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Y Jigami

Publications and source records attributed to Y Jigami.

At least 37 records · Page 2Linked to original sources
PubMed1998

The involvement of mnn4 and mnn6 mutations in mannosylphosphorylation of O-linked oligosaccharide in yeast Saccharomyces cerevisiae.

The structure of O-linked acidic oligosaccharide from Saccharomyces cerevisiae was analyzed. The chitinase, exclusively O-glycosylated extracelluar protein, was purified from strains mnn1, mnn1 mnn4, mnn1 mnn6 and deltakre2 and the oligosaccharides were hydrolyzed by O-linked sugar chain specific hydrazinolysis. The mannosylphosphorylated mannotriose (M3-P-M) was detected in strain mnn1, but not in the other three strains (mnn1n mnn4, mnn1 mnn6 and deltakre2). alpha-Mannosidase treatment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of mannosylphosphorylated mannotriose revealed that mannosylphosphate was attached to a middle mannose of alpha-1,2-linked mannotriose. This result indicates that the mnn4 and mnn6 mutations affect the mannosylphosphorylation of O-linked oligosaccharide, together with that of N-linked oligosaccharide. The amount of mannosylphosphorylated mannotriose was 7% of total O-linked oligosaccharides (20% of neutral mannotriose) of chitinase in strain mnn1.

Alkaline Phosphatase↗
PubMed1998

Functional evidence for UDP-galactose transporter in Saccharomyces cerevisiae through the in vivo galactosylation and in vitro transport assay.

The oligosaccharide profiles in glycoproteins are determined by a series of processing reactions catalyzed by Golgi glycosyltransferases and glycosidases. Recently in vivo galactose incorporation in Saccharomyces cerevisiae has been demonstrated through the expression of human beta-1,4-galactosyltransferase in an alg1 mutant, suggesting the presence of a UDP-galactose transporter in S. cerevisiae (Schwientek, T., Narimatsu, H., and Ernst, J. F. (1996) J. Biol. Chem. 271, 3398-3405). However, this is quite unexpected, because S. cerevisiae does not have galactose residues in its glycoproteins. To address this question we have constructed S. cerevisiae mnn1 mutant strains expressing Schizosaccharomyces pombe alpha-1,2-galactosyltransferase. The mnn1 mutant of S. cerevisiae provides endogenous acceptors for galactose transfer by the expressed alpha-1,2-galactosyltransferase. We present here three lines of evidences for the existence of UDP-galactose transporter in S. cerevisiae. (i) About 15-20% of the total transformed mnn1 cells grown in a galactose medium were stained with fluorescein isothiocyanate-conjugated alpha-galactose-specific lectin, indicating the presence of alpha-galactose residues on the cell surface. (ii) Galactomannan proteins can be precipitated with agarose-immobilized alpha-galactose-specific lectin from a whole cell lysate prepared from transformed mnn1 cells grown in a galactose medium. (iii) The presence of UDP-galactose transporter was demonstrated by direct transport assay. This transport in S. cerevisiae is dependent on time, temperature, and protein concentration and is inhibited by nucleotide monophosphate and Triton X-100. The overall UDP-galactose transport in S. cerevisiae is comparable with that in S. pombe, indicating a more or less similar reaction velocity, while the rate of GDP-mannose transport is higher in S. pombe than in S. cerevisiae.

Biological Transport↗
PubMed1998

Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli.

If ribozymes are to be exploited in vivo, it is necessary to select ribozymes that are functional in the intracellular environment. Ribozymes selected in the intracellular environment should retain their function in vivo as well as in vitro. We have devised a novel system for selection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our first attempt, a sequence encoding either an active or an inactive ribozyme was connected upstream of the gene for DHFR. Each plasmid was designed such that, when the ribozyme was active, the ribozyme would cleave the target site and, as a result, the rate of production of DHFR would be high enough to endow resistance to trimethoprim (TMP). However, a critical defect may be associated with introduction of a ribozyme upstream of the DHFR gene because, during actual screening for active ribozymes on the 5' side from a pool of random sequences, there is the danger of selecting sequences that are not related to the activity of ribozymes. Indeed, some upstream linker sequences affected the level of expression of the DHFR protein and, as a result, the resistance of Escherichia coli to TMP. Therefore, we newly constructed a 3'-connected ribozyme system, and activities in vivo of 5'-connected and 3'-connected ribozymes were compared. We found that the cleavage efficiencies in vivo were nearly identical for the two types of ribozyme, 24% for the 5'-side ribozyme and 23% for the 3'-side ribozyme, indicating that polysomes did not seem to inhibit the action of the 3'-connected ribozyme. In both cases, when cells were transformed with a 1 : 1 mixture of active and inactive ribozyme-coding plasmids, it was mainly the cells that harbored the active ribozyme that survived in the presence of TMP.

