Language：English   Publishing type：Research paper (scientific journal)  

In sake brewing, the steamed rice is used in 2 ways, added to sake-mash and making rice-koji. Rice-koji is made from the steamed rice by using koji starter, and its quality is an important determinant of the aroma/taste of sake. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by crossing 2 sake rice varieties, Gohyakumangoku and Yamadanishiki. Recently, we reported the characteristic components/metabolites in sake made from KOS by conducting metabolome analysis using UPLC-QTOF-MS. In this study, to investigate the effect of koji starter and sake rice cultivars on the sake metabolites, we performed small-scale sake-making tests using the above 3 rice cultivars and 3 koji starters. Finally, we demonstrated that some of the characteristic components/metabolites of sake from KOS are affected by the koji starter. Thus, in addition to rice cultivar, koji starter plays an important role for establishment/maintenance of the quality of the final product.

DOI： 10.1080/09168451.2020.1763154

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Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Sko1 plays a key role in the control of gene expression by osmotic and oxidative stress in yeast. We demonstrate that the decrease in chronological lifespan (CLS) of hog1 Delta cells was suppressed by SKO1 deletion. sko1 Delta single mutant cells were shown to have a longer CLS, thus implicating Sko1 in the regulation of their CLS.

DOI： 10.1080/09168451.2019.1571901

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Language：English   Publishing type：Research paper (scientific journal)  

Mutations frequently occur during breeding of sake yeasts and result in unexpected phenotypes. Here, genome editing tools were applied to develop an ideal nonfoam-forming sake yeast strain, K7GE01, which had homozygous awa1∆/awa1∆ deletion alleles that were responsible for nonfoam formation and few off-target mutations. High-dimensional morphological phenotyping revealed no detectable morphological differences between the genome-edited strain and its parent, while the canonical nonfoam-forming strain, K701, showed obvious morphological changes. Small-scale fermentation tests also showed differences between components of sake produced by K7GE01 and K701. The K7GE01 strain produced sake with significant differences in the concentrations of ethyl acetate, malic acid, lactic acid, and acetic acid, while K701 produced sake with more differences. Our results indicated genuine phenotypes of awa1∆/awa1∆ in sake yeast isolates and showed the usefulness of genome editing tools for sake yeast breeding.

DOI： 10.1080/09168451.2019.1631146

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Language：English   Publishing type：Research paper (scientific journal)  

In sake brewing, the steamed rice is used in two ways, added to sake-mash (as kake-mai) and making koji. The rice is an important determinant for the quality of sake, as the metabolites in sake affect its taste/aroma. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by genetically crossing two sake rice, Gohyakumangoku and Yamadanishiki. However, the metabolites in sake from KOS have not been analyzed. Here, to investigate the characteristic metabolites in sake from KOS, we performed two types of small-scale sake-fermentation tests changing only the rice used for kake-mai or total rice (both kake-mai and koji) by these three rice cultivars and examined the effect of KOS on sake metabolites by the metabolome analysis method using UPLC-QTOF-MS. We identified the peaks/metabolites, whose intensity in sake from KOS was higher/lower than those from the other cultivars. The brewing properties of KOS were partially characterized by this analysis.

DOI： 10.1080/09168451.2019.1608804

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Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The proper organization of microtubules is essential for many cellular functions. Microtubule organization and reorganization are highly regulated during the cell cycle, but the underlying mechanisms remain elusive. Here we characterized unusual interphase microtubule organization in fission yeast nuclear export mutant crm1-124. The mutant cells have an intranuclear microtubule bundle during interphase that pushes the nuclear envelope to assume a protruding morphology. We showed that the formation of this protruding microtubule bundle requires the nuclear accumulation of two microtubule-associated proteins (MAPs), Alp14/TOG and Mal3/EB1. Interestingly, the forced accumulation of Alp14 in the nucleus of wild type cells is sufficient to form the intranuclear microtubule bundle. Furthermore, the frequency of the intranuclear microtubule formation by Alp14 accumulated in the nucleus is prominently increased by a reduction in the nucleation activity of interphase cytoplasmic microtubules. We propose that properly regulated nucleocytoplasmic transport and maintained activity of cytoplasmic microtubule nucleation during interphase are important for the proper organization of interphase cy

DOI： 10.1016/j.bbrc.2018.06.135

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本醸造協会  

type:論文
「おいしさ」は，「食」に豊かさを付与する楽しみや喜びをともなう感覚的なもの(快感)である。ヒトが感じるおいしさは，複数の因子によって構成され，それらは物性的な要因と非物性的な要因に大別される。前者に関するおいしさの評価法は，味覚の受容体から脳内情報処理に至る経路などの分子基盤も含め，多くの知見が得られ研究が進展している。一方，後者に関するおいしさの評価法はいまだ不明な点が多い。このたび，我々は，チーズにおいて提案された複数の非物性的な要因を統合する総合的なおいしさの評価法を，日本酒のおいしさの評価に適用した。本稿では，料理とともに味わう日本酒のおいしさの評価，その新たな試みについて概略と展望を紹介したい。
identifier:922470
identifier:ZZ00020787

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Other Link： http://id.ndl.go.jp/bib/029066545

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We investigated the global landscape of Ca2+ homeostasis in budding yeast based on high-dimensional chemical-genetic interaction profiles. The morphological responses of 62 Ca2+-sensitive (cls) mutants were quantitatively analyzed with the image processing program CalMorph after exposure to a high concentration of Ca2+ After a generalized linear model was applied, an analysis of covariance model was used to detect significant Ca2+-cls interactions. We found that high-dimensional, morphological Ca2+-cls interactions were mixed with positive (86%) and negative (14%) chemical-genetic interactions, whereas one-dimensional fitness Ca2+-cls interactions were all negative in principle. Clustering analysis with the interaction profiles revealed nine distinct gene groups, six of which were functionally associated. In addition, characterization of Ca2+-cls interactions revealed that morphology-based negative interactions are unique signatures of sensitized cellular processes and pathways. Principal component analysis was used to discriminate between suppression and enhancement of the Ca2+-sensitive phenotypes triggered by inactivation of calcineurin, a Ca2+-dependent phosphatase. Finally, similarity of the interaction profiles was used to reveal a connected network among the Ca2+ homeostasis units acting in different cellular compartments. Our analyses of high-dimensional chemical-genetic interaction profiles provide novel insights into the intracellular network of yeast Ca2+ homeostasis.

DOI： 10.1091/mbc.E17-04-0216

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Cell polarity is coordinately regulated with the cell cycle. Growth polarity of the fission yeast Schizosaccharomyces pombe transits from monopolar to bipolar during G2 phase, termed NETO (new end take off). Upon perturbation of DNA replication, the checkpoint kinase Cdsl/CHK2 induces NETO delay through activation of Ca2+/calmodulin-dependent protein phosphatase calcineurin (CN). CN in turn regulates its downstream targets including the microtubule (MT) plus-end tracking CLIP170 homologue Tipl and the Casein kinase 1 gamma Cki3. However, whether and which Ca2+ signaling molecules are involved in the NETO delay remains elusive. Here we show that 3 genes (trp1322, vcxl and SPAC6c3.06c encoding TRP channel, antiporter and P-type ATPase, respectively) play vital roles in the NETO delay. Upon perturbation of DNA replication, these 3 genes are required for not only the NETO delay but also for the maintenance of cell viability. Trp1322 and Vcxl act downstream of Cds1 and upstream of CN for the NETO delay, whereas SPAC6c3.06c acts downstream of CN. Consistently, Trp1322 and Vcxl, but not SPAC6c3.06c, are essential for activation of CN. Interestingly, we have found that elevated extracellular Ca2+ per se induces a NETO delay, which depends on CN and its downstream target genes. These findings imply that Ca2+-CN signaling plays a central role in cell polarity control by checkpoint activation. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2017.07.129

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Sake yeast was developed exclusively in Japan. Its diversification during breeding remains largely uncharacterized. To evaluate the breeding processes of the sake lineage, we thoroughly investigated the phenotypes and differentiation of 27 sake yeast strains using high-dimensional, single-cell, morphological phenotyping. Although the genetic diversity of the sake yeast lineage is relatively low, its morphological diversity has expanded substantially compared to that of the Saccharomycescerevisiae species as a whole. Evaluation of the different types of breeding processes showed that the generation of hybrids (crossbreeding) has more profound effects on cell morphology than the isolation of mutants (mutation breeding). Analysis of phenotypic robustness revealed that some sake yeast strains are more morphologically heterogeneous, possibly due to impairment of cellular network hubs. This study provides a new perspective for studying yeast breeding genetics and micro-organism breeding strategies.

DOI： 10.1534/g3.117.044099

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Many factors contribute to palatability. In order to evaluate the palatability of Japanese alcohol sake paired with certain dishes by integrating multiple factors, here we applied an evaluation method previously reported for palatability of cheese by multiple regression analysis based on 3 subdomain factors (rewarding, cultural, and informational). We asked 94 Japanese participants/subjects to evaluate the palatability of sake (1st evaluation/E1 for the first cup, 2nd/E2 and 3rd/E3 for the palatability with aftertaste/afterglow of certain dishes) and to respond to a questionnaire related to 3 subdomains. In E1, 3 factors were extracted by a factor analysis, and the subsequent multiple regression analyses indicated that the palatability of sake was interpreted by mainly the rewarding. Further, the results of attribution-dissections in E1 indicated that 2 factors (rewarding and informational) contributed to the palatability. Finally, our results indicated that the palatability of sake was influenced by the dish eaten just before drinking.

DOI： 10.1080/09168451.2017.1336924

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DOI： 10.1073/pnas.1604047113

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Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

In high-quality sake brewing, the cerulenin-resistant sake yeast K1801 with high ethyl caproate-producing ability has been used widely; however, K1801 has a defective spindle assembly checkpoint (SAC). To identify the mutation causing this defect, we first searched for sake yeasts with a SAC-defect like K1801 and found that K13 had such a defect. Then, we searched for a common SNP in only K1801 and K13 by examining 15 checkpoint-related genes in 23 sake yeasts, and found 1 mutation, R48P of Cdc55, the PP2A regulatory B subunit that is important for the SAC. Furthermore, we confirmed that the Cdc55-R48P mutation was responsible for the SAC-defect in K1801 by molecular genetic analyses. Morphological analysis indicated that this mutation caused a high cell morphological variation. But this mutation did not affect the excellent brewing properties of K1801. Thus, this mutation is a target for breeding of a new risk-free K1801 with normal checkpoint integrity.

DOI： 10.1080/09168451.2016.1184963

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Publishing type：Research paper (scientific journal)   Publisher：ELIFE SCIENCES PUBLICATIONS LTD  

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.

