• 한국미생물·생명공학회지
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• 제20권6호
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• pp.625-629
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• 1992

서로 다른 성접합형 A와 a를 가지는 Rhodosporidium toruloides가 생산하는 invertase를 조사한 결과, 접합형 A는 세포내, 세포벽 결합, 세포의 분비성 invertase의 3가지를 생산하였으나, 접합형 a는 세포외 invertase를 생산하지 않는 접합형 특이성이 있는 것을 알았다. 양 접합형 세포로부터 세포내 invertase를 부분정제하여 이들의 성질을 비교한 결과, 접합형 A세포 유래의 효소는 $Zn^{2+}$에 의하여 11의 활성 상승효과와Mn^{2+}$에 의한 42.2%의 감소를 보이는 반면, a세포 유래의 효소는 $Mn^{2+}$에 의한 15의 활성 감소만을 보였다. 효소 저해제의 효과에서 a세포의 invertase는 2-mercaptoethanol, sodium dodecyl sulfate, pheonl에만 약간 저해를 받을 뿐이었다. 이들 양 효소의 열안정성은 모두 $80^{\circ}C$ 이상에서 급격히 실활되는 양상을 보였으며 최적 온도가 $60^{\circ}C$로 둘다 비슷하였다. a세포 유래의 invertase는 pH 3에서 pH 10까지 넓은 pH의 범위에서 안정하며 최적 pH가 4.0인반면, A 세포의 invertase는 pH 3에서 pH 6까지 산성의 조건에서만 안정하였으며 최적 pH는 5.0으로 나타났다.그리고, A 세포와 atpvh 유래 invertase의 $K_m$값은 각각 $2.5{\times}10^3$M 과 $3.4{\times}10^3$M과 이였다.

• 한국균학회지
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• 제19권4호
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• pp.266-275
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• 1991

효모의 mating pheromone에 의한 세포내 Slgnal을 전달하는 물질을 coding 하거나 조절할 수 있는 유전자에 대한 정보를 얻기 위해서 효모에서 G-protein의 ${\alpha}-subunit$를 coding 하는 유전자 CDC70에 돌연변이가 얼어난 균주 $A14-3(MAT{\alpha},\;cdc70-5)$에 UV를 조사하여 돌연변이 cdc70-5를 억제할수 있는 또 다른 돌연변이를 획득하였다 . 돌연변이 cdc70-5의 표현형은 온도 감수성 (temperature sensitivity)이며, $38^{\circ}C$에서 배양하면 세포주기가 G1에서 정지되고 shmoo모양을 보인다 . 균주 A14-3에서 UV를 조사하였을 때 $38^{\circ}C$에서 colony를 형성할 수 있고 세포분열이 정상적으로 진행되어 출아를 하고 있다는 것은 새로운 돌연변이가 cdc70-5 의 표현형을 억제 한다는 사실을 의미한다 . 이러한 억제돌연변이 중 signal transducer와 직접적으로 관련이 없는 유전자들인 $sir^-$ 및 $mat{\alpha}2^-$ 돌연변이를 배제한 다음 선택된 15개의 돌연변이들를 중심으로 분석을 행하였다 . 어느 유전자에서 유발 되었는지 그리고 유전자 종류는 몇 가지나 되는지를 암기 위하여 tetrad analysis를 통해 연관여부를 조사하여 분리된 15개의 돌연변이중 12개는 4개의 연관군(sga1, sga2, sga3, sga4)에 속함을 알았다.

• Journal of Microbiology
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• 제34권1호
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• pp.30-36
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• 1996

