinguishable from that observed in cut3-477 and cut14-208, known mutations in SMC4 and SMC2-like subunits. Condensin subunits tagged with GFP were observed to alter dramatically their localization during the cell cycle, enriched in the nucleus during mitosis, but cytoplasmic during other stages. This stage-specific alteration in localization requires mitosis-specific phosphorylation of the T19 Cdc2 site in Cut3. The T19 site is phosphorylated in vitro by Cdc2 kinase and shows the maximal phosphorylation in metaphase in vivo. Its alanine substitution mutant fails to suppress the temperature-sensitive phenotype of cut3-477, and shows deficiency in condensation, probably because Cut3 T19A remains cytoplasmic. Therefore, direct Cdc2 phosphorylation of fission yeast condensin may facilitate its nuclear accumulation during mitosis.

Amino Acid Sequence↗
PubMed1999

Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation.

High-fidelity chromosome transmission is fundamental in controlling the quality of the cell division cycle. The spindle pole-to-pole distance remains constant from metaphase to anaphase A. We show that fission yeast sister centromere-connecting proteins, Mis6 and Mis12, are required for correct spindle morphogenesis, determining metaphase spindle length. Thirty-five to sixty percent extension of metaphase spindle length takes place in mis6 and mis12 mutants. This may be due to incorrect spindle morphogenesis containing impaired sister centromeres or force unbalance between pulling by the linked sister kinetochores and kinetochore-independent pushing. The mutant spindle fully extends in anaphase, although it is accompanied by drastic missegregation by aberrant sister centromere separation. Hence, metaphase spindle length may be crucial for segregation fidelity. Suppressors of mis12 partly restore normal metaphase spindle length. In mis4 that is defective in sister chromatid cohesion, metaphase spindle length is also long, but anaphase spindle extension is blocked, probably due to the activated spindle checkpoint. Extensive missegregation is caused in mis12 only when Mis12 is inactivated from the previous M through to the following M, an effective way to avoid missegregation in the cell cycle. Mis12 has conserved homologs in budding yeast and filamentous fungi.

Amino Acid Sequence↗
PubMed1999

Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments.

The small GTPase Rho and one of its targets, Rho-associated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can specifically recognize distinct phosphorylated sites on glial fibrillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate filament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of anti-Rho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/moesin (ERM) proteins and Ser19-phosphorylated myosin light chain. These results raise the possibility that Rho-kinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

Cell Division↗
PubMed1999

IgE reactivity to alpha1 and alpha2 chains of bovine type 1 collagen in children with bovine gelatin allergy.

BACKGROUND: Anaphylactic reactions to measles, mumps, and rubella vaccines, including gelatin as a stabilizer, have been reported. It had been found that most of these reactions to live vaccines are caused by the bovine gelatin included in these vaccines. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. OBJECTIVE: The current study was designed to investigate the IgE reactivity to alpha1 and alpha2 chains of bovine type I collagen in gelatin-sensitive children. METHODS: Serum samples were taken from 10 children who had anaphylaxis to the vaccines and high levels of specific IgE to bovine gelatin. Bovine type I collagen was isolated from bovine skin and then separated to alpha1 and alpha2 chains by column chromatography. IgE reactivity to denatured type I collagen and its alpha1 and alpha2 chains was analyzed by immunoblotting, ELISA, and histamine release from the mast cells passive sensitized with IgE antibodies in pooled serum of the children. RESULTS: All children had specific IgE to bovine type I collagen. Furthermore, IgE antibodies in their sera reacted with the alpha;2 chain but not with the alpha1 chain. Similarly, the mast cells sensitized with pooled sera in the children showed alpha2 chain-specific histamine release but not alpha1 chain-specific histamine release. CONCLUSION: In gelatin allergy denatured bovine type I collagen is a major allergen and IgE-binding sites exist in the alpha2 chain of type I collagen.

Anaphylaxis↗
PubMed1999

Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage checkpoint.

DNA damage induces cell cycle arrest (called the damage checkpoint), during which cells carry out actions for repair. A fission yeast protein, Crb2/Rhp9, which resembles budding yeast Rad9p and human BRCA1, promotes checkpoint by activating Chk1 kinase, which restrains Cdc2 activation. We show here that phosphorylation of the T215 Cdc2 site of Crb2 is required for reentering the cell cycle after the damage-induced checkpoint arrest. If this site is nonphosphorylatable, irradiated cells remain arrested, though damage is repaired, and maintain the phosphorylated state of Chk1 kinase. The T215 site is in vitro phosphorylated by purified Cdc2 kinase. Phosphorylation of T215 occurs intensely in response to DNA damage at a late stage, suggesting an antagonistic role of Cdc2 phosphorylation toward checkpoint.