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Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Identification of biologically active natural compounds that promote health and longevity, and understanding how they act, will provide insights into aging and metabolism, and strategies for developing agents that prevent chronic disease. The garlic-derived thioallyl compounds S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) have been shown to have multiple biological activities. Here we show that SAC and SAMC increase lifespan and stress resistance in Caenorhabditis elegans and reduce accumulation of reactive oxygen species (ROS). These compounds do not appear to activate DAF-16 (FOXO orthologue) or mimic dietary restriction (DR) effects, but selectively induce SKN-1 (Nrf1/2/3 orthologue) targets involved in oxidative stress defense. Interestingly, their treatments do not facilitate SKN-1 nuclear accumulation, but slightly increased intracellular SKN-1 levels. Our data also indicate that thioallyl structure and the number of sulfur atoms are important for SKN-1 target induction. Our results indicate that SAC and SAMC may serve as potential agents that slow aging.

DOI： 10.1038/srep21611

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Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Fission yeast undergoes growth polarity transition from monopolar to bipolar during G2 phase, designated NETO (New End Take Off). It is known that NETO onset involves two prerequisites, the completion of DNA replication and attainment of a certain cell size. However, the molecular mechanism remains unexplored. Here, we show that casein kinase 1 gamma, Cki3 is a critical determinant of NETO onset. Not only did cki3 Delta cells undergo NETO during G1- or S-phase, but they also displayed premature NETO under unperturbed conditions with a smaller cell size, leading to cell integrity defects. Cki3 interacted with the polarity factor Tea1, of which phosphorylation was dependent on Cki3 kinase activity. GFP nanotrap of Tea1 by Cki3 led to Tea1 hyperphosphorylation with monopolar growth, whereas the same entrapment by kinase-dead Cki3 resulted in converse bipolar growth. Intriguingly, the Tea1 interactor Tea4 was dissociated from Tea1 by Cki3 entrapment. Mass spectrometry identified four phosphoserine residues within Tea1 that were hypophosphorylated in cki3 Delta cells. Phosphomimetic Tea1 mutants showed compromised binding to Tea4 and NETO defects, indicating that these serine residues a

DOI： 10.1111/gtc.12309

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

In the brewing of high-quality sake such as Daiginjo-shu, the cerulenin-resistant sake yeast strains with high producing ability to the flavor component ethyl caproate have been used widely. Genetic stability of sake yeast would be important for the maintenance of both fermentation properties of yeast and quality of sake. In eukaryotes, checkpoint mechanisms ensure genetic stability. However, the integrity of these mechanisms in sake yeast has not been examined yet. Here, we investigated the checkpoint integrity of sake yeasts, and the results suggested that a currently used cerulenin-resistant sake yeast had a defect in spindle assembly checkpoint (SAC). We also isolated a spontaneous cerulenin-resistant sake yeast FAS2-G1250S mutant, G9CR, which showed both high ethyl caproate-producing ability and integrity/intactness of the checkpoint mechanisms. Further, morphological phenotypic robustness analysis by use of CalMorph supported the genetic stability of G9CR. Finally, we confirmed the high quality of sake from G9CR in an industrial sake brewing setting.

DOI： 10.1080/09168451.2015.1020756

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Calcineurin, which is a Ca2+/calmodulin-dependent protein phosphatase, is a key mediator in calcium signaling in diverse biological processes and of clinical importance as the target of the immunosuppressant FK506. To identify a mutant(s) in which calcine

DOI： 10.1080/09168451.2014.1003132

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Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Cell polarity is essential for various cellular functions during both proliferative and developmental stages, and it displays dynamic alterations in response to intracellular and extracellular cues. However, the molecular mechanisms underlying spatiotemporal control of polarity transition are poorly understood. Here, we show that fission yeast Cki3 (a casein kinase 1 gamma homolog) is a critical regulator to ensure persistent monopolar growth during S phase. Unlike the wild type, cki3 mutant cells undergo bipolar growth when S phase is blocked, a condition known to delay transition from monopolar to bipolar growth (termed NETO [new end takeoff]). Consistent with this role, Cki3 kinase activity is substantially increased, and cells lose their viability in the absence of Cki3 upon an S-phase block. Cki3 acts downstream of the checkpoint kinase Cds1/Chk2 and calcineurin, and the latter physically interacts with Cki3. Autophosphorylation in the C terminus is inhibitory toward Cki3 kinase activity, and calcineurin is responsible for its dephosphorylation. Cki3 localizes to the plasma membrane, and this localization requires the palmitoyltransferase complex Erf2-Erf4. Membrane localizati

DOI： 10.1128/MCB.01465-14

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Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature-sensitive fission yeast strains, rrs1-D14/22G and rrs1-L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1-84 (D22/30G) and rrs1-124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two-hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts. Copyright (c) 2015 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.3083

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Low protein content and sufficient grain rigidity are desired properties for the rice used in high-quality sake brewing such as Daiginjo-shu (polishing ratio of the rice, less than 50%). Two kinds of rice, sake rice (SR) and cooking rice (CR), have been used for sake brewing. Compared with those of SR, analyses of CR for high-quality sake brewing using highly polished rice have been limited. Here we described the original screening of late-maturing CR Sensyuraku (SEN) as rice with low protein content and characterization of its properties for high-quality sake brewing. The protein content of SEN was lower than those of SR Gohyakumangoku (GOM) and CR Yukinosei (YUK), and its grain rigidity was higher than that of GOM. The excellent properties of SEN with respect to both water-adsorption and enzyme digestibility were confirmed using a Rapid Visco Analyzer (RVA). Further, we confirmed a clear taste of sake produced from SEN by sensory evaluation. Thus, SEN has excellent properties, equivalent to those of SR, for high-quality sake brewing.

DOI： 10.1080/09168451.2014.930329

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We applied Chrysanthemum flower oil (CFO) to a hyperuricemia model by feeding rats a hyperuricemia-inducing diet (HID) and investigated its effect on serum uric acid (SUA) levels and its mode of action. CFO is the oily fraction that contains polyphenols derived from chrysanthemum flowers. Oral administration of CFO to HID-fed rats significantly decreased their SUA levels. It also inhibited xanthine oxidase activities in the liver and increased urine uric acid levels. The effects of CFO on the renal gene expressions that accompanied the induction of hyperuricemia were comprehensively confirmed by DNA microarray analysis. The analysis showed up-regulation of those genes for uric acid excretion by CFO administration. These results suggest that CFO suppresses the increase in SUA levels via two mechanisms: suppression of uric acid production by inhibition of xanthine oxidase in the liver and acceleration of its excretion by up-regulation of uric acid transporter genes in the kidney.

DOI： 10.1080/09168451.2014.890028

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The Japanese high-quality sake Daiginjo-shu is made from highly polished rice (polishing ratio, less than 50%). Here we showed that the sake rice Koshitanrei (KOS) has an excellent polishing property. Rice grains of KOS had the same lined white-core region as the sake rice Yamadanishiki (YAM). The grain rigidity/hardness of KOS was higher than that of the sake rice Gohyakumangoku (GOM). The loss ratio of KOS after high polishing by an industrial polishing machine was lower than that of GOM. Further, a clear taste of sake produced from KOS was confirmed by sensory evaluation.

DOI： 10.1271/bbb.130515

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Hog1 of Saccharomyces cerevisiae is activated by hyperosmotic stress, and this leads to cell-cycle delay in G(1), but the mechanism by which cells restart from G(1) delay remains elusive. We found that Whi3, a negative regulator of G(1) cyclin, counteracted Hog1 in the restart from G(1) delay caused by osmotic stress. We have found that phosphorylation of Ser-568 in Whi3 by RAS/cAMP-dependent protein kinase (PKA) plays an inhibitory role in Whi3 function. In this study we found that the phosphomimetic Whi3 S568D mutant, like the Delta whi3 strain, slightly suppressed G(1) delay of Delta hog1 cells under osmotic stress conditions, whereas the non-phosphorylatable S568A mutation of Whi3 caused prolonged G(1) arrest of Delta hog1 cells. These results indicate that Hog1 activity is required for restart from G(1) arrest under osmotic stress conditions, whereas Whi3 acts as a negative regulator for this restart mechanism.

DOI： 10.1271/bbb.130260

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

In eukaryotes, cell morphogenesis is regulated coordinately with the cell cycle. In fission yeast, the morphogenesis network MOR (morphogenesis Orb6 network) consists of 5 conserved proteins, Pmo25, Nak1, Mor2, Orb6, and Mob2, and is essential for cell polarity control and cell separation following cytokinesis. Here we show that the conserved leucine-rich repeat protein Lrp1 is required for cell morphogenesis as a newly recognized component of MOR. Lrp1 has 4 leucine-rich repeats in its N-terminus and is a homolog of the budding yeast Sog2, which is a component of the RAM network (regulation of Ace2 activity and cellular morphogenesis). Lrp1 was essential for both cell growth and cell morphogenesis as were the other MOR components. Lrp1 was localized to the SPBs (spindle pole bodies, the yeast equivalent of the animal centrosome) throughout the cell cycle and to the medial ring during cytokinesis. Lrp1 interacted with Nak1 and was important for Orb6 kinase activity. Thus Lrp1 proved to function upstream of Orb6 in cell morphogenesis.

DOI： 10.1271/bbb.130064

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Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The Start/G(1) phase in the cell cycle is an important period during which cells determine their developmental fate, onset of mitotic progression, or the switch to developmental stages in response to both external and internal signals. In the budding yeast Saccharomyces cerevisiae, Whi3, a negative regulator of the G(1) cyclins, has been identified as a positive regulator of cell size control and is involved in the regulation of Start. However, the regulatory pathway of Whi3 governing the response to multiple signals remains largely unknown. Here, we show that Whi3 is phosphorylated by the Ras/cAMP-dependent protein kinase (PKA) and that phosphorylation of Ser-568 in Whi3 by PKA plays an inhibitory role in Whi3 function. Phosphorylation of Whi3 by PKA led to its decreased interaction with CLN3 G(1) cyclin mRNA and was required for the promotion of G(1)/S progression. Furthermore, we demonstrate that the phosphomimetic S568D mutation of Whi3 prevented the developmental fate switch to sporulation or invasive growth. Thus, PKA modulated the function of Whi3 by phosphorylation, thus implicating PKA-mediated modulation of Whi3 in multiple cellular events.

DOI： 10.1074/jbc.M112.402214

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Language：English   Publishing type：Research paper (scientific journal)  

In the fermentation industry, the traceability of microorganisms during the process is important to ensure safety and efficacy. Ethyl carbamate, a group-2A carcinogen, is produced from ethanol and urea during the storage of food/alcoholic beverages. We isolated non-urea-producing sake yeast car1 mutants carrying a discriminable molecular marker, and demonstrated, by the use of PCR assays, that these mutants are useful for traceability analysis and identification during the sake brewing process.

DOI： 10.1271/bbb.130621

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To investigate the biological activity of a novel 24-membered macrolide compound, JBIR-19, isolated from the culture broth of the entomopathogenic fungus Metarhizium sp. fE61, morphological changes in yeast cells were examined using the automated image-processing program CalMorph. Principal components analysis was used to elucidate dynamic changes in the phenotypes, revealing two independent effects of JBIR-19 in yeast cells: bud elongation and increased size of the actin region. Using a fitness assay, we identified the genes required for robust growth in the presence of JBIR-19. Among these were CCW12, YLR111W, and DHH1, which are also involved in abnormal bud morphology. Based on these results and others, we predict intracellular targets of JBIR-19 and its functional interactions. © 2011 Federation of European Microbiological Societies.