We have exmained the re-establishment of HIMRE mediated silencing function on the transcriptional activity of yeast heast shock gene HSP82. To test whether the onset of SIR repression can occur in growing cells in the rpesence of a potent inhibitor of DNA replication, HMRa/HSP82 strains with SIR4- and SIR4S$^{+}$ genetic backgrounds were arrested in S phase by incubation of a culture in 200 mM hydroxyurea for 120 min. It was clear that following a 20 minute heat shock, silencing of the HMRa/HSP82 allele in cells pretreated with hydroxyurea does occur in a SIR4-dependen fashion, even though the kinetics of repression appears to be substantially delayed. We also have tested whether re- establishement of silencing at the HMR/hsp82 locus can occur in G1-arrested cells. Cell cycle arrest at G1 phase was achieved by treatment of early log a cell cultures with .alpha.-factor mating pheromone, which induces G1 arrest. The result suggests that passage through S phase (and therefore DNA replication) is nor required for re-establishing silencer-mediated repression at the HMNRa/HSP82 locus. Finally, to test whether de nono protein synthesis is required for re-establishment of silencer-mediated repression, cells were pretreated with cycloheximide (500 /.mu.g/ml) 120 min. It was apparent that inhibiting protein synthesis delays, but does not prevent, re-establishment of silencer-mediated repression. Altogether, these results indicate that re-establishment of silencer-mediated repression is not dependent on the DNA replication and has no requirement for protein synthesis.s.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.340-344
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• 2002

A gene, npl, encoding neopullulanase from Paenibacillus sp. KCTC 8848P was cloned and expressed in Escherichia coli. It consisted of an open reading frame of 1,530 bp for a protein that consisted of 510 amino acids with a molecular weight of 58,075 Da. The deduced amino acid sequence of the neopullulanase gene had $92\%$ identity with the neopullulanase of Bacillus polymyxa. The npl gene was also expressed in Saccharomyces cerevisiae secreting Schwanniomyces occidentalis glucoamylase (GAM1) under the control of the yeast actin gene (ACT1) promoter. Secretion of the neopullulanase was directed by the yeast mating pheromone ${\alpha}$ -factor ($MF{\alpha}1$) prepro region. Enzyme assays confirmed that co-expression of npl and GAM1 enhanced starch and pullulan degradation by S. cerevisiae.

• Molecules and Cells
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• 제26권1호
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• pp.41-47
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• 2008

Mitogen-activated protein kinase (MAPK) signaling is a crucial component of eukaryotic cells; it plays an important role in responses to extracelluar stimuli and in the regulation of various cellular activities. The signaling cascade is evolutionarily conserved in the eukaryotic kingdom from yeast to human. In response to a variety of extracellular signals, MAPK activity is known to be regulated via phosphorylation of a conserved $T{\times}Y$ motif at the activation loop in which both threonine and tyrosine residues are phosphorylated by the upstream kinase. However, the mechanism by which both residues are phosphorylated continues to remain elusive. In the budding yeast, Saccharomyces cerevisiae, Fus3 MAPK is involved in the mating signaling pathway. In order to elucidate the functional mechanism of MAPK activation, we quantitatively profiled phosphorylation of the $T{\times}Y$ motif in Fus3 using mass spectrometry (MS). We used synthetic heavy stable isotope-labeled phosphopeptides and nonphosphopeptides corresponding to the proteolytic $T{\times}Y$ motif of Fus3 and accompanying data-dependent tandem MS to quantitatively monitor dynamic changes in the phosphorylation events of MAPK. Phosphospecific immunoblotting and the MS data suggested that the tyrosine residue is dynamically phosphorylated upon stimulation and that this leads to dual phosphorylation. In contrast, the magnitude of threonine phosphorylation did not change significantly. However, the absence of a threonine residue leads to hyperphosphorylation of the tyrosine residue in the unstimulated condition, suggesting that the threonine residue contributes to the control of signaling noise.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.966-971
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• 2009

Peroxidase from Coprinus cinereus (CiP) has attracted attention for its high specific activity and broad substrate spectrum compared with other peroxidases. In this study, the functional expression of this peroxidase was successfully achieved in the methylotrophic yeast Pichia pastoris. The expression level of CiP was increased by varying the microbial hosts and the expression promoters. Since a signal sequence, such as the alpha mating factor of Saccharomyces cerevisiae, was placed preceding the cDNA of the CiP coding gene, expressed recombinant CiP (rCiP) was secreted into the culture broth. The Mut Pichia pastoris host showed a 3-fold higher peroxidase activity, as well as 2-fold higher growth rate, compared with the $Mut^s $ Pichia pastoris host. Furthermore, the AOX1 promoter facilitated a 5-fold higher expression of rCiP than did the GAP promoter.

