• Journal of Microbiology and Biotechnology
• /
• 제20권2호
• /
• pp.356-362
• /
• 2010

Ceramide is important not only for the maintenance of the barrier function of the skin but also for the water-binding capacity of the stratum corneum. Although the exact role of ceramide in the human skin is not fully understood, ceramide has become a widely used ingredient in cosmetic and pharmaceutical industries. Compared with other microorganisms, yeast is more suitable for the production of ceramide because yeast grows fast and is non-toxic. However, production of ceramide from yeast has not been widely studied and most work in this area has been carried out using Saccharomyces cerevisiae. Regulating the genes that are involved in sphingolipid synthesis is necessary to increase ceramide production. In this study, we investigated the effect of the genes involved in the synthesis of ceramide, lcb1, lcb2, tsc10, lac1, lag1, and sur2, on ceramide production levels. The genes were cloned into pYES2 high copy number vectors. S. cerevisiae was cultivated on YPDG medium at $30^{\circ}C$. Ceramide was purified from the cell extracts by solvent extraction and the ceramide content was analyzed by HPLC using ELSD. The maximum production of ceramide (9.8 mg ceramide/g cell) was obtained when the tsc10 gene was amplified by the pYES2 vector. Real-time RT-PCR analysis showed that the increase in ceramide content was proportional to the increase in the tsc10 gene expression level, which was 4.56 times higher than that of the control strain.

• 미생물학회지
• /
• 제46권4호
• /
• pp.401-404
• /
• 2010

출아효모 Saccharomyces cerevisiae에서 RNA-binding 단백질인 Tho1은 mRNA가 전사되는 동안 초기 mRNA에 결합하여 mRNP 생성과 성숙한 mRNA의 핵에서 세포질로의 방출에 관여하는 것으로 여겨진다. 분열효모 Schizosaccharomyces pombe에서도 Tho1과 유사한 단백질을 암호화하는 유전자(spTho1로 명명)를 찾아 그 특성을 조사하였다. 이배체 S.pombe 균주에 하나의 spTho1 유전자만을 결실시킨 후 4분체분석을 수행한 결과, 이 유전자는 생장에 반드시 필요하지 않았다. 또한 spTho1 결실 돌연변이는 mRNA의 핵에서 세포질로의 방출도 정상적으로 보였다. 그러나 티아민에 의해 발현이 조절되는 강력한 프로모터를 이용하여 spTho1를 과발현시키면, 세포의 생장이 억제되었으며 $poly(A)^+$ RNA가 핵 안에 축적되었다. 이와 같은 결과들은 spTho1 유전자가 필수적이지는 않지만 mRNA의 핵에서 세포질로의 방출에 관여하고 있음을 시사한다.

• 한국미생물·생명공학회지
• /
• 제14권3호
• /
• pp.213-218
• /
• 1986

B. amyloliquefaciens의 $\alpha$-amylase 유전자가 S. cerevisiae 내에서 형질발현하는 가를 조사하기 위하여 본, 연구에서 YRp7 plasmid에 B. amyloliquefaciens amylase유전자를 cloning하여 만든 pEA24를 형질전환시켰다. 먼저 YRp7 plasmid를 이용하여 형질전환 최적 조건을 검토하여 본 바, PH 7과 8사이, 반응온도 3$0^{\circ}C$에서 40%의 polyethylene glycol(MW 4,000)을 처리한 후 2 %의 agar를 함유한 재생배지에 중층도말 하였을 때 형질전환율이 가장 높았다. 형질전환주로부터 생성된 amylase의 활성을 측정한 결과, S. cerevisiae에서 약간의 amylase활성을 나타내어 최고 B. amyloliquefaciens의 2% 정도였고, 세포외효소는 검출되지 않았다. 이들 형질전환 주가 가지고 있는 pEA24 plasmid의 안정성을 조사한 결과 YRp7보다 불안정하였으며, 추출한 DNA를 전기영동하여 그 band를 확인하였다.