Base Sequence↗
PubMed1997

Mannosylphosphate transfer to cell wall mannan is regulated by the transcriptional level of the MNN4 gene in Saccharomyces cerevisiae.

Mannosylphosphorylation is a major oligosaccharide modification that provides negative charge in the Saccharomyces cerevisiae cell wall. Although two genes, MNN6 and MNN4, which encode a mannosylphosphate transferase and its putative positive regulator, respectively, are involved in this modification, the amount of Mnn4p has been found to be a limiting factor for mannosylphosphorylation. The level of mannosylphosphorylation increased at late-logarithmic and stationary phases of cell growth, and this was correlated to the transcriptional enhancement of MNN4. We also find that mannosylphosphate transfer to mannan is negatively regulated by the protein kinase A pathway, while the presence of 0.5 M potassium chloride enhanced MNN4 transcription. This type of transcriptional regulation is observed in many stress response genes, implying that mannosylphosphate transfer is involved in the cellular response to a variety of stresses.

Alcian Blue↗
PubMed1997

Substrate specificity of alpha-1,6-mannosyltransferase that initiates N-linked mannose outer chain elongation in Saccharomyces cerevisiae.

Yeast Saccharomyces cerevisiae OCH1 gene encodes the mannosyltransferase that is essential for the outer chain elongation of N-linked oligosaccharides. Mannosyltransferase activity of OCH1 gene product (Och1p) was measured on HPLC by using pyridylaminated Man8GlcNAc2 (Man8GlcNAc2-PA) as an acceptor and the reaction product was observed at the retention time corresponding to Man9GlcNAc2-PA. 1H-NMR and fast atom bombardment mass spectrometry (FAB-MS) fragmentation analysis of Man9GlcNAc2-PA showed that the additional mannose was attached with an alpha-1,6 linkage at the site where mannose outer chain elongation initiates. Substrate specificity of Och1p was investigated by using various high mannose-type oligosaccharides as acceptors. Man8GlcNAc2 was the best acceptor for Och1p. The loss of one or two alpha-1,2-mannoses from Man8GlcNAc2 reduced the mannosyltransferase activity and the Man5GlcNAc2 completely lacking alpha-1,2-mannose residues did not serve as an acceptor. Man8GlcNAcOH that involves an open sugar ring by reduction of reducing terminal GlcNAc residue did not serve as an acceptor for Och1p. The loss of three mannoses at the alpha-1,6-branch also reduced the Och1p activity. These results suggest that Och1p is an initiation specific alpha-1,6-mannosyltransferase that requires the intact structure of Man8GlcNAc for efficient mannose outer chain initiation.

Acetylglucosamine↗
PubMed1997

MNN6, a member of the KRE2/MNT1 family, is the gene for mannosylphosphate transfer in Saccharomyces cerevisiae.

