• 한국식품과학회지
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• 제35권3호
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• pp.488-492
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• 2003

S. cereviaiae KCTC 7911에 돌연변이를 유도하고 selective pressure로서 세포벽 분해효소인 zymolsae와 mechanical stress인 glass bead를 차례로 처리하여 효모변이주를 S. cerevisiae IS2를 선발하였다. S. cerevisiae IS2는 세포벽 분해효소인 zymolase의 농도별 내성실험 결과 wild-type에 비해 훨씬 강한 내성을 보여 세포벽에 변화가 일어난 균주로 예상된다. 효모변이주와 wild-type으로부터 베타글루칸을 추출하여 면역활성에 미치는 영향을 조사하기 위해 생쥐의 복강에 주사하고 생성되는 면역세포의 수, NO 생성능, 및 면역세포의 대다수를 차지하는 대식세포의 탐식능을 측정하였다. 베타글루칸을 쥐의 복강에 주사하였을 때 베타글루칸의 종류에 상관없이 면역세포의 수, NO 생성능 및 대식세포의 활성도가 증가하는 결과를 얻을 수 있었다. 특히 변이주 베타글루칸을 주사하였을 경우 wild-type 베타글루칸에 비해 면역세포의 수는 1.40배, NO 생성능은 1.12배, 대식세포의 활성도와 탐식능은 각각 1.18배와 1.43배 높은 수치를 얻을 수 있었다. 이러한 결과들로 미루어 변이주 베타글루칸이 wild-type 베타글루칸보다 우수한 면역활성 촉진능력을 가지고 있음을 증명할 수 있었으며, 고부가가치 기능성 면역물질로서의 응용 가능성을 확인할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.247-255
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• 2006

Yeast cell wall matrix particles are composed entirely of mannoprotein and ${\beta}-glucan$. The mannoproteins of yeast cell wall can systemically enhance the immune system. We previously purified and analyzed alkali-soluble ${\beta}-glucans$ [${\beta}$-(1,3)- and ${\beta}$-(1,6)-glucans] [10]. In the present study, a wild-type strain was first mutagenized with ultraviolet light, and the cell wall mutants were then selected by treatment with 1.0 mg/ml laminarinase (endo-${\beta}$-(1,3)-D-glucanase). Mannoproteins of Saccharomyces cerevisiae were released by laminarinase, purified by concanavalin-A affinity and ion-exchange chromatography. The results indicated that the mutants yielded 3-fold more mannoprotein than the wild-type. The mannoprotein mass of mutant K48L3 was 2.25 mg/100 mg of yeast cell dry mass. Carbohydrate analysis revealed that they contained mannose, glucose, and N-acetylglucosamine. Saccharomyces cerevisiae cell wall components, mannoproteins, are known to interact with macrophages through receptors, thereby inducing release of tumor necrosis factor alpha ($TNF-{\alpha}$) and nitric oxide. Mannoprotein tractions in the present study had a higher macrophage activity of secretion of $TNF-{\alpha}$ and nitric oxide and direct phagocytosis than positive control ($1{\mu}g$ of lipopolysaccharide). In particular, F1 and F3 fractions in mannoproteins of K48L3 enhanced and upregulated the activity of nitric oxide secretion and macrophage phagocytosis by approximately two- and four-fold, respectively.

• BMB Reports
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• 제30권3호
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• pp.167-172
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• 1997

The yeast Saccharomyces cerevisiae, mutant CH117, shows a drug-hypersensitivity (dhs) to cycloheximide, bleomycin, actinomycin D, 5-fluorouracil. nystatin, nigericin and several other antibiotics. CH 117 was also temperature-sensitive (ts). being unable to grow at $37^{\circ}C$ and secreted more invertase and acid phosphatase into the medium than the parent yeast. CH117 grows very slowly and the cell shape is somewhat larger and more sensitive to zymolyase than the wild type cells. Light microscopic and electron microscopic observation also revealed abnormality of the mutant cell wall. These characteristics indicate that CH117 has a defect in an essential component of the cell surface and that the cell wall which performs barrier functions has become leaky in the mutant. We screened a genomic library of wild type yeast for clones that can complement the mutation of CH117. A plasmid, pCHX1, with an insert of 3.6 kilobases (kbs) could complement the dhs and ts of CH117. Deletion and subcloning of the 3.6 kb insert showed that a gene for the complementation of mutant phenotypes was located in 1.9 kbs Puvll-Hindlll fragment.