• Animal Bioscience
• /
• 제35권6호
• /
• pp.858-868
• /
• 2022

Objective: This study was conducted to evaluate the effects of dietary yeast hydrolysate (YH) supplementation on intestinal morphology, barrier, and anti-inflammatory functions of broilers. Methods: A total of 320 one day old male broilers were randomly allocated into four groups with eight replicates of ten broilers each. The broilers were supplemented with a basal diet (the control group) or basal diets adding 50, 100, 150 mg/kg YH, respectively. This trial lasted for 42 days. The orthogonal polynomial contrasts were used to determine the linear and quadratic effects of increasing levels of YH. Results: In our previous research, supplementing YH improved growth performance by enhancing body weight gain but decreased feed-to-gain ratio. In this study, compared with the control group, dietary YH addition linearly and quadratically decreased serum diamine oxidase activity (p<0.05). Additionally, supplementing YH linearly and/or quadratically decreased jejunal crypt depth (CD), tumor necrosis factor-alpha (TNF-α) concentration as well as mucin 2, interleukin-6 (IL-6), IL-1β, TNF-α, nuclear factor kappa B, and myeloid differentiation factor 88 gene expression levels (p<0.05). Whereas the jejunal villus height (VH), VH/CD, IL-10 concentration as well as zonula occludens-1 and IL-10 gene expression levels were linearly and/or quadratically increased by YH supplementation (p<0.05). Conclusion: Dietary YH supplementation improved intestinal morphology, barrier and anti-inflammatory functions while decreased intestinal permeability of broilers, which might be related with altering pertinent genes expression. This study provides evidence of YH as a promising feed additive for broilers.

• KSBB Journal
• /
• 제19권5호
• /
• pp.352-357
• /
• 2004

Human ferritin H- and L-chain genes (hfH and hfL) were cloned into the yeast shuttle vector YEp352 containing the GAL1 (galactokinase) and GAL10 (epimerase) divergent promoters and the vectors constructed were used to transform Saccharomyces cerevisiae 2805. SDS-PAGE displayed expression of the introduced hfH and hfL in both recombinant strains of Y1H10L and Y1L10H. The ferritin subunits, that represented ca. $22\%$ and $15\%$ of the soluble proteins in Y1H10L and Y1L10H, were spontaneously assembled into active ferritin heteropolymers. The H subunit content of the purified recombinant human ferritin heteropolymers was proven to reflect the relative expression yield of the subunits. When the cells of 2d culture were incubated with 14.3 mM Fe(2), the cellular iron concentration of Y1H10L and Y1L10H was 1.7 and 2.0 times, respectively, that of the control strain. It is assumed that increase in the iron uptake of the recombinant yeasts is closely related to ferritin expression and H subunit content.

• 한국생물정보학회:학술대회논문집
• /
• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
• /
• pp.313-318
• /
• 2005

Estimating the reliability of protein-protein interaction data sets obtained by high-throughput technologies such as yeast two-hybrid assays and mass spectrometry is of great importance. We develop a maximum likelihood estimation method that uses both protein localization and gene expression data to estimate the reliability of protein interaction data sets. By integrating protein localization data and gene expression data, we can obtain more accurate estimates of the reliability of various interaction data sets. We apply the method to protein physical interaction data sets and protein complex data sets. The reliability of the yeast two-hybrid interactions by Ito et al. (2001) is 27%, and that by Uetz et at.(2000) is 68%. The reliability of the protein complex data sets using tandem affinity purification-mass spec-trometry (TAP) by Gavin et at. (2002) is 45%, and that using high-throughput mass spectrometric protein complex identification (HMS-PCI) by Ho et al. (2002) is 20%. The method is general and can be applied to analyze any protein interaction data sets.