In yeast Saccharomyces cerevisiae the N-linked sugar chain is modified at different positions by the addition of mannosylphosphate. The mnn6 mutant is deficient in the mannosylphosphate transferase activity toward mannotetraose (Karson, E. M., and Ballou, C. E. (1978) J. Biol. Chem. 253, 6484-6492). We have cloned the MNN6 gene by complementation. It has encoded a 446-amino acid polypeptide with the characteristics of type II membrane protein. The deduced Mnn6p showed a significant similarity to Kre2p/Mnt1p, a Golgi alpha-1, 2-mannosyltransferase involved in O-glycosylation. The null mutant of MNN6 showed a normal cell growth, less binding to Alcian blue, hypersensitivity to Calcoflour White and hygromycin B, and diminished mannosylphosphate transferase activity toward the endoplasmic reticulum core oligosaccharide acceptors (Man8GlcNAc2-PA and Man5GlcNAc2-PA) in vitro, suggesting the involvement of the MNN6 gene in the endoplasmic reticulum core oligosaccharide phosphorylation. However, no differences were observed in N-linked mannoprotein oligosaccharides between Deltaoch1 Deltamnn1 cells and Deltaoch1Deltamnn1Deltamnn6 cells, indicating the existence of redundant genes required for the core oligosaccharide phosphorylation. Based on a dramatic decrease in polymannose outer chain phosphorylation by MNN6 gene disruption and a determination of the mannosylphosphorylation site in the acceptor, it is postulated that the MNN6 gene may be a structural gene encoding a mannosylphosphate transferase, which recognizes any oligosaccharides with at least one alpha-1,2-linked mannobiose unit.

Amino Acid Sequence↗
PubMed1997

A defect in GTP synthesis affects mannose outer chain elongation in Saccharomyces cerevisiae.

We have found that yeast mutants that are defective in mannose outer chain elongation of N-linked glycoproteins show higher cell wall porosity than normal cells, and are hypersensitive to antibiotics with a large molecular weight; such as neomycin and geneticin. Wild-type yeast cells also showed enhanced sensitivity to neomycin in the presence of tunicamycin, an inhibitor of N-glycosylation, suggesting that the extent of N-glycosylation may affect the sensitivity of yeast cells to drugs and that sensitivity to neomycin may be an effective method for screening for yeast mutants defective in N-glycosylation. Pursuing this logic, we isolated neomycin-sensitive yeast mutants and screened them for defects in N-glycosylation. The neomycin-sensitive, N-glycosylation-defective mutants fell into 15 complementation groups including alleles of the previously isolated temperature-sensitive nes mutants nes10, nes17, and nes25. Gene cloning revealed that NES10 was identical to SEC20, which is involved in ER-Golgi protein transport, NES17 was identical to ALG1, which encodes a beta-1,4-mannosyltransferase present in the ER, MSN17, a multicopy suppressor of nes17/alg1, was also isolated and found to be an allele of PSA1, which is involved in GDP-mannose synthesis, NES25 was identical to GUK1, which encodes a GMP kinase. Overexpression of MSN17 increased the GDP-mannose level in a wild-type strain by about threefold, and guk1 decreased the GDP-mannose level to one-fourth, suggesting a close relationship between GTP metabolism and mannose outer chain elongation; the link is presumably provided by the process of GDP-mannose transport in the Golgi membranes.

Anti-Bacterial Agents↗
PubMed1996

Molecular cloning of a cDNA encoding enolase from the filamentous fungus, Aspergillus oryzae.

A 1.6-kbp full-length cDNA for the Aspergillus oryzae enolase gene (enoA) was cloned. The sequenced insert contained a continuous open reading frame of 1314 bp encoding a protein of molecular weight 47 405. Among all enolases sequenced to-date, the deduced amino-acid sequence showed the highest homology (74.9%) with Candida albicans enolase (ENO1). Strong codon biases and multiple transcription start sites downstream from CT-blocks in the 5'-flanking region suggested strong expression. enoA mRNA was found to occupy approximately 3% (w/w) of total mRNA of A. oryzae by quantitative RT-PCR. This strong transcription was dependent on the carbon source in the medium and correlated with the growth rate of the mycelium.

Amino Acid Sequence↗
PubMed1996

Mutations in GCR3, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, suppress the temperature-sensitive growth of hpr1 mutants.