• Journal of Microbiology
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• 제40권3호
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• pp.219-223
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• 2002

In order to investigate the function of Soo1p/${\alpha}$-COP during post-translational modification and intra-cellular transport of cell wall proteins in Saccharomyces cerevisiae, cell wall proteins from the soo1-1/ret1-1 mutant cells were analyzed. SDS-PAGE analysis of biotin labeled cell wall proteins suggested that the soo1-1 mutation impairs post-translational modification of cell wall proteins, such as N- and/ or Ο-glycosylation. Analysis of cell wall proteins with antibodies against ${\beta}$-1,3-glucan and ${\beta}$-1,6-glucan revealed alteration of the linkage between cell wall proteins and ${\beta}$-glucans in the soo1-1 mutant cells. Compositional sugar analysis of the cell wall proteins also suggested that the soo1-1 mutation impairs glycosylation of cell wall protein in the ER, which is crucial for the maintenance of cell wall integrity.

• Journal of Microbiology and Biotechnology
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• 제16권4호
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• pp.576-583
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• 2006

The alkali-soluble glucan of the yeast cell wall contains $\beta-(1,3)-$ and (1,6)-D-linkages and is known to systemically enhance the immune system. In the previous study [6], in order to isolate cell wall mutants, a wild-type strain was mutagenized by exposure to ultraviolet light, and the mutants were then selected via treatment with laminarinase $(endo-\beta-(1,3)-D-glucanase)$. The mass of alkali- and water-soluble glucans produced by the mutant was measured to be 33.8 mg/g of the dry mass of the yeast cell. Our results showed that the mutants generated the amount of alkali-soluble glucan 10-fold higher than that generated by the wild-type. Structural analysis showed that the alkali-soluble glucan from the mutants was associated with a higher degree of $\beta-(1,6)-D-linkage$ than was observed in conjunction with the wild-type. Yeast cell wall $\beta-glucan$ was shown to interact with macrophages via receptors, thereby inducing the release of tumor necrosis factor alpha $(TNF-\alpha)$ and nitric oxide. Alkali-soluble $\beta-glucans$, both from water-soluble and water-insoluble glucan, exhibited a higher degree of macrophage activity with regard to both the secretion of tumor necrosis factor alpha $(TNF-\alpha)$ and nitric oxide and direct phagocytosis, than did the positive control ($1{\mu}g$ of lipopolysaccharide).

• 한국균학회소식:학술대회논문집
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• 한국균학회 2018년도 춘계학술대회 및 임시총회
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• pp.11-11
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• 2018

Cryphonectria parasitica, chestnut blight fungus, has a characteristic of decreasing pathogenicity when infected with Cryphonectria hypovirus 1. C. parasitica is known to be one of the most representative model systems used to observe the interaction between viruses, plants and fungi. The mitogen-activated protein kinase (MAPK) pathway, which is well conserved in various organisms ranging from yeast to humans, functions in relaying phosphorylation-dependent signals within MAPK cascades to diverse cellular functions involved in the regulation of pheromone, cell wall integrity, and osmotolerance in filamentous fungi. Several genes in the MAPK pathway were revealed to be regulated by hypovirus, or to be involved in pathogenicity in C. parasitica. Among these pathways, the CWI pathway has aroused interest because CpBck1, an ortholog of yeast Bck1 (a CWI MAPKKK), was previously reported to be involved in cell wall integrity and sectorization. Interestingly, sporadic sectorization was observed in the CpBck1 mutant and sectored phenotypes were stably inherited in the progeny that were successively transferred from sectored mycelia. In this study, we analyzed the biological function of CpSlt2, downstream gene of CpBck1, to confirm whether the sectorization phenomenon occurred in the specific single gene or cell wall integrity (CWI) pathway. As results, the CpSlt2-null mutant exhibited marked changes in colonial growth, near absence of conidiation and aerial hyphae, abnormal pigmentation, CWI-related phenotypic defects, and dramatically impaired virulence. As cultivation of the mutant strains progressed, the majority of the colonies showed sporadic sectorization and mycelia from the sectored area stably maintained the sectored phenotype. These results suggest that the unique sectorization is CWI pathway-specific, though the components in the same CWI pathway have common and specific functions.

• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.263-268
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• 2003

Recently, we identified a domain, termed MIRC3-4, for the protein-protein interaction between yeast chitin synthase 3 (CHS3) and chitin synthase 4 (CHS4). In this study, the functional roles of MIRC3-4 were examined at the G1 phase and cytokinesis of the cell cycle by Calcofluor staining and FISH. Some mutations in MIRC3-4 resulted in disappearance of the chitin ring in the early G1 phase, but did not affect chitin synthesis in the cell wall at cytokinesis. The chitin distribution in chs4 mutant cells indicated that CHS4 was involved in the synthesis of chitinring in the G1 phase and in the synthesis of cell wall chitin after cytokinesis, suggesting that Chs4p regulates chitin synthase 3 activity differently in G1 and cytokinesis. Absence of the chitin ring could be caused either by delocalization of Chs3p to the bud-neck or by improper interaction with Chs4p. When mutant cells were immunostained with a Chs3p-specific antibody to discriminate between these two alternatives, the mutated Ch3p was found to localize to the neck in all MIRC3-4 mutants. These results strongly irdicate that Chs4p regulates Chs3p as an activator but not a recruiter.

• Mycobiology
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• 제38권2호
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• pp.108-112
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• 2010

To investigate the possible roles of LAMMER kinase homologue, Lkh1, in Schizosaccharomyces pombe, whole proteins were extracted from wild type and lkh1-deletion mutant cells and subjected to polyacrylamide gel electrophoresis. Differentially expressed proteins were identified by tandem mass spectrometry (MS/MS) and were compared with a protein database. In whole-cell extracts, 10 proteins were up-regulated and 9 proteins were down-regulated in the mutant. In extracellular preparations, 6 proteins were up-regulated in the lkh1+ null mutant and 4 proteins successfully identified: glycolipid anchored surface precursor, $\beta$-glucosidase (Psu1), cell surface protein, glucan 1,3-$\beta$-glucosidase (Bgl2), and exo-1,3 $\beta$-glucanase (Exg1). These results suggest that Lkh1 is involved in regulating cell wall assembly.

• Mycobiology
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• 제45권4호
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• pp.362-369
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• 2017

We assessed the regulation of cryparin, a class II hydrophobin, using three representative mitogen-activated protein kinase (MAPK) pathways in Cryphonectria parasitica. Mutation of the CpSlt2 gene, an ortholog of yeast SLT2 in the cell wall integrity (CWI) pathway, resulted in a dramatic decrease in cryparin production. Similarly, a mutant of the CpBck1 gene, a MAP kinase kinase kinase gene in the CWI pathway, showed decreased cryparin production. Additionally, mutation of the cpmk1 gene, an ortholog of yeast HOG1, showed decreased cryparin production. However, mutation of the cpmk2 gene, an ortholog of yeast Kss1/Fus3, showed increased cryparin production. The easy-wet phenotype and accumulation of the cryparin transcript in corresponding mutants were consistent with the cryparin production results. In silico analysis of the promoter region of the cryparin gene revealed the presence of binding motifs related to downstream transcription factors of CWI, HOG1, and pheromone responsive pathways including MADS-box- and Ste12-binding domains. Real-time reverse transcriptase PCR analyses indicated that both CpRlm1, an ortholog of yeast RLM1 in the CWI pathway, and cpst12, an ortholog of yeast STE12 in the mating pathway, showed significantly reduced transcription levels in the mutant strains showing lower cryparin production in C. prasitica. However, the transcription of CpMcm1, an ortholog of yeast MCM1, did not correlate with that of the mutant strains showing downregulation of cryparin. These results indicate that three representative MAPK pathways played a role in regulating cryparin production. However, regulation varied depending on the MAPK pathways: the CWI and HOG1 pathways were stimulatory, whereas the pheromone-responsive MAPK was repressive.

• 미생물학회지
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• 제37권1호
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• pp.42-48
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• 2001

출아 효모인 Saccharomyces cerevisiae의 베타-1,3-글루칸 생합성의 결함을 초래하는 돌연변이 유전자(sool-1)를 분리하여 돌연변이 부위의 염기서열을 결정하고 그 특성을 분석하였다. cool-1 유전자의 염기서열 분석 결과, 681번의 염기인 G가 A로 치환되어 Soolp의 $Gly^{227}$이 Asp로 치환되는 결과를 나타내는 것으로 판명되었고, cool-1 유전자는 기존에 보고된 retl-1 유전자와 동일한 돌연변이 유전자로 판명되었다. 그러나, ret1-1이 나타내는 온도감수성 형질은 배지에 1.2 M sorbitol 등의 삼투안정제를 첨가하면 극복될 수 있으며, cool-l/retl-1의 돌연변이 부위가 세포벽합성 관련 단백질의 번역 후 수식과정에 영향을 미친 것임을 확인하였다. 한편, Soolp/$\alpha$-COP의 N-말단에 존재하는 6개의 WD40 domain중 5번째 WD40 domain이 효모의 세포벽 합성이나 구조유지에 중요한 역할을 담당할 것임을 시사하는 결과를 얻었다.