DOI： 10.1111/j.1567-1364.2011.00770.x

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We show that the concentration of total free fatty acids (FFAs) in sake produced by yeast with high productivity of ethyl caproate could be approximated by the concentration of 2 FFAs, caproic and caprylic acids. Measurement of the total FFAs concentration by an enzymatic method proved useful for both estimating the ethyl caproate concentration in sake and also for yeast breeding. © 2012 W. S. Maney &amp
Son Ltd.

DOI： 10.1271/bbb.110698

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We conducted a mitotic localization study on gene products encoded by 56 uncharacterized fission yeast ORFs that were transcriptionally up-regulated during meiotic division. Despite meiotic gene induction, these genes were expressed during mitosis as well. Seven gene products were localized in the nucleus and/or chromatin; another one was a mitosis-specific spindle pole body component and, intriguingly, its human homologue was also localized in the centrosome of cultured HeLa cells. Two products appeared to be localized in cytoplasmic microtubules, whereas four were mitochondrial proteins. Three other proteins were found in the medial ring upon cytokinesis and another was localized on the entire cell periphery. The remaining 38 proteins were detected in the cytoplasm and showed varied spatial patterns. This systematic study helps our integrated understanding of all the protein functions in the fission yeast as a eukaryotic model.

DOI： 10.1271/bbb.110558

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

In eukaryotic cells, Ca2+-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca2+/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca2+ sensitivity of zds1 Delta cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca2+ signaling and life span in yeast. Here we show that Ca2+ affected the replicative life span (RLS) of yeast. Increased external and intracellular Ca2+ levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1 Delta cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted aging without impairing the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a regulation of RLS even under normal growth condition. Thus, our results indicate that Ca2+ homeostasis/Ca2+ signaling are required to regulate longevity in budding yeast.

DOI： 10.1074/jbc.M111.231415

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We tested the effect of oral administration of fermented sake lees with lactic acid bacteria (FESLAB) on a murine model of allergic rhinitis upon immunization and nasal sensitization with ovalbumin (OVA). We used Lactobacillus paracasei NPSRIk-4 (isolated from sake lees), and L. brevis NPSRIv-8 (from fermented milk) as starter strains to produce the FESLAB. Oral FESLAB administration resulted in the development of significantly fewer sneezing symptoms than those seen in sham control animals given sterile water. We also found that FESLAB suppressed the allergen-induced degranulation of RBL2H3 rat basophilic leukemia cells.

DOI： 10.1271/bbb.100541

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Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

How cell morphology and the cell cycle are coordinately regulated is a fundamental subject in cell biology. In fission yeast, 2 germinal center kinases (GCKs), Sid1 and Nak1, play an essential role in septation/cytokinesis and cell separation/cell polarity control, respectively, as components of the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR). Here we show that a third GCK, Ppk11, is also required for efficient cell separation particularly, at a high temperature. Although Ppk11 is not essential for cell division, this kinase plays an auxiliary role in concert with MOR in cell morphogenesis. Ppk11 physically interacts with the MOR component Pmo25 and is localized to the septum, by which Ppk11 is crucial for Pmo25 targeting/accumulation to the septum. The conserved C-terminal WDF motif of Ppk11 is essential for both septum accumulation of Pmo25 and efficient cell separation. In contrast its kinase activity is required only for cell separation. Thus, both interaction of Ppk11 with Pmo25 and Ppk11 kinase activity are critical for efficient cell separation.

DOI： 10.1074/jbc.M110.176867

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Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

The mechanisms that regulate cytoskeletal remodeling during the transition between mitosis and interphase are poorly understood. In fission yeast the MOR pathway promotes actin polarization to cell tips in interphase, whereas the SIN signaling pathway drives actomyosin ring assembly and cytokinesis. We show that the SIN inhibits MOR signaling in mitosis by interfering with Nak1 kinase-mediated activation of the most downstream MOR component, the NDR family kinase Orb6. Inactivation of the MOR may be a key function of the SIN because attenuation of MOR signaling rescued the cytokinetic defects of SIN mutants and allowed weak SIN signaling to trigger ectopic cytokinesis. Furthermore, failure to inhibit the MOR is toxic when the cell division apparatus is compromised. Together, our results reveal a mutually antagonistic relationship between the SIN and MOR pathways, which is important for completion of cytokinesis and coordination of cytoskeletal remodeling at the mitosis-to-interphase transition.

DOI： 10.1083/jcb.201002055

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

In eukaryotes, cell polarity is essential for cell proliferation, differentiation, and development. It is regulated in 3 steps: establishment, maintenance, and transition. Compared to current knowledge of establishment and maintenance, the mechanism regulating the transition of cell polarity is poorly understood. In fission yeast during the G2 phase, growth polarity undergoes a dramatic transition, from monopolar to bipolar growth (termed NETO: new end take off). In this study, we screened systematically for protein kinases related to NETO using a genome-wide kinase deletion library. Analysis of these deletions suggested that 35 and 2 kinases had a putative positive and a negative role, respectively, in NETO. Moreover, 5 kinases were required for NETO-delay in the G1-arrested cdc10 mutant. These results suggest that many signaling pathways are involved in the regulation of NETO.

DOI： 10.1271/bbb.100223

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Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general transcriptional corepressor. This repression is mediated by recruitment of the Tup1-Cyc8 complex to target promoters through sequence-specific DNA-binding proteins such as Sko1, which mediates the HOG pathway-dependent regulation. We identified tup1 and cyc8 mutant alleles as the suppressor of osmo-sensitivity of the hog1 Delta strain. In these mutants, although the expression of the genes under the control of DNA-binding proteins other than Sko1 was apparently normal, the Sko1-regulated genes GRE2 and AHP1 were derepressed under non-stress conditions, suggesting that the Tup1 and Cyc8 mutant proteins were specifically defective in the repression of the Sko1-dependent genes. Chromatin immunoprecipitation analyses of the GRE2 promoter in the mutants demonstrated that the Sko1-Tup1-Cyc8 complex was localized to the promoter, together with Gcn5/SAGA, suggesting that the erroneous recruitment of SAGA to the promoter led to the derepression. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.01.033

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Yeast cell morphology can be treated as a quantitative trait using the image processing software CalMorph. In the present study, we investigated Ca2+-induced morphological changes in Ca2+-sensitive (cls) mutants of Saccharomyces cerevisiae, based on the discovery that the characteristic Ca2+-induced morphological changes in the Ca2+-sensitive mutant zds1 reflect changes in the Ca2+ signaling-mediated cell cycle control pathway. By applying hierarchical cluster analysis to the quantitative morphological data of 58 cls mutants, 31 of these mutants were classified into seven classes based on morphological similarities. The patterns of morphological change induced by Ca2+ in one class differed from those of another class. Based on the results obtained using versatile methods for phenotypic analysis, we conclude that a high concentration of Ca2+ exerts a wide variety of effects on yeast and that there are multiple Ca2+-regulatory pathways that are distinct from the Zds1p-related pathway.

DOI： 10.1128/EC.00012-07

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

In fission yeast, the conserved proteins, MO25/ Pmo25, GC kinase/Nak1, Furry/Mor2, NDR kinase/ Orb6, and Mob2, constitute the morphogenesis Orb6 network (MOR). Previously we showed that Pmo25 functions as an upstream component of MOR and that it plays a connecting role between the septation initiation network (SIN) and MOR. Here we establish a Pmo25-associated kinase assay and show that the activity is dependent on Nak1/MOR and Sid1/SIN.

DOI： 10.1271/bbb.60574

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The Ca2+-activated pathways in Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1p, a negative regulatory kinase that inhibits the Cdc28p/Clb complex. We isolated the YAP1 gene as a multicopy suppressor of calcium sensitivity owing to the loss of ZDS1, a negative regulator of SWE1 and CLN2 gene expression. YAP1 deletion on a zds1 Delta background exacerbated the Ca2+-related phenotype. Yap1p was degraded in a calcineurin-dependent manner when cells were exposed to calcium. In yap1 Delta cells, the expression level of the RPN4 gene encoding a transcription factor for the subunits of the ubiquitin-proteasome system was diminished. The deletion of YAP1 gene or RPN4 gene led to the accumulation of Swe1p and Cln2p. Yap1p was a substrate of calcineurin in vivo and in vitro. The calcineurin-mediated Yap1p degradation seems to be a long adaptive response that assures a G(2) delay in response to a stress that causes the activation of the calcium signalling pathways.

DOI： 10.1038/sj.embor.7400647

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DOI： 10.1091/mbc.E05-08-0802

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DOI： 10.1007/s00294-005-0051-0

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DOI： 10.1038/sj.embor.7400540

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Protein kinase C, a highly conserved signaling molecule among eukaryotes, has been implicated in the regulation of cellular processes such as cell proliferation and polarized growth. In Saccharomyces cerevisiae, the unique protein kinase C Pkc1p is thought to have multiple functions, including the activation of the Mpk1p (Slt2p) MAP kinase pathway, which is essential for cell wall construction and bud emergence. However, little is known about the other functions of Pkc1p. In the course of screening for the mutants that suppress the Ca2+-sensitivity phenotype of the Ca2+-sensitive strain zds Delta, we isolated a novel mutant allele (scz6/pkc1-834) of PKC1. Unlike the previously characterized PKC1 allele stt1-1, heat-shock-induced Mpk1p activation and cell-wall integrity were not impaired in the pkc1-834 mutant. By contrast, the mutant was defective in the maintenance of Ca2+-induced F-actin polarization in a manner independent of Mpk1p activation. This phenotype was caused by a decreased expression level of the G(1) cyclin Cln2p. The Rho1 small G protein molecular switch was suggested to be involved in the novel Pkc1p function. The Pkc1p novel function was required for posttranscriptional upregulation of Cln2p and appeared to be important for the coordinated regulation of polar bud growth and the cell cycle.

DOI： 10.1242/jcs.02535

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Multidrug resistance ABC transporter Pdr5p of Saccharomyces cerevisiae is particularly important due to its ability to export a wide range of unrelated substrates. To clarify its function, we generated Pdr5p mutants by random mutagenesis and screened for mutants with altered drug specificity in vivo by using 5 drug compounds. Nine point mutations that caused significant changes in drug specificity distributed throughout the length of Pdr5p, namely, in the extracellular, transmembrane or cytoplasmic regions of the transporter. We then investigated their effects upon drug resistance, using 36 chemically related or distinct substrates. From this study, overall geometry of the Pdr5p was suggested to contribute in acquiring the enormous range of drug specificity. Based on their ability to inhibit the growth of the mutant strains, the 36 tested drugs were classified into: drugs to which the mutants responded differently (Group 1), drugs to which all the mutants showed sensitivity (Group 2), and drugs to which all the mutants exhibited resistance (Group 3). The ability of the compounds to be partitioned to the plasma membrane seemed an important factor for recognition by Pdr5p.