To study the functions of DNA topoisomerase I and Hpr1 protein, a suppressor mutant of the temperature-sensitive growth of an hpr1 top1-5ts double mutant was isolated. The isolated triple mutant showed cold-sensitive growth. By complementation of this phenotype, the suppressor gene was cloned. DNA sequencing showed it to be GCR3, a gene involved in the expression of glycolytic genes. Further analysis showed that gcr3 mutations also suppressed the temperature-sensitive growth of hpr1 single mutants. Experiments with gcr3 truncation mutants also suggested a genetic interaction between GCR3 and HPR1. The fact that top1 suppressed the growth defect of gcr3 suggested an interaction between those two genes also. Plasmid DNA isolated from gcr3 mutants was significantly more negatively supercoiled than normal, suggesting that Gcr3 protein, like topoisomerase I and Hpr1p, affects chromatin structure, perhaps during transcription.

Alleles↗
PubMed1996

Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae MNN4 gene, which is involved in mannosylphosphate transfer from GDP-mannose to N-linked oligosaccharide, has been cloned from a lambda phage containing a yeast chromosome XI DNA fragment. The MNN4 ORF encodes a protein of 1178 amino acids. The deduced amino acid sequence shows a topology of type II membrane proteins and has a unique repeated sequence of lysine and glutamic acid at the C-terminus. Disruption and overexpression of MNN4 led to a decrease and increase, respectively, of the mannosylphosphate content in cell wall mannans prepared from both mnn4 and wild type strains. A dramatic decrease of mannosylphosphate occurs in delta mnn4 mutans. The results from genetic and biochemical experiments combine to suggest that Mnn4p is required to mediate mannosylphosphate transfer in both the core and outer chain portions of N-linked oligosaccharides.

Alleles↗
PubMed1996

Application of long-distance PCR to restriction site mapping of a cloned DNA fragment on the lambda EMBL3 phage vector.

Long-distance PCR was applied to rapidly map the restriction sites of long inserts cloned on lambda EMBL3 phage vector. The restriction sites of 9 of 15 enzymes were completely assigned in a model experiment within 14 h, including 8h for the PCR amplification. This method was found particularly useful for genomic DNA cloning when the partial sequence of the corresponding cDNA is known.

Bacteriophage lambda↗
PubMed1996

Molecular cloning of a genomic DNA for enolase from Aspergillus oryzae.

We have isolated an enolase gene (enoA) from Aspergillus oryzae by heterologous hybridization using the corresponding Saccharomyces cerevisiae ENO2 gene as a probe. A 2.9-kb BglII-fragment contained the entire structural gene enoA including 5'- and 3'- flanking regions. The homology between A. oryzae enoA and S. cerevisiae ENO2 genes is 66.9% when introns are removed. Genomic Southern analysis indicated that there is only one enolase gene in A. oryzae.

Amino Acid Sequence↗
PubMed1995

X-ray structure of wheat germ agglutinin isolectin 3.

Wheat germ agglutinin isolectin 3 (WGA3) was crystallized from 10 mM acetate buffer at pH 4.9 containing 6 mM CaCl(2) and 4%(v/v) ethanol. The crystal belongs to monoclinic space group P2(1) with unit-cell dimensions a = 44.86, b = 91.02, c = 44.86 A, and beta = 110.22 degrees. The asymmetric unit contains two molecules (V(m) = 2.51 A(3) Da(-1)). The crystal structure was solved by the molecular-replacement method and was refined by the simulated-annealing method. The conventional R value was 0.191 for 19713 reflections [|F(o)| > 3sigma(F)] in the resolution range 8-1.9 A. The r.m.s. deviations from the ideal bond distances and angles were 0.014 A, and 3.0 degrees, respectively, and the estimated coordinate error was 0.2-0.25 A. The two molecules in the asymmetric unit are related by the pseudo twofold symmetry and form a dimer structure. The backbone structures of the two subunits are nearly identical with the r.m.s. difference of 0.36 A for the superposition of equivalent C(alpha) atoms. The dimer structure is very similar to those of isolectins 1 and 2 with the r.m.s. difference of 0.35-0.39 A for the C(alpha) superposition. Since amino-acid residues which differ from those of isolectin 1 or 2 are not involved in the contact between the two subunits, the subunit-subunit interaction is not significantly affected by the replacement of these residues. As a result, the geometry of the sugar-binding sites which are located at the interface between the two subunit molecules is basically conserved among three isolectins.