• 응용통계연구
• /
• 제20권2호
• /
• pp.409-422
• /
• 2007

시간경로 유전자 발현자료에 대한 본격적인 통계분석을 수행하기에 앞서 의미있는 정보를 제공하지 못할 것으로 여겨지는 유전자들은 선별하여 미리 제거함으로서 자료의 차원을 축소시킬 수 있을 뿐 아니라, 잡음이나 변이가 낮은 자료로 인한 잘못된 판단을 감소시킬 수 있다. 본 논문에서는 관측표본에 대한 백분위수 기준과 붓스트랩 표본에 대한 백분위수 기준 하에서 무 변화 패턴을 갖는 유전자들을 제거시킬 수 있는 기존의 필터링 함수들을 비교하였다. 이스트(yeast) 자료에 적용하여 두 가지 필터링 방식에 대해 가장 유사한 결과를 보인 것은 분산 함수였다.

• Journal of Microbiology and Biotechnology
• /
• 제25권1호
• /
• pp.50-56
• /
• 2015

During pretreatment of lignocellulosic biomass, a variety of fermentation inhibitors, including acetic acid and vanillin, are released. Using DNA microarray analysis, this study explored genes of the budding yeast Saccharomyces cerevisiae that respond to vanillin-induced stress. The expression of 273 genes was upregulated and that of 205 genes was downregulated under vanillin stress. Significantly induced genes included MCH2, SNG1, GPH1, and TMA10, whereas NOP2, UTP18, FUR1, and SPR1 were down regulated. Sequence analysis of the 5'-flanking region of upregulated genes suggested that vanillin might regulate gene expression in a stress response element (STRE)-dependent manner, in addition to a pathway that involved the transcription factor Yap1p. Retardation in the cell growth of mutant strains indicated that MCH2, SNG1, and GPH1 are intimately involved in vanillin stress response. Deletion of the genes whose expression levels were decreased under vanillin stress did not result in a notable change in S. cerevisiae growth under vanillin stress. This study will provide the basis for a better understanding of the stress response of the yeast S. cerevisiae to fermentation inhibitors.

• BMB Reports
• /
• 제41권8호
• /
• pp.581-586
• /
• 2008

The pre-replication complex (pre-RC), including the core hexameric MCM2-7 complex, ensures that the eukaryotic genome is replicated only once per cell division cycle. In this study, we identified a rice $\underline{m}ini\underline{c}hromosome$ $\underline{m}aintenance$ (MCM) homologue (OsMCM2) that functionally complemented fission yeast MCM2 (CDC19) mutants. We found OsMCM2 transcript expression in roots, leaves, and seeds, although expression levels differed slightly among the organs. Likewise, the OsMCM2 protein was ubiquitously expressed, but it was downregulated when nutritients were limiting, indicating that MCM2 expression (and therefore cell cycle progression) requires adequate nutrition. Yeast two-hybrid and GST pull-down assays demonstrated that OsMCM2 interacted with the COP9 signalosome 5 (CSN5). Taken as a whole, our results indicated that OsMCM2 functions as a subunit of the rice MCM complex and interacts with CSN5 during developmental regulation.

• Journal of Microbiology
• /
• 제44권1호
• /
• pp.35-41
• /
• 2006

The unique gene encoding thioredoxin reductase (TrxR) was previously cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its expression was induced by oxidative stress. To elucidate tbe regulatory mechanism of the S. pombe TrxR gene, three fusion plasmids were generated using polymerase chain reaction: pYUTR20, pYUTR30, and pYUTR40. Plasmid pYUTR20 has an upstream region of 891 base pairs, pYUTR30 has 499 in this region, and pYUTR40 has an 186 bp upstream region. Negatively acting sequence is located between $-1,526\;\~\;-891bp$ upstream of the gene. The upstream sequence, responsible for the induction of TrxR by menadione (MD), is situated on the $-499\;\~\;-186bp$ region, which is also required for TrxR induction by mercuric chloride. The same region also appeared to be required for Pap1-mediated transcriptional regulation of the TrxR gene, which contains the two plausible Papl binding sites, TTACGAAT and TTACGCGA. Consistently, basal and inducible expression of the TrxR gene was markedly lower in the Pap1-negative TP108-3C cells than in wild-type yeast cells. In summary, up-regulation of the S. pombe TrxR gene is mediated by Pap1 via the transcriptional motif(s) located on the $-499\;\~\;-186bp$ region.