DOI： 10.1111/j.1365-2443.2005.00847.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Deletion of PDR5 gene (Delta pdr5) in Saccharomyces cerevisiae led to increased resistance to calcium. The cellular Ca2+ level in the presence of high calcium as estimated by reporter assay in Delta pdr5 cells was significantly lower than that in wild-type cells. Membrane Pdr5p levels diminished rapidly during incubation with high calcium in a manner dependent on calcineurin and Pep4p, suggesting a feedback regulatory mechanism for Pdr5p abundance.

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/nogeikagaku1924.76.734

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

To investigate the mechanism coupling growth (protein synthesis) with cell division, we examined the relationship between the tyrosine kinase Wee1 that inhibits Cdc2-Cdc13 mitosis-inducing kinase by phosphorylating it, and protein synthesis inhibition in fission yeast. The wee1-50 mutant showed supersensitivity to protein synthesis inhibitor, cycloheximide, Wee1 was essential for the G(2) delay upon a partial inhibition of protein synthesis. Indeed, the protein synthesis inhibition caused an increase in the Wee1 protein by the Sty1/Spc1 MAPK-dependent transcriptional and the Sty1/Spc1 MAPK-independent post-transcriptional regulations. Further, the results indicated that the post-transcriptional regulation is important for the G(2) delay. (C) 2000 Federation of European Biochemical Societies, Published by Elsevier Science B.V. All rights reserved.

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The Saccharomyces cerevisiae crv mutants (crv1, 2, 3 and 4) exhibit phenotypes, such as calcium resistance and vanadate sensitivity, which are apparently similar to those of calcineurin-deficient mutants, We have cloned and characterized the CRV4 gene that complements all the phenotypes of the crv4 mutant. DNA sequencing revealed that CRV4 is identical to the previously cloned gene TTP1, which encodes a type II membrane protein of unknown function. Deletion of CRV4/TTP1 causes no obvious phenotype except for Ca2+ resistance and vanadate sensitivity, but is synthetically lethal in combination with a deletion of MPK1, in a manner which is suppressible by the addition of an osmotic stabilizer. In medium containing sorbitol as an osmotic stabilizer, the cnb1 mpk1 ttp1 triple mutant exhibits a more severe growth defect than does any of the double mutants cnb1 ttp1, cnb1 mpk1 or mpk1 ttp1. A high Ca2+ concentration (50 mM) or a constitutively active form of calcineurin partially suppresses the growth defect of the mpk1 ttp1 double mutant. These results indicate that Ttp1 participates in a cellular event essential for growth and morphogenesis, in parallel with the pathways involving Mpk1 MAP kinase and calcineurin.

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：日本醸造協会・日本醸造学会  

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The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.

DOI： 10.1271/bbb.68.968

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The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.

DOI： 10.1271/bbb.68.968

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Because Ca2+ signaling of budding yeast, through the activation of calcineurin and the Mpk1/Slt2 mitogen-activated protein kinase cascade, performs redundant function(s) in the events essential for growth, the simultaneous deletion of both these pathways (Deltacnb1 Deltampk1) leads to lethality. A PTC4 cDNA that encodes a protein phosphatase belonging to the PP2C family was obtained as a high dosage suppressor of the lethality of Deltacnb1 Deltampk1 strain. Overexpression of PTC4 led to a decrease in the high osmolarity-induced Hog1 phosphorylation, and HOG1 deletion remarkably suppressed the synthetic lethality, indicating an antagonistic role of the high osmolarity glycerol (HOG) pathway and the Ca2+ signaling pathway in growth regulation. The calcineurin-Crz1 pathway was required for the down-regulation of the HOG pathway. Analysis of the time course of actin polarization, bud formation, and the onset of mitosis in synchronous cell cultures demonstrated that calcineurin negatively regulates actin polarization at the bud site, whereas the HOG pathway positively regulates bud formation at a later step after actin has polarized.

DOI： 10.1074/jbc.M306098200

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Language：Japanese   Publisher：日本農芸化学会  

DOI： 10.1271/kagakutoseibutsu1962.41.651

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JOHN WILEY & SONS LTD  

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

To dissect the action mechanism of reveromycin A (RM-A), a G(1)-specific inhibitor, a Saccharomyces cerevisiae dominant mutant specifically resistant to RM-A, was isolated from a strain in which the genes implicated in nonspecific multidrug resistance had been deleted. The mutant gene (YRR2-1) responsible for the resistance was identified as an allele of the ILS1 gene encoding tRNA(Ile) synthetase (MeRS). The activity of HeRS, but not several other aminoacyl-tRNA synthetases examined in wild type cell extract, was highly sensitive to RM-A (IC50 = 8 ng/ml). The IleRS activity of the YRR2-1 mutant was 4-fold more resistant to the inhibitor compared with that of wild type. The mutation IleRS(N660D), near the KMSKS consensus sequence commonly found in the class I aminoacyl transferases, was found to be responsible for RM-A resistance. Moreover, overexpression of the ILS1 gene from a high-copy plasmid conferred RM-A resistance. These results indicated that IleRS is a target of RM-A in vivo. A defect of the GCN2 gene led to decreased RM-A resistance. IleRS inhibition by RM-A led to transcriptional activation of the ILS1 gene via the Gcn2-Gcn4 general amino acid control pathway, and this autoregulation seemed to contribute to RM-A resistance.

DOI： 10.1074/jbc.M203827200

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：化学同人  

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Accurate chromosome segregation is dependent upon the integrity of mitotic spindles, which pull each pair of sister chromatids towards opposite poles. In this study, we have characterised fission yeast pop3-5235, a diploidising mutant that is impaired in genome stability. Pop3 is the same as Wat1, a conserved protein containing 7 WD repeats. Pop3/Wat1 has also been isolated from a two-hybrid screen as a binding partner to Prp2, the large subunit of the essential splicing factor U2AF. In wat1 mutants, the cellular amount of alpha -tubulin is decreased to very low levels, which results in compromised microtubules and spindles, consequently leading to unequal chromosome separation. Further analysis shows that, in spite of the binding between Wat1 and Prp2, Wat1 may not be involved directly in splicing reactions per se. Instead, we find that Wat1 is required for the maintenance of alpha -tubulin mRNA levels; moreover, transcript levels of genes other than the alpha -tubulin gene are also equally decreased in this mutant. Wild-type Wat1, but not the mutant protein, forms a large complex in the cell with several other proteins, suggesting that Wat1 functions as a structural linker in the complex. The results suggest that Wat1 plays a role in mRNA maturation as a coupling protein between splicing and synthesis and/or stabilisation.

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Background: Protein phosphatase 2A (PP2A) is serine/threonine phosphatase distributed in eukaryotes from yeast to human, and plays pivotal roles in diverse cellular functions such as metabolism, cell cycle progression, gene expression and development. PP2A holoenzyme is a heterodimer of a catalytic subunit C and a regulatory subunit A, or a heterotrimer of C, A and a variable regulatory subunit consisting of three families; B, B', and PR72. Specific functions for each variable subunit are not well understood.
Results: Two fission yeast genes pbp1(+) and pbp2(+) homologous to the regulatory subunit B' were isolated. Physical in vivo interaction of the gene products with the catalytic subunit was demonstrated. A double disruption haploid mutant (Delta pbp1 Delta pbp2) showed growth defect, cell shape and size abnormality, multiseptation and anucleated cell formation due to abnormality in septum positioning. These phenotypes were suppressed by human B' cDNA, indicating the striking conservation of the B' function from yeast to human. Over-expression of fission yeast B' led to growth defects, a loss of cell shape polarity, septal abnormality and anucleated cell formation. Delta pbp1 Delta pbp2 and pbp1 null haploids were hypersensitive to calcineurin inhibitors, cyclosporin A and FK506, with which the mutants underwent arrest at post-anaphase and cell lysis. Double disruption of calcineurin and pbp1(+'), but not pbp2(+), genes led to synthetic lethality.
Conclusion: The fission yeast B' subunit of PP2A plays critical roles in cell shape control and septum formation, and shares essential functions with calcineurin for viability, possibly through their role in cytokinesis and cell wall integrity.

DOI： 10.1046/j.1365-2443.2001.00429.x

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：日本生物工学会  

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The Ca2+-activated pathways of Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1, a negative regulatory kinase that inhibits the Cdc28-Clb complex. Calcineurin and Mpk1 activate Swe1 at the transcriptional and post-translational level, respectively, and both pathways are essential for the cell cycle delay. Our genetic screening identified the MCK1 gene, which encodes a glycogen synthetase kinase-3 family protein kinase, as a component of the Ca2+ signaling pathway. Genetic analyses indicated that Mck1 functions downstream of the Mpk1 pathway and down-regulates Hsl1, an inhibitory kinase of Swe1, In medium with a high concentration of Ca2+, Hsl1 was delocalized from the bud neck and destabilized in a manner dependent on both calcineurin and Mck1, Calcineurin was required for the dephosphorylation of autophosphorylated Hsl1. The E3 ubiquitin ligase complex SCFCdc4, but not the anaphase-promoting complex (APC), was essential for Hsl1 destabilization. The Ca2+-activated pathway may play a role in the rapid inactivation of Hsl1 at the cell cycle stage(s) when APC activity is low.

DOI： 10.1093/emboj/20.5.1074

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DOI： 10.1046/j.1365-2443.2001.00429.x

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To investigate the mechanism coupling growth (protein synthesis) with cell division, we examined the relationship between the tyrosine kinase Wee1 that inhibits Cdc2-Cdc13 mitosis-inducing kinase by phosphorylating it, and protein synthesis inhibition in fission yeast. The wee1-50 mutant showed supersensitivity to protein synthesis inhibitor, cycloheximide. Wee1 was essential for the G2 delay upon a partial inhibition of protein synthesis. Indeed, the protein synthesis inhibition caused an increase in the Wee1 protein by the Sty1/Spc1 MAPK-dependent transcriptional and the Sty1/Spc1 MAPK-independent post-transcriptional regulations. Further, the results indicated that the post-transcriptional regulation is important for the G2 delay. Copyright (C) 2000 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(00)02299-7

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An inappropriate activation of a signaling pathway in yeast often has a deleterious physiological effect and causes various defects, including growth defects. In a certain genetic background (deltazds1) of Saccharomyces cerevisiae, the cell-cycle progression in G2 is specifically blocked in the medium with CaCl2 by the hyperactivation of the Ca2+-signaling pathways. Here, we developed a novel drug screening procedure designed to detect the active compounds that specifically attenuate the Ca2+-signaling activity on the basis of the ability to abrogate the growth defect of the cells suffering from the hyperactivated Ca2+ signal. Using known calcineurin inhibitors as model compounds, we have established the screening conditions for the drugs that suppress the Ca2+-induced growth inhibition. An indicator strain with an increased drug sensitivity was constructed with a syr1/erg3 null mutation.