Journal Article↗
PubMed1995

Site-directed mutagenesis and sugar-binding properties of the wheat germ agglutinin mutants Tyr73Phe and Phe116Tyr.

Wheat germ agglutinin is a dimeric lectin composed of two identical subunits. Each subunit consists of four homologous hevein-like domains of 42 or 43 amino acids each. Amino acid residues at the same position in each domain involved in sugar binding are thought to play a similar role in sugar binding. In order to clarify the role of the amino acid residue at domain position 30 of wheat germ agglutinin isolectin 2 (WGA2) in sugar binding, two WGA2 variants each containing a mutation, either Tyr73-->Phe (domain B) or Phe116-->Tyr (domain C), were produced. The binding activity for (GlcNAc)3 and the three-dimensional structure of these mutants were characterized by comparing with the properties of wild-type WGA2. Equilibrium dialysis experiments using (GlcNAc)3 indicated that the mutation Tyr73-->Phe reduced the overall sugar-binding activity at both pH 5.9 and pH 4.7. In addition, positive cooperativity toward (GlcNAc)3 binding was observed at pH 4.7. In contrast, the mutation of Phe116-->Tyr increased the overall sugar-binding activity at pH 5.9, but reduced this activity at pH 4.7 without changing the number of sugar-binding sites. Positive cooperativity was not observed at pH 5.9 or pH 4.7. X-ray crystallographic analysis of mutant WGA2 revealed that the mutation of Tyr73-->Phe caused a side chain movement of the Glu115 residue of the opposite subunit that formed a hydrogen bond with Tyr73 in wild-type WGA2. No changes were observed in the backbone structure and the disposition of the benzene ring of Phe73. The mutation Phe116-->Tyr caused the formation of a new hydrogen bond between Tyr116 and Glu72 of the opposite subunit. The changes in the sugar-binding properties in WGA2 mutants are discussed in relation to the structural change at the binding site.

Base Sequence↗
PubMed1995

A new essential gene GCR4 located in the upstream region of GCR1.

A new essential gene of Saccharomyces cerevisiae was found upstream of GCR1. Its cloning and sequencing predict a 280 amino acid protein (32,577 Da). The predicted protein is fairly hydrophobic, and a search of the database did not identify any homologous proteins. A LEU2 disruption at codon 104 was lethal, but disruption at codon 221 showed a temperature-sensitive conditional growth phenotype. Abnormalities were observed in some glycolytic enzyme levels.

Amino Acid Sequence↗
PubMed1995

Mutations in GCR1, a transcriptional activator of Saccharomyces cerevisiae glycolytic genes, function as suppressors of gcr2 mutations.

The Saccharomyces cerevisiae GCR1 and GCR2 genes affect expression of most of the glycolytic genes. Evidence for Gcr1p/Gcr2p interaction has been presented earlier and is now supported by the isolation of mutations in Gcr1p suppressing gcr2, as assessed by growth and enzyme assay. Four specific mutation sites were identified. Together with use of the two-hybrid system of Fields and Song, they show that Gcr1p in its N-terminal half has a potential transcriptional activating function as well as elements for interaction with Gcr2p, which perhaps acts normally to expose an otherwise cryptic activation domain on Gcr1p. Complementation of various gcr1 mutant alleles and results with the two-hybrid system also indicate that Gcr1p itself normally functions as an oligomer.

DNA-Binding Proteins↗
PubMed1994

Alteration of the substrate specificity of human lysozyme by site-specific intermolecular cross-linking.

Human lysozyme dimers were prepared by the intermolecular cross-linking of the monomer that contained the mutation of either Arg41 to Cys or Ala73 to Cys with a divalent maleimide compound. Among the three kinds of possible dimers only R41C-R41C dimer, in which the two catalytic clefts can come close to each other due to the proximity of the conjugation site to the active sites, turned out to be 2.3 times more specific to a polymer substrate, ethylene glycol chitin, as compared to an oligomer substrate, PNP-(GlcNAc)5. The result indicates that it is possible to alter the substrate specificity of an enzyme by artificially controlling the orientation of the active sites.

Alanine↗