BRCA1 Protein↗
PubMed1999

Involvement of CRM1, a nuclear export receptor, in mRNA export in mammalian cells and fission yeast.

BACKGROUND: CRM1, an evolutionarily conserved protein, was shown to be a receptor for leucine-rich nuclear export signal (NES)-dependent protein transport. In lower eukaryotes CRM1 is reported to be required for the export of mRNA, however, involvement of the NES-dependent transport pathway in mRNA export in higher eukaryotes has not been established. RESULTS: We have found that treatment of mammalian cells with leptomycin B (LMB), a specific inhibitor of CRM1, induces the nuclear accumulation of endogenous mRNA, probably due to the inhibition of its export. In fission yeasts, the nuclear accumulation of mRNA also occurred in cells treated with LMB or in a temperature-sensitive crm1 mutant at a restrictive temperature. A synthetic mRNA that was injected into the nucleus of mammalian cultured cells was exported from the nucleus within 5 h. This export was inhibited by both wheat germ agglutinin and a temperature of 4 degrees C. Importantly, this mRNA export was inhibited by LMB or by an excess amount of the NES peptide-conjugates. LMB treatment, on the other hand, rapidly induced the nuclear entry of RanBP1, a factor involved in the active nucleocytoplasmic transport, although the treatment did not interfere with a nuclear localization signal-dependent transport system within 7 h. CONCLUSION: These results suggest that CRM1 is involved in mRNA export in eukaryotic cells.

Animals↗
PubMed1999

Fission yeast APC/cyclosome subunits, Cut20/Apc4 and Cut23/Apc8, in regulating metaphase-anaphase progression and cellular stress responses.

BACKGROUND: The 20S cyclosome/APC complex promotes metaphase-anaphase transition by ubiquitinating its specific substrates such as mitotic cyclins and anaphase inhibitor Cut2/Pds1/securin. The complex has been shown to contain more than 10 proteins in budding yeast and frog. In fission yeast, however, only five (Cut4, Cut9, Nuc2, Apc10, Hcn1) have been identified. RESULTS: More than five hundred temperature-sensitive mutants were screened for identifying those defective in mitotic anaphase. Fifty-five showed the cut (cell untimely torn) phenotype or metaphase-arrest phenotypes, 27 of them locating at new loci. Their extracts were run in sucrose gradient centrifugation, and four showed alterations in the sedimentation profiles. The gene products of cut20+ and cut23+ were thus identified. Phenotypes of cut20-100 mutant highly resemble cut4-533 in many ways: they are hypersensitive to canavanine and CdCl2, and suppressed by PKA-inactivating regulators, cAMP-dependent phosphodiesterase and PKA regulatory subunits. Cut20 interacts closely with Cut4 in the assembly process of cyclosome. But cut20 mutant differs from cut4, as a novel gene stw1+ suppresses cut20 mutant but not cut4. cut23-194 mutant cells are sterile and blocked at metaphase, but does not show sensitivity to the stress and cAMP. TPR repeat-containing Cut23 may not be the stable component of APC/cyclosome, and its level significantly fluctuates during cell cycle. Cut23 may be ubiquitinated and degraded in a cell cycle dependent fashion. CONCLUSIONS: We identified two new subunits of fission yeast cyclosome/APC complex. Our observations indicate that cyclosome components are divided into several subgroups with distinctly different roles.

Adenomatous Polyposis Coli Protein↗
PubMed1999

Reactivity of the immunoglobulin E in bovine gelatin-sensitive children to gelatins from various animals.

It has been reported that most children who showed anaphylaxis to measles, mumps and rubella vaccines containing bovine gelatin as a stabilizer have anti-bovine gelatin IgE. The present study was designed to investigate the reactivity of IgE in bovine gelatin-sensitive children to gelatins from various animals, and the antigenic cross-reactivity between the gelatins. Serum samples taken from 10 children who showed anaphylaxis to vaccines containing bovine gelatin were used in this study. The level of anti-bovine gelatin IgE in these serum samples ranged from 11.0 to 251 Ua/ml. The IgE in most of the children reacted to kangaroo and mouse gelatins, to which they had had little or no exposure as a food or a vaccine stabilizer. The IgE binding to kangaroo and mouse gelatins was completely inhibited by bovine gelatin, whereas reciprocal inhibition was not complete, indicating that antigenic cross-reactivity is present between the mammalian gelatins. Only one child had strong IgE reactivity to fish gelatins, and this reactivity was not inhibited by bovine gelatin, indicating that no antigenic cross-reactivity exists between bovine and fish gelatins. Most of the children who displayed sensitivity to bovine gelatin showed IgE reactivity to other mammalian gelatins. This reactivity may be due primarily to the antigenic cross-reactivity between mammalian gelatins.

Anaphylaxis↗