DOI： 10.1271/bbb.64.1942

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DOI： 10.1016/S0014-5793(00)02299-7

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To identify new proteins involved in Mn2+ homeostasis, we isolated Mn2+-resistant mutants of Saccharomyces cerevisiae starting from a calcineurin-deficient, Mn2+ hypersensitive strain (Delta cmp1 Delta cmp2). The mutations were found to lie in the PMR1 gene, known to encode a "P-type" Ca2+-ATPase that transports Ca2+ and Mn2+ from the cytosol to the Golgi apparatus. A second gene, AHP1, was cloned as a suppressor of the Mn2+ tolerance of a Delta cmp1 Delta cmp2 pmr1 mutant. Ahp1p was recently described as a thioredoxin peroxidase type II, an antioxidant protein with alkyl hydroperoxide defense properties in yeast. AHP1 disruption in strain W303 decreased tolerance to Mn2+ and H2O2. We found that a GFP-Ahp1p fusion construct was in the cytosol when cells were grown in glucose, and in the mitochondria when cells were grown in oleate. Based on Mn2+ transport data, we concluded that Ahp1p is involved in cellular Mn2+ homeostasis in trafficking of Mn2+ from cytosol to mitochondria and from cytosol for export across the plasma membrane.

DOI： 10.1271/bbb.63.1871

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Language：English   Publisher：AMER SOC CELL BIOLOGY  

We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules. Alp11B contains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21(E) does not. both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11(B) interacts with both a-tubulin and Alp21(E), but not with the cofactor D homologue Alp1, whereas Alp21(E) also interacts with Alpl(D). The cellular amount of a-tubulin is decreased in both alp1 and alp(11) mutants. Overproduction of Alp11(B) results in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of alpha-tubulin. Both hull-length Alp11(B) and the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to alpha-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21(+) or alp(1+), whereas alp(21) deletion is rescued by overexpression of either alp11(+) or alp21(+). The alp1(+) but not alp11(+). Finally, the alp1 mutant is not complemented by either alp11(+) or alp21(+). The results suggest that cofactors operate in a linear pathway (Alp11(B)-Alp21(E)-Alp(D)), each with distinct roles.

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We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules. Alp11B contains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21(E) does not. both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11(B) interacts with both a-tubulin and Alp21(E), but not with the cofactor D homologue Alp1, whereas Alp21(E) also interacts with Alpl(D). The cellular amount of a-tubulin is decreased in both alp1 and alp(11) mutants. Overproduction of Alp11(B) results in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of alpha-tubulin. Both hull-length Alp11(B) and the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to alpha-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21(+) or alp(1+), whereas alp(21) deletion is rescued by overexpression of either alp11(+) or alp21(+). The alp1(+) but not alp11(+). Finally, the alp1 mutant is not complemented by either alp11(+) or alp21(+). The results suggest that cofactors operate in a linear pathway (Alp11(B)-Alp21(E)-Alp(D)), each with distinct roles.

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Background: Elongation factor lo (EF1 alpha), an essential component of the eukaryotic translational machinery, has been shown to possess various biochemical and biological activities, including F-actin-binding and -bundling, microtubule-severing, and the activity of making fibroblasts highly susceptible to transformation. However, our understanding of the biological significance of EF1 alpha with respect to these various biochemical or biological activities remains Limited. Here we report the identification of EF1 alpha-encoding genes as genes whose over-expression causes aberrant cell morphology in fission yeast.
Results: Overproduction of EF1 alpha caused aberrant cell morphology-elliptic, curved or branched-and growth defects in yeast cells at high temperatures. EF1 alpha-overproducing cells showed a supersensitivity to the actin inhibitor cytochalasin D and to the tubulin inhibitor thiabendazole. Genetic analyses using cdc mutants suggested that excess EF1 alpha disturbed the establishment and the maintenance of growth polarity in the G1 phase by preventing the localization of F-actin to the polarized growing site and the organization of microtubules. Results from DNase I column chromatography indicated that EF1 alpha was bound to G-actin. Indeed, the fission yeast actin was immunoprecipitated along with EF1 alpha. Moreover, the temperature sensitivity caused by the overproduction of EF1 alpha was restored by co-overproduction of actin.
Conclusions: Fission yeast EF1 alpha has the ability to alter the cell morphology of yeast by affecting the control of actin and microtubule cytoskeletons.

DOI： 10.1046/j.1365-2443.1999.00279.x

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In fission yeast protein kinase C homologues (Pck1 and Pck2) are essential for cell morphogenesis. We have isolated mok1(+) in a genetic screen to identify downstream effecters for Pck1/2. mok1(+) is essential for viability and encodes a protein that has several membrane-spanning domains and regions homologous to glucan metabolic enzymes. mok1 mutant shows abnormal cell shape, randomization of F-actin and weak cell wall. Biochemical analysis shows that Mok1 appears to have a-glucan synthase activity. Mok1 localization undergoes dramatic alteration during the cell cycle. It localizes to the growing tips in interphase, the medial ring upon mitosis, a double ring before and dense dot during cytokinesis. Double immunofluorescence staining shows that Mok1 exists in close proximity to actin. The subcellular localization of Mok1 is dependent upon the integrity of the F-actin cytoskeleton. Conversely, overexpression of mok1(+) blocks the translocation of cortical actin from one end of the cell to the other. pck2 mutant is synthetically lethal with mok1 mutant, delocalizes Mok1 and shows a lower level of alpha-glucan. These results indicate that Mok1 plays a crucial role in cell morphogenesis interdependently of the actin cytoskeleton and works as one of downstream effecters for Pck1/2.

DOI： 10.1083/jcb.144.6.1173

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Language：English   Publisher：ROCKEFELLER UNIV PRESS  

In fission yeast protein kinase C homologues (Pck1 and Pck2) are essential for cell morphogenesis. We have isolated mok1(+) in a genetic screen to identify downstream effecters for Pck1/2. mok1(+) is essential for viability and encodes a protein that has several membrane-spanning domains and regions homologous to glucan metabolic enzymes. mok1 mutant shows abnormal cell shape, randomization of F-actin and weak cell wall. Biochemical analysis shows that Mok1 appears to have a-glucan synthase activity. Mok1 localization undergoes dramatic alteration during the cell cycle. It localizes to the growing tips in interphase, the medial ring upon mitosis, a double ring before and dense dot during cytokinesis. Double immunofluorescence staining shows that Mok1 exists in close proximity to actin. The subcellular localization of Mok1 is dependent upon the integrity of the F-actin cytoskeleton. Conversely, overexpression of mok1(+) blocks the translocation of cortical actin from one end of the cell to the other. pck2 mutant is synthetically lethal with mok1 mutant, delocalizes Mok1 and shows a lower level of alpha-glucan. These results indicate that Mok1 plays a crucial role in cell morphogenesis interdependently of the actin cytoskeleton and works as one of downstream effecters for Pck1/2.

DOI： 10.1083/jcb.144.6.1173

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：羊土社  

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The yeast gene SNQ2, which encodes a multidrug resistance ABC superfamily protein, is required for resistance to the mutagen 4-nitroquinoline N-oxide (4-NQO). The expression of the SNQ2 gene is under the control of a regulatory network that involves the transcription factor Yrr1p, as well as Pdr1p/Pdr3p (Cui et al., Mel. Microbiol., 29, 1307-1315 (1998)). By 5'-deletion analysis of the promoter by using SNQ2-lacZ fusion constructs, four regions: -745 to -639 (region I), -639 to -578 (region II), -548 to -533 (region III) and -533 to -485 (region IV) were found to be important for SNQ2 expression. Genetic analysis suggested that the site in region IV was responsible for the Yrr1p-mediated SNQ2 expression. A consensus motif known for the binding of Pdr1p/Pdr3p (PDRE) was not found in region IV.

DOI： 10.1271/bbb.63.162

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DOI： 10.1271/nogeikagaku1924.73.887

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=235685

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In a search for components involved in Mn2+ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn2+ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1-272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn2+ resistance, whereas the corresponding null mutation did not. Both hip1-272 cells and the null mutant exhibited low tolerance to divalent cations such as Co2+, Ni2+, Zn2+, and Cu2+. The Mn2+ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn2+ content of the hip1-272 mutant was lower than that of wild type of null mutant, due to increase rates of Mn2+ efflux. We propose that Hip1p is involved in Mn2+ transport, carrying out a function related to Mn2+ export.

DOI： 10.1007/s004380050846

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Language：English   Publisher：BLACKWELL SCIENCE LTD  

We have cloned and characterized a Saccharomyces cerevisiae gene YRR1 that is important for resistance to the mutagen 4-nitroquinoline N-oxide (4-NQO). The wild-type YRR1 gene encodes a protein that contains a Zn(II)(2)Cys(6)-type zinc-finger motif. Disruption of the YRR1 gene leads to hypersensitivity to 4-NQO. A dominant mutation (YRR1-1) that confers strong resistance to 4-NQO has been identified. Epistasis analysis demonstrated that 4-NQO resistances mediated by the YRR1 and YRR1-1 alleles require the presence of the SNQ2 gene that encodes a multidrug resistance ATP binding cassette superfamily protein responsible for 4-NQO export. Northern blot analysis of SNQ2 mRNA levels indicated that Yrr1p is involved in basal and drug-induced transcriptional activation of SNQ2, whereas Pdr1p/Pdr3p transcription factors are mainly involved in basal SNQ2 expression. In the YRR1-1 mutant, the level of SNQ2 mRNA is constitutively elevated. These results establish that Yrr1p is important for 4-NQO resistance by mediating transcriptional activation of the SNQ2 gene in response to the stress imposed by 4-NQO. The gain-of-function mutation of Yrr1-1p was attributable to the duplication of a 12-amino-acid sequence generated near the carboxy terminus.

DOI： 10.1046/j.1365-2958.1998.01027.x

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Language：English   Publisher：AMER SOC CELL BIOLOGY  

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1-15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two alpha-tubulin genes) and alp12 to nda3 (the single beta-tubulin gene), atb2(+) is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between alpha/beta-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2(+); overexpression of atb2(+) lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

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Language：English   Publisher：TAYLOR & FRANCIS LTD  

Staurosporine is a potent inhibitor of protein kinase C. To identify the genes that functionally interact with the Pkc1 pathway of the yeast Saccharomyces cerevisiae, we screened for the genes that cause induced staurosporine sensitivity when overexpressed from a galactose-inducible promoter. The novel gene ISR1 encodes a predicted protein kinase with the highest sequence similarity to mammalian Raf in the kinase domain. Drug sensitivity induced by ISR1 overexpression is specific to staurosporine. Although ISR1 disruption causes no obvious phenotype, it does exacerbate the phenotypes of a temperature-sensitive allele (stt1-1) of PKC1, but not of the mpk1 and bck1 mutants of the Mpk1 MAP kinase pathway. These results suggest that Isr1 functions in an event important for growth in a manner redundant with a Mpk1-independent branch of the Pkc1 signalling pathways.