• Journal of Microbiology and Biotechnology
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• 제15권1호
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• pp.202-205
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• 2005

Our previous study showed that the overexpression of carboxypeptidase Y (CPY) of Saccharomyces cerevisiae in Escherichia coli resulted in the formation of insoluble inclusion bodies. To produce soluble CPY, we designed a novel Pichia pastoris expression system, in which the following were inserted into expression vectors: three different signal sequences derived from the mating factor a1 of S. cerevisiae, an inulinase of Kluyveromyces marxianus, and the endogenous signal sequence of CPY. The expression vector pHIL-D2-SSinul-proCPY was the most effective in the production of proCPY among the vectors examined. The purified active CPY was obtained from proCPY by treating with proteinase K, followed by QExcellose ion-exchange column chromatography.

• 생명과학회지
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• 제23권10호
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• pp.1192-1198
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• 2013

바이오 에탄올 생산을 위한 최적 효모균주의 개량을 위해 효모 genome shuffling 법을 이용하여 에탄올내성, 내열성 및 ${\beta}$-1,3-glucanase 활성을 가진 효모균주의 육종을 계획하였다. 본 연구에서는 세포 외 ${\beta}$-1,3-glucanase 활성을 가진 Saccharomyces cerevisiae $BY4742{\Delta}exg1$/pAInu-exgA 균주와 에탄올내성 및 내열성을 가진 S. cerevisiae YKY020 균주를 효모 protoplast fusion을 통하여 융합시켰다. 세포융합에 의해 $40^{\circ}C$에서 내열성을 보이는 네 개의 후보 균주(No. 3, 9, 11, 12)를 선별한 다음, 7% 에탄올 농도에서의 에탄올내성 및 ${\beta}$-1,3-glucanase 활성을 조사하였다. 두 모균주의 모든 표현형을 보이는 하나의 균주(No. 11)가 선별되었고, 이 균주를 BYK-F11이라고 명명하였다. BYK-F11 융합균주는 $BY4742{\Delta}exg1$/pAInu-exgA와 YKY020균주에 비해서 증가된 세포성장속도, 에탄올 내성, ${\beta}$-1,3-glucanase 활성 및 에탄올 생산성을 보임을 알 수 있었다. 따라서 본 연구에서는 다양한 특성을 가지지만 같은 접합형을 가진 효모균주들을 protoplast fusion법을 사용하여 손쉽게 새로운 산업용 효모균주로 육종시킬 수 있다는 것을 증명하였다.

• BMB Reports
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• 제35권5호
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• pp.476-481
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• 2002

The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae $\alpha$-factor or Pem-CMG was employed. The results demonstrated that ${\alpha}Pem$-CMG, either with (${\alpha}2EACMG$) or without (${\alpha}CMG$) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either ${\alpha}2EACMG$ or ${\alpha}CMG$ was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both ${\alpha}2EACMG$ and ${\alpha}CMG$ was correctly processed. This produced the mature Pem-CMG peptide.

• 미생물학회지
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• 제32권2호
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• pp.102-108
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• 1994

ABF1(Autonomously replicating sequence Binding Factor 1)은 효모 genome에서 $RTCRYN_5ACG$의 염기 서열을 가지고 있는 promoter, mating-type silencer, ARS에 결합하는 DNA binding 단백질이다. E. coli 에서 ABF1 유전자를 발현하기 위하여, ABF1 유전자를 pMAL-c2 벡터에 cloning하였다.(pMAHW). pMAHW를 E. coli에 형질전환하여, ABF1 융합단백질을 발현시키고, amylose resin affinity chromatography에 의하여 분리하였다. Factor Xa protease를 이용하여 분리된 융합단백질로부터 maltose binding protein을 잘라낸 후에 gel retardation analysis 방법으로 분리된 ABF1이 ARS1에 결합하는 능력을 지니고 있음을 확인하였다. DNA 결합에 관련된 부위를 찾기 위하여, 비전형적인 zinc finger motif가 위치하는 자리에서 pMAHW의 ABF1 유전자에 His-61을 다른 아미노산으로 치환하였다. DNA binding 부위로 추정되는 ABF1 단백질의 중간지역에 Leu-353, Leu-360를 다른 아미노산으로 치환하였다. Site-specific mutagenesis 를 통해 만들어진 mutant를 gel retardation analysis와 complementation test를 통해서 비전형적인 zinc finger motif이외에 다른 DNA binding motif가 있는 것을 알 수 있었다.