DOI： 10.1271/bbb.62.1376

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：日本農芸化学会  

DOI： 10.1271/nogeikagaku1924.72.772

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Language：English   Publisher：COMPANY OF BIOLOGISTS LTD  

To identify the genes involved in cell morphogenesis in Schizosaccharomyces pombe, we screened for the genes that cause aberrant cell morphology by overexpression, The isolated genes were classified on the basis of morphology conferred, One of the genes causing a rounded morphology was identified as the rho2(+) gene encoding a small GTP-binding protein. The overexpression of rho2(+) resulted in a randomized distribution of cortical F-actin and formation of a thick cell wall. Analyses using cdc mutants suggested that the overexpression of rho2(+) prevents the establishment of growth polarity in G(1). The rho2(+) gene was not essential, but among cells deleted for rho2(+), those with an irregular shape were observed, The disruptant also showed a defect in cell wall integrity, An HA-Rho2 expressed in the cell was suggested to be present as a membrane-bound form by a cell fractionation experiment. A GFP-Rho2 was localized at the growing end(s) of the cell and the septation site, The localization of GFP-Rho2 during interphase was partially dependent on sts5(+), These results indicate that Rho2 is involved in cell morphogenesis, control of cell wall integrity, control of growth polarity, and maintenance of growth direction, Analysis of functional overlapping between Rho2 and Rho1 revealed that their functions are distinct from each other, with partial overlapping.

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To identify the genes involved in cell morphogenesis in Schizosaccharomyces pombe, we screened for the genes that cause aberrant cell morphology by overexpression. The isolated genes were classified on the basis of morphology conferred. One of the genes causing a rounded morphology was identified as the rho2+ gene encoding a small GTP-binding protein. The overexpression of rho2+ resulted in a randomized distribution of cortical F-actin and formation of a thick cell wall. Analyses using cdc mutants suggested that the overexpression of rho2+ prevents the establishment of growth polarity in G1. The rho2+ gene was not essential, but among cells deleted for rho2+, those with an irregular shape were observed. The disruptant also showed a defect in cell wall integrity. An HA-Rho2 expressed in the cell was suggested to be present as a membrane-bound form by a cell fractionation experiment. A GFP-Rho2 was localized at the growing end(s) of the cell and the septation site. The localization of GFP-Rho2 during interphase was partially dependent on sts5+. These results indicate that Rho2 is involved in cell morphogenesis, control of cell wall integrity, control of growth polarity, and maintenance of growth direction. Analysis of functional overlapping between Rho2 and Rho1 revealed that their functions are distinct from each other, with partial overlapping.

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Staurosporine is a potent inhibitor of protein kinase C. To identify the genes that functionally interact with the Pkc1 pathway of the yeast Saccharomyces cerevisiae, we screened for the genes that cause induced staurosporine sensitivity when overexpressed from a galactose-inducible promoter. The novel gene ISR1 encodes a predicted protein kinase with the highest sequence similarity to mammalian Raf in the kinase domain. Drug sensitivity induced by ISR1 overexpression is specific to staurosporine. Although ISR1 disruption causes no obvious phenotype, it does exacerbate the phenotypes of a temperature-sensitive allele (stt1-1) of PKC1, but not of the mpk1 and bck1 mutants of the Mpk1 MAP kinase pathway. These results suggest that Isr1 functions in an event important for growth in a manner redundant with a Mpk1-independent branch of the Pkc1 signalling pathways. © 1998, Taylor &amp
Francis Group, LLC. All rights reserved.

DOI： 10.1271/bbb.62.1376

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DOI： 10.1046/j.1365-2958.1998.01027.x

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The Saccharomyces cerevisiae crv mutants (crv1, 2, 3 and 4) exhibit phenotypes, such as calcium resistance and vanadate sensitivity, which are apparently similar to those of calcineurin-deficient mutants. We have cloned and characterized the CRV4 gene that complements all the phenotypes of the crv4 mutant. DNA sequencing revealed that CRV4 is identical to the previously cloned gene TTP1, which encodes a type II membrane protein of unknown function. Deletion of CRV4/TTP1 causes no obvious phenotype except for Ca2+ resistance and vanadate sensitivity, but is synthetically lethal in combination with a deletion of MPK1, In a manner which is suppressible by the addition of an osmotic stabilizer. In medium containing sorbitol as an osmotic stabilizer, the cnb1 mpk1 ttp1 triple mutant exhibits a more severe growth defect than does any of the double mutants cnb1 ttp1, cnb1 mpk1 or mpk1 ttp1. A high Ca2+ concentration (50 mM) or a constitutively active form of calcineurin partially suppresses the growth defect of the mpk1 ttp1 double mutant. These results indicate that Ttp1 participates in a cellular event essential for growth and morphogenesis, in parallel with the pathways involving Mpk1 MAP kinase and calcineurin.

DOI： 10.1007/s004380050592

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Language：English   Publisher：ELSEVIER SCIENCE BV  

To define the essential amino acid residues of Yap1 in stress response, we generated yap1 mutations by in vitro mutagenesis, which cause defects in mediating resistance to the stress of H2O2, hut not of CdCl2. Sequence analysis of the mutant yap1 genes revealed three point mutations and two truncation mutations near the carboxy-terminus. The truncation mutations resulted in hyperresistance to cadmium, Northern blot analysis of stress-induced levels of TRX2 and GSH1 mRNAs indicated that the ability of the mutant Yap1 protein to induce transcriptional activation of target genes correlates well with its ability to confer stress resistance, The carboxy-terminal domain of Yap1 appears to act negatively in cadmium resistance. (C) 1997 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(97)01233-7

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To define the essential amino acid residues of Yap1 in stress response, we generated yap1 mutations by in vitro mutagenesis, which cause defects in mediating resistance to the stress of H2O2, hut not of CdCl2. Sequence analysis of the mutant yap1 genes revealed three point mutations and two truncation mutations near the carboxy-terminus. The truncation mutations resulted in hyperresistance to cadmium, Northern blot analysis of stress-induced levels of TRX2 and GSH1 mRNAs indicated that the ability of the mutant Yap1 protein to induce transcriptional activation of target genes correlates well with its ability to confer stress resistance, The carboxy-terminal domain of Yap1 appears to act negatively in cadmium resistance. (C) 1997 Federation of European Biochemical Societies.

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Language：Japanese   Publishing type：Book review, literature introduction, etc.   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

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DOI： 10.1271/nogeikagaku1924.71.808

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：共立出版  

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The ATP-binding cassette superfamily proteins Pdr5p and Snq2p of Saccharomyces cerevisiae are implicated in multidrug resistance. Here, we show that these transporters are also involved in cation resistance. Null mutants of PDR5 and SNQ2 genes exhibit increased sensitivity to NaCl, LiCl and MnCl2. The mutant cells grown in the presence of high concentrations of these metal salts contain higher levels of the metals than wild-type cells. The expression of PDR5 and SNQ2 is induced by the metal salts. These results provide evidence that the yeast drug transporters contribute to cation resistance by regulating cellular cation homeostasis under ionic stress conditions.

DOI： 10.1016/S0014-5793(96)01353-1

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Language：English   Publisher：NATL ACAD SCIENCES  

The Schizosaccharomyces pombe mutant, ban5-4, displays aberrant mitochondrial distribution. Incubation of this conditional-lethal mutant at the nonpermissive temperature led to aggregated mitochondria that were distributed asymmetrically within the cell. Development of this mitochondrial asymmetry but not mitochondrial aggregation required progression through the cell division cycle. Genetic analysis revealed that ban5-4 is an allele of atb2 encoding alpha 2-tubulin. Consistent with this finding, cells with the cold-sensitive nda3 mutation in beta-tubulin displayed aggregated and asymmetrically distributed mitochondria after incubation at lowered temperatures. These results indicate that microtubules mediate mitochondrial distribution in fission yeast and provide the first genetic evidence for the role of microtubules in mitochondrial movement.

DOI： 10.1073/pnas.93.21.11664

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Cell morphogenesis is a fundamental phenomenon that involves understanding a number of biological processes including the developmental program, polarity and cell division, Fission yeast sts5 mutant cells are round rather than cylindrical with cortical actin randomly dispersed, Genetic analyses demonstrate that the sts5(+) gene is required for maintenance of cell shape during interphase when the cell normally exhibits polarised growth, The sts5 mutant is not defective in cell wall integrity, Deletion of ppe1(+), which encodes a type 2A-like protein phosphatase, shows similar phenotypes to the sts5 mutant and these two mutations are synthetically lethal, Multicopy plasmids containing either the protein kinase C-like gene pck1(+) or the protein tyrosine phosphatase pyp1(+), an inhibitor of an osmosensing Sty1/Spc1 MAP-kinase, are capable of suppressing the sts5 mutation, Consistent with this, we have found that the wis1 mutation, which is defective in a MAP-kinase kinase of the pathway, suppresses the sts5 mutation, The predicted sts5(+) gene product exhibits sequence similarity to two yeast proteins, Dis3 and Ssd1 and a nematode protein, F46E8.6, where the former two yeast proteins have been shown to be involved in cell cycle control and cell morphogenesis, The sts5(+) gene is not essential for cell viability, but is absolutely required for polarised growth as the gene disruption showed the same phenotypes as those of the original mutants, Overexpression of the sts5(+) gene resulted in altered cell morphology and, cortical actin in these overproducing cells was also abnormal, fainter and often dispersed, Anti-Sts5 antibody specifically detected a 130 kDa protein by western blotting, A green fluorescent protein-Sts5 fusion protein localised in the cytoplasm with a discrete punctate pattern, suggesting that the Sts5 protein is a component of a novel structure, These results have indicated that the Sts5 protein is a crucial determinant of polarised growth and that it functionally interacts with the serine/threonine phosphatase, protein kinase C, and an osmosensing MAP-kinase to maintain cell morphology.

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have cloned and characterized a Saccharomyces cerevisiae gene YRS1 that complements the phenotype of the mutant sensitive to the anionic drug reveromycin A, The YRS1 gene, which is identical to the recently identified YOR1 gene, encodes a protein with extensive homology to the human multidrug resistance-associated protein (MRP) and the yeast cadmium factor (Ycf1), A chromosomal deletion of YRS1 lead to viable Delta yrs1 cells, which exhibited hypersensitivity to reveromycin A. Elevation of the YRS1 gene dosage in wild type cells conferred increased resistance to reveromycin A, By analyzing the effect of YRS1 disruption and overexpression it was demonstrated that Yrs1 is involved in the detoxification of a wide range of the organic anions that con tain carboxyl group(s) but none of the other type of toxic compounds examined, Fluorescence-activated cell sorter analysis indicated the increased accumulation of the anionic fluorescent compound rhodamine B in Delta yrs1 cells, The expression of YRS1 was induced strikingly by reveromycin A. These results suggest that Yrs1 is a multispecific organic anion transporter important for tolerance against toxic environmental organic anions, Yrs1 had an overlapping specificity with Ycf1 in the resistance to cadmium.

DOI： 10.1074/jbc.271.25.14712

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Saccharomyces cerevisiae mutants which exhibit phenotypes (calcium resistance and vanadate sensitivity) similar to those of calcineurin-deficient mutants were isolated. The mutants were classified into four complementation groups (crv1,2,3 and 4). crv1 was allelic to cnb1, a mutation in the regulatory subunit of calcineurin. The nucleotide sequences of CRV2 and CRV3 genes which complemented the crv2 and crv3 mutations, respectively, are identical to those of BCK1/SLK1/SKC1/SSP31 and MPK1/SLT2, respectively, which are both involved in the MAP kinase cascade. A calcineurin-deletion mutation (Delta cnb1), which by itself has no detectable effect on growth and morphology, enhanced some phenotypes (slow growth and morphological abnormality) of crv2 and crv3 mutants. These phenotypes of crv2 and crv3 mutants were partially suppressed by Ca2+ or by overproduction of the calcineurin subunits (Cmp2 and Cnb1). Like the calcineurin-deficient mutant, crv2 and crv3 mutants were defective in recovery from alpha-factor-induced growth arrest. The defect in recovery of the Delta cnb1 mutant was suppressed by overexpression of MPK1. These results indicated that the calcineurin-mediated and the Mpk1- (Bck1-) mediated signaling pathways act in parallel to regulate functionally redundant cellular events important for growth.

DOI： 10.1007/s004380050159

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We have examined whether the stress-induced transcriptional activation of YDR1/PDR5/STS1 is mediated by yAP-1 and yAP-2. Of the stresses examined, heat shock-induced, rapid and transient PDR5 expression became very low in a yap1 yap2 double-gene disruptant, indicating that the yAP proteins mediate the response. Similar results were obtained with SNQ2, a close homologue of PDR5. A set of 5'-truncation derivatives of the PDR5 gene identified the region from -484 to -434 as being sufficient for the response. A sequence similar to the yAP-1 recognition element recently identified in the stress-responsive yeast genes was found in this region and in the 5'-flanking sequences of SNQ2.

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Ca2+/calmodulin-dependent phosphoprotein phosphatase (calcineurin, PP2B) of Saccharomyces cerevisiae is implicated in adaptation to high-salt conditions. Calcineurin mediates high salt-induced expression of the ENA1/PMR2 gene encoding the P-type ATPase, which is suggested to be involved in Na+ efflux. We identified the PDE1 gene encoding the low-affinity cAMP phosphodiesterase as a multicopy suppressor of the Li+- and Na+-sensitive calcineurin null mutant suggesting that cAMP is a negative regulator of adaptation to high-salt stress. Genetic analysis indicated that calcineurin and cAMP act antagonistically in a common pathway for adaptation. The bcy1 disruption, which leads to constitutive cAMP-dependent protein kinase (PKA) activity, inhibited high NaCl-induced expression of the ENA1/PMR2 gene, caused an elevation of the intracellular Na+ level and a growth defect in high-NaCl medium, all of which were analogous to the defects of a calcineurin mutant. A reduced cAMP level resulting from multiple copies of the PDE1 gene caused increased expression of the ENA1/PMR2 gene in response to high NaCl. We propose a model for the regulation of cation homeostasis, in which calcineurin antagonizes PKA to activate transcription of the ENA1/PMR2 gene in response to high-salt conditions.

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A novel Saccharomyces cerevisiae (Sc) SVS1 gene was cloned as a multicopy suppressor of vanadate (Vn) sensitivity (Vn(s)) due to a calcineurin (CaN) null mutation. SVS1 encoded a 260-amino-acid protein abundant in Ser and Thr residues, with a putative signal sequence at the N terminus. Deletion of SVS1 resulted in increased sensitivity to Vn, but not to other metallic ions or drugs. Northern analysis of the SVS1 mRNA indicated that the induction of the gene occurred specifically in the response to Vn. These results suggested that Sc has a mechanism to enhance the tolerance to Vn by increasing the expression of SVS1. The results of genetic experiments suggested that CaN and the Svs1 proteins act in separate pathways to enhance the tolerance to Vn.

DOI： 10.1016/0378-1119(95)00503-X

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The role of protein phosphatase 2B (PP2B/calcineurin) of Saccharomyces cerevisiae in the tolerance to divalent cations was investigated. PP2B-deficient mutants were found to be sensitive to MnCl2, but not to ZnCl2, CuCl2, NiCl2 and CoCl2. By measuring both manganese uptake and its efflux, it was found that the sensitivity of the mutant cells was due to an increase in manganese uptake and that the wild-type cells were able to prevent manganese entry into the cells, rather than export it in a more efficient manner. In the presence of the immunosuppressant FK506, the behavior of wild-type cells became similar to that of PP2B mutants. Out of various divalent cations tested, externally added magnesium ions were able to block manganese uptake in both wild-type and PP2B mutant strains.

DOI： 10.1111/j.1432-1033.1995.tb20865.x

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Language：Japanese   Publisher：社団法人日本農芸化学会  

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CiNii Books

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Language：Japanese   Publisher：社団法人日本農芸化学会  

CiNii Article

CiNii Books

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Language：Japanese   Publisher：社団法人日本農芸化学会  

CiNii Article

CiNii Books

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Language：Japanese   Publisher：社団法人日本農芸化学会  

CiNii Article

CiNii Books

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Language：Japanese   Publisher：社団法人日本農芸化学会  

CiNii Article

CiNii Books

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Language：Japanese   Publisher：社団法人日本農芸化学会  

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Language：English   Publisher：WILEY  

Temperature-sensitive suppressor mutants were isolated from two fission yeast mutants defective in cell shape control: ppe1, encoding a type 2A-like protein phosphatase, and sts5, one of 11 staurosporine-supersensitive mutants, Complementation tests showed that suppression was due to two chromosomal loci, ssp1 and ssp2, Cells of the ssp1 mutant grown at the restrictive temperature arrested uniformly with an elongated cell body and a 2C content of DNA, Interestingly, these mutant cells grew only in a monopolar manner, At a specific point in the G(2) phase of the cell cycle, wild-type cells exhibit a drastic alteration in growth polarity, from mono- to bipolar, This Change coincides with the distribution of cortical actin from one end of the cell to both ends, In the ssp1 mutant cells, cortical actin was localized only at one end, suggesting that the mutant fails to change growth polarity. Nucleotide sequence determination showed that ssp1(+) encodes a novel protein kinase, Ectopic overexpression of ssp1(+) resulted in an altered cell morphology and cortical actin was randomly dispersed within the cells, Immunocytological analysis revealed that the protein was primarily localized in the cytoplasm and that half of the protein existed in an insoluble fraction, These results show that the dynamics of actin-based growth polarity during the cell cycle are regulated, at least in part, by a novel set of protein kinases and phosphatases.

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Language：English   Publisher：TAYLOR & FRANCIS LTD  

Ergosterol is a major sterol component of fungal plasma membranes. The effects of disrupting the Saccharomyces cevevisiae SYR1/ERG3 gene, which encodes sterol C-5 desaturase, an enzyme of ergosterol biosynthesis pathway, were markedly different for different S. cerevisiae strains and growth temperatures. The null mutation of SYR1 (Delta syr1) in strain RAY-3A had only a slight effect on the growth rate at 28 degrees C. However, at this temperature, the same mutation caused poor growth in strain KA-311A and no growth in strain W303-1A. The Delta syr1 disruptant of these strains were able to grow at 37 degrees C, as,yell as their parental strains. Moreover, the growth of the Delta syr1 disruptant of W303-1A and KA-311A strains were severely inhibited at 16 degrees C. These results indicated that ergosterol is essential for growth at low temperatures, and the effects of the gene disruption are variable by the genetic background. The growth defect at low temperatures appeared to be due to the defect of tryptophan uptake in the Delta syr1 mutants. The Delta syr1 mutants were sensitive to a wide variety of drugs, chemicals, and ions, suggesting that yeast ergosterol is important as permeability barrier against various chemical stresses.

DOI： 10.1271/bbb.59.482

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The Saccharomyces cerevisiae gene YDR1/PDR5/STS1, which encodes a member of the ATP binding cassette (ABC) superfamily, is important for cross-resistance to apparently unrelated drugs. The expression of YDR1 is induced by various drugs and heat shock [Hirata et al., Guru, Genet., 26, 285-294 (1994)]. By deletion analysis of the 5'-noncoding region of the YDB1 gene, two drug responsive regions (-604 to -485 and -335 to -275) were identified with fluphenazine and cycloheximide. Three conserved palindrome sequences were found in these regions.

DOI： 10.1271/bbb.59.147

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Language：English  

Ergosterol is a major sterol component of fungal plasma membranes. The effects of disrupting the Saccharomyces cerevisiae SYR1/ERG3 gene, which encodes sterol C-5 desaturase, an enzyme of ergosterol biosynthesis pathway, were markedly different for different S. cerevisiae strains and growth temperatures. The null mutation of SYR1 (⊿syrl) in strain RAY-3 A had only a slight effect on the growth rate at 28°C. However, at this temperature, the same mutation caused poor growth in strain KA-311A and no growth in strain W303-1A. The ⊿syrl disruptant of these strains were able to grow at 37°C, as well as their parental strains. Moreover, the growth of the ⊿syrl disruptant of W303-1A and KA-311A strains were severely inhibited at 16°C. These results indicated that ergosterol is essential for growth at low temperatures, and the effects of the gene disruption are variable by the genetic background. The growth defect at low temperatures appeared to be due to the defect of tryptophan uptake in the ⊿syrl mutants. The ⊿syrl mutants were sensitive to a wide variety of drugs, chemicals, and ions, suggesting that yeast ergosterol is imnortant as oermeabilitv barrier against various chemical stresses. © 1995, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.

DOI： 10.1271/bbb.59.482

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Language：English   Publisher：Springer-Verlag  

A multidrug resistance gene, YDR1, of Saccharomyces cerevisiae, which encodes a 170-kDa protein of a member of the ABC superfamily, was identified. Disruption of YDR1 resulted in hypersensitivity to cycloheximide, cerulenin, compactin, staurosporine and fluphenazine, indicating that YDR1 is an important determinant of cross resistance to apparently-unrelated drugs. The Ydr1 protein bears the highest similarity to the S. cerevisiae Snq2 protein required for resistance to the mutagen 4-NQO. The drug-specificity analysis of YDR1 and SNQ2 by gene disruption, and its phenotypic suppression by the overexpressed genes, revealed overlapping, yet distinct, specificities. YDR1 was responsible for cycloheximide, cerulenin and compactin resistance, whereas, SNQ2 was responsible for 4-NQO resistance. The two genes had overlapping specificities toward staurosporine and fluphenazine. The transcription of YDR1 and SNQ2 was induced by various drugs, both relevant and irrelevant to the resistance caused by the gene, suggesting that drug specificity can be mainly attributed to the functional difference of the putative transporters. The transcription of these genes was also increased by heat shock. The yeast drug-resistance system provides a novel model for mammalian multidrug resistance. © 1994 Springer-Verlag.

DOI： 10.1007/BF00310491

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：共立出版  

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Language：Japanese   Publisher：共立出版  

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Language：English   Publisher：Springer-Verlag  

The Saccharomyces cerevisiae YAP2 gene encoding an AP-1-like transcriptional activator protein was cloned by selection for genes that confer pleiotropic drug resistance when present in high copy number. The novel YAP2 gene encodes a protein of 45827 daltons and is homologous in part to a known transcriptional activator protein encoded by YAP1/PDR4/SNQ3/PAR1. Homology was found only in both terminal regions. The N-terminal portion contains a region rich in basic amino acids, followed by a "leucine zipper" motif. Overexpression of YAP2 led to the induction of expression of an AP-1 recognition element (ARE)-dependent promoter. The yap1 disruptant has been shown to be sensitive to H2O2. In this study, we demonstrated that the yap1 disruptant is also unable to grow in medium containing 150 μM cadmium, whereas the yap2 disruptant exhibited no significant phenotypes. However, YAP2 in high copy number did suppress cadmium sensitivity, but not H2O2 sensitivity of the yap1 disruptant. YAP1 was able to mediate both cadmium- and H2O2-induced transcriptional activation of an ARE-dependent promoter. A high-copy-number plasmid bearing YAP2 mediated cadmium-induced transcriptional activation of this promoter. The inductions were prevented by the antioxidant N-acetyl-l-cysteine. © 1994 Springer-Verlag.

DOI： 10.1007/BF00280413

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Language：English   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

Saccharomyces cerevisiae transformants which secrete high levels of cellulolytic enzymes, with chromosome-integrated multicopies of heterologous DNA sequences encoding the cellulolytic enzymes were constructed. An expression construct of beta-glucosidase and carboxymethyl cellulase directed by the GAP promoter was integrated into the chromosomes of the haploid S. cerevisiae using the a sequence-mediated integration system. Southern blot analysis of the chromosomes prepared from various integrants and separated by pulse-field gel electrophoresis demonstrated that the integration occurred mainly in a particular chromosome and the copy number of the integration was variable. The amount of enzymes secreted by the transformants correlated with the copy number of integration. For each enzyme, the highest activity was about 1.4-fold that produced by the transformant harboring the same expression cassette on a YEp-type plasmid. The delta-integrated exogenous DNA was mitotically stable in rich medium. A haploid double transformant which coexpresses and secretes beta-glucosidase and carboxymethyl cellulase was further constructed by genetic crossing of the haploid transformant that produces a high level of the enzyme, followed by meiotic segregation of the resulting diploid strain. The haploid double transformant, but neither of the single transformant, could grow on a plate containing carboxymethyl cellulose as a sole carbon source. It is suggested that the delta-sequence-mediated integration system is a very useful means for the genetic engineering of yeast, especially when overproduction and secretion of multiple heterologous enzymes are desired.

DOI： 10.1016/0922-338X(94)90112-0

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Language：English   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

To assess the physiological function of Ca2+-dependent protein phosphatase (PP2B) in the yeast Saccharomyces cerevisiae, the phenotypes of PP2B-deficient mutants were investigated. Although PP2B was dispensable for growth under normal conditions, the mutations did, however, cause growth inhibition under certain stress circumstances. The growth of the mutants was inhibited by NaCl and LiCl, but not by KCl, CaCl2, MgCl2 or nonspecific osmotic stresses. Upon shift to high NaCl medium, intracellular Na+ levels of both wild type yeast and the mutants initially increased at a comparable rate. However, internal Na+ in wild type cells started to decline more rapidly than the mutant cells during cultivation in high NaCl medium, indicating that PP2B is important in maintaining a gradient across the membrane. The protection against salt stress was achieved, at least in part, by the stimulation of Na+ export. The maintenance of a high level of internal K+ in high NaCl medium was also PP2B-dependent. In the presence of the immunosuppressant FK506, the growth behaviour and intracellular Na+ and K+ of wild type cells in high NaCl medium became very similar to those of the PP2B-deficient mutant in a manner dependent on the presence of the FK506 binding protein.

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Two new members of the Saccharomyces cerevisiae heat-shock protein 70 multigene (HSP70) family were isolated from a yeast expression library using antisera made against a yeast calmodulin-binding fraction. They are designated as SSE1 and, SSE2, because their predicted amino acid (aa) sequences are highly homologous to each other (76% identical), and share homology with known members of the yeast HSP70 multigene family, but their homologies (13 to 28% identity) are not high enough to place them in known subfamilies. SSE1 and SSE2 are thought to encode polypeptides of 693 aa with calculated M(r)'s of 77408 and 77619, respectively. The SSE1 mRNAs were moderately abundant during steady-state growth at 23-degrees-C, and increased a few-fold upon upshift to 37-degrees-C. SSE2 mRNAs were present at low level during steady-state growth at 23-degrees-C, and greatly increased upon upshift to 37-degrees-C. Disruption of SSE1 results in slow-growing cells at any temperature. No phenotypic effects of the mutation in SSE2 were detected, and the growth property of the sse1sse2 double mutant was the same as that of the sse1 single mutant.

DOI： 10.1016/0378-1119(93)90514-4

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Two new members of the Saccharomyces cerevisiae heat-shock protein 70 multigene (HSP70) family were isolated from a yeast expression library using antisera made against a yeast calmodulin-binding fraction. They are designated as SSE1 and, SSE2, because their predicted amino acid (aa) sequences are highly homologous to each other (76% identical), and share homology with known members of the yeast HSP70 multigene family, but their homologies (13 to 28% identity) are not high enough to place them in known subfamilies. SSE1 and SSE2 are thought to encode polypeptides of 693 aa with calculated M(r)'s of 77408 and 77619, respectively. The SSE1 mRNAs were moderately abundant during steady-state growth at 23-degrees-C, and increased a few-fold upon upshift to 37-degrees-C. SSE2 mRNAs were present at low level during steady-state growth at 23-degrees-C, and greatly increased upon upshift to 37-degrees-C. Disruption of SSE1 results in slow-growing cells at any temperature. No phenotypic effects of the mutation in SSE2 were detected, and the growth property of the sse1sse2 double mutant was the same as that of the sse1 single mutant.

DOI： 10.1016/0378-1119(93)90514-4

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

DOI： 10.1271/bbb.56.1682

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

A useful sake-fermenting yeast for brewing ginjo-sake was bred by protoplast fusion. First, 61 native strains were isolated from ginjo-sake mash. From these isolates, two strains, YNS50 and YNS59, were selected. These two strains possessed many of the characteristics required for ginjo-sake brewing in a complementary manner. Strain YNS50 produced high amounts of the flavor components isoamyl acetate and ethyl caproate and low amounts of acids. Strain YNS59, on the other hand, exhibited good growth and fermentation at low temperatures (approximately 10-degrees-C) and had high resistance to ethyl alcohol. Next, a lysine auxotrophic mutant (YDH17) of YNS50 and rho-mutant (YDH8) of YNS59 with a marker useful for the selection of the fusants were constructed. Finally, prototrophs arising from the complementation of each marker by protoplast fusion were obtained. A strain (strain YNF74) that stably maintained all of the useful characteristics derived from each wild type strain was screened from 120 fusants. In a sub-industrial scale sake brewing test, we demonstrated that strain YNF74 exhibited its superior characteristics in brewing ginjo-sake. The sake produced by strain YNF74 achieved high evaluation in a tasting test.

DOI： 10.1016/0922-338X(92)90145-K

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

A useful sake-fermenting yeast for brewing ginjo-sake was bred by protoplast fusion. First, 61 native strains were isolated from ginjo-sake mash. From these isolates, two strains, YNS50 and YNS59, were selected. These two strains possessed many of the characteristics required for ginjo-sake brewing in a complementary manner. Strain YNS50 produced high amounts of the flavor components isoamyl acetate and ethyl caproate and low amounts of acids. Strain YNS59, on the other hand, exhibited good growth and fermentation at low temperatures (approximately 10-degrees-C) and had high resistance to ethyl alcohol. Next, a lysine auxotrophic mutant (YDH17) of YNS50 and rho-mutant (YDH8) of YNS59 with a marker useful for the selection of the fusants were constructed. Finally, prototrophs arising from the complementation of each marker by protoplast fusion were obtained. A strain (strain YNF74) that stably maintained all of the useful characteristics derived from each wild type strain was screened from 120 fusants. In a sub-industrial scale sake brewing test, we demonstrated that strain YNF74 exhibited its superior characteristics in brewing ginjo-sake. The sake produced by strain YNF74 achieved high evaluation in a tasting test.

DOI： 10.1016/0922-338X(92)90145-K

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Saccharomycopsis fibuligera glucoamylase (GLU1) and Escherichia coli β-lactamase (bla) fusion gene was constructed and expressed in Saccharomyces cerevisiae. S. cerevisiae transformants with the plasmid harboring the GLU1-bla fusion gene produced in the culture medium a 90 Kd protein with both glucoamylase and β-lactamase activities, in agreement with the size of the chimera protein expected from the gene construct. © 1992.

DOI： 10.1016/0922-338X(92)90122-B

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

Saccharomycopsis fibuligera glucoamylase (GLU1) and Escherichia coli beta-lactamase (bla) fusion gene was constructed and expressed in Saccharomyces cerevisiae. S. cerevisiae transformants with the plasmid harboring the GLU1-bla fusion gene produced in the culture medium a 90 Kd protein with both glucoamylase and beta-lactamase activities, in agreement with the size of the chimera protein expected from the gene construct.

DOI： 10.1016/0922-338X(92)90122-B

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Language：English   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

Isoamyl acetate, one of the major components of flavor in a Japanese brewing product, sake, contributes to the fruity flavor of sake. We cloned Saccharomyces cerevisiae genes that cause overproduction of isoamyl alcohol, the precursor of isoamyl acetate, by increased gene-dosage effect. Three plasmids, pScFlr1, pScFlr2, and pScFlr3, were obtained by selection from the yeast genomic DNA which, on a multicopy plasmid, confer a phenotype resistant to 5,5,5-trifluoro-DL-leucine, an analogue of leucine. The cloned DNA fragments in the three plasmids had different restriction maps. Transformed cells with each of the plasmids produced large amounts of both isoamyl alcohol and isoamyl acetate. The gene cloned in the plasmid pScFlr1 was identified as the LEU4 gene encoding alpha-isopropylmalate synthase.

DOI： 10.1271/bbb1961.55.919

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Language：English   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

Isoamyl acetate, one of the major components of flavor in a Japanese brewing product, sake, contributes to the fruity flavor of sake. We cloned Saccharomyces cerevisiae genes that cause overproduction of isoamyl alcohol, the precursor of isoamyl acetate, by increased gene-dosage effect. Three plasmids, pScFlr1, pScFlr2, and pScFlr3, were obtained by selection from the yeast genomic DNA which, on a multicopy plasmid, confer a phenotype resistant to 5,5,5-trifluoro-DL-leucine, an analogue of leucine. The cloned DNA fragments in the three plasmids had different restriction maps. Transformed cells with each of the plasmids produced large amounts of both isoamyl alcohol and isoamyl acetate. The gene cloned in the plasmid pScFlr1 was identified as the LEU4 gene encoding alpha-isopropylmalate synthase.

DOI： 10.1271/bbb1961.55.919

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

DOI： 10.1271/bbb1961.52.2647

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

DOI： 10.1271/bbb1961.52.2647

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Language：English   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

DOI： 10.1271/bbb1961.50.109

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Language：English   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

DOI： 10.1271/bbb1961.50.109

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