• 한국미생물·생명공학회지
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• 제25권3호
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• pp.258-265
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• 1997

The effects of medium pH and culture temperature on the expression and secretion of inulinase and invertase were investigated with recombinant Saccharomyces cerevisiae cells. These cells were obtained by transformation of 2$\mu$-based plasmids pYI10 and pYS10 which contain Kluyveromyces marxianus inulinase gene (INU1A) and S. cerevisiae invertase gene (SUC2), respectively, in the downstream of GAL1 promoter. The expression level and localization of inulinase and invertase were not affected significantly by the initial medium pH: secretion efficiencies of inulinase and invertase into the medium were about 90% and 60%, respectively, in the pH ranges of 4.0 to 6.5. However, the expression and secretion of both enzymes were strongly dependent on the culture temperature. The highest expression (7.7 units/mL) and secretion (6.7 units/mL) of inulinase were observed at 28$\circ$C and 30$\circ$C. As a consequence of decreased localization of inulinase in the periplasmic space, the secretion efficiency increased from 68% at 20$\circ$C, to 95% at 35$\circ$C,. The total expression level and secretion efficiency of invertase increased from 19 units/mL and 55% at 20$\circ$C to 25 units/mL and 68% at 35$\circ$C, respectively. Irrespective of the culture temperature, the invertase activity in the cellular fraction (periplasmic space and cytoplasmic fractions) was kept constant at around 33-45%.

• Journal of Microbiology and Biotechnology
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• 제3권3호
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• pp.168-173
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• 1993

The expression kinetics of human lipocortin I (LCI), a potential anti-inflammatory agent, was studied in the shake-flask and fermenter cultures of Saccharomyces cerevisiae carrying a galactose-inducible expression system. The cell growth, expression level of LCI, and the plasmid stability were investigted under various galactose induction conditions. The expression of LCI was repressed by the presence of a very small amount of dextrose in the culture medium, but it was induced by galactose after dextrose became completely depleted. The optimal ratio of dextrose to galactose for lipocortin I production was found to be 1.0 (10 g/l dextrose and 10 g/l galactose). With optimal D/G ratio of 1.0 and the addition of galactose prior to dextrose depletion, LCI of about 100~130 mg/l was produced. LCI at a concentration of 174 mg/l was porduced in the fed-batch culture, which was nearly a twice as much of that produced in the batch culture. The plasmid stability was very high in all culture cases, and thus was considered to be not an important parameter in the expression of LCI.

• Molecules and Cells
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• 제25권2호
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• pp.172-177
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• 2008

Eukaryotic translation initiation factor 5B (eIF5B) plays a role in recognition of the AUG codon in conjunction with translation factor eIF2, and promotes joining of the 60S ribosomal subunit. To see whether the eIF5B proteins of other organisms function in Saccharomyces cerevisiae, we cloned the corresponding genes from Oryza sativa, Arabidopsis thaliana, Aspergillus nidulans and Candida albican and expressed them under the control of the galactose-inducible GAL promoter in the $fun12{\Delta}$ strain of Saccharomyces cerevisiae. Expression of Candida albicans eIF5B complemented the slow-growth phenotype of the $fun12{\Delta}$ strain, but that of Aspergillus nidulance did not, despite the fact that its protein was expressed better than that of Candida albicans. The Arabidopsis thaliana protein was also not functional in Saccharomyces. These results reveal that the eIF5B in Candida albicans has a close functional relationship with that of Sacharomyces cerevisiae, as also shown by a phylogenetic analysis based on the amino acid sequences of the eIF5Bs.

• 한국동물학회지
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• 제38권2호
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• pp.196-203
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• 1995

The c-myc proto-oncogene, one of the immediately earlY genes, is expressed in various mammalian cell types and heavily involved in the regulation of cell proliferation and differentiation. To determine endogeneous expression pattern of c-myc gene in preimpBantation mouse embwos, we employed a reverse transcription coupled to polvrnerase chain reaction (RT-PCR). Transcript of c-myc was detected at fertilized embryos as a maternal transcript. At the early two-cell stave, transcript of c-myc gene was hardly detected, bu, appeared at late two-cell embryos as a zygotic transcript. The level of c-myc expresion was increased at later stases and peaked at blastocvst stage. To examine the functional role of promoter region for c-myc gene transcription, we fused the 5'upstream region (1.8 kb) including econ 1 of c-myc genomic DNA with E. coli lacE gene fnamed as pcMYC-laczl. pcMYC-lacZ was microiniected into the pronscleus of mouse one-cell embryovs, and p·salactosidase activity was determined tv histochemical staining with X-gal at different stases. f-galactosidase activity was detected only at blastocyst, but not at the earlier stage embryos. This result indicates that c-myc gene is transcriptionallv active during mouse preimplantation development.

• Journal of Chest Surgery
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• 제30권2호
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• pp.125-130
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• 1997

저체온 심폐바이패스후에 환자를 적절히 재가온시키지 못하면 수술직후 중환자실에서 종종 후하강 (afterdrop)현상이 발생할 수있다. 후하강이 생기면 전해질 장애, 부정맥, 혈역학적 불안정을 일으킬 수 있을뿐 아니라 전율에 의해 산소소모량을 크게 증가시킴으로서 수술 직후 불안정한 심근에 악영향을 미칠 수 있다. 따라서 본 연구는 소아 심장수술환자에서 적정 재가온 종료 온도를 찾기 위한 노력의 일환으로 50명의 환자들을 직장 온도를 기준으로 섭씨 35.5도에서 재가온을 종료한 군과 37도에서 재가온을 종료한 군으로 나눈뒤 두 군 사이에 후하강의 발생 여부를 비교 분석하였다. 그리고 후하강의 여부와 함께 심박수, 수축기 및 확장기 혈압 등 활력 증후도 함께 측정하였다. 그밖에 체온측정 장소에 따른 온도 의 차이를 관찰하기 위 해 심폐바이패스 중에는 직장 온도와 함께 식도 온도를 그리고 중환실에서는 직장 온도와 함께 액와 온도를 각각 측정하였다. 환자들은 전례에서 롤러 펌프에 의한 비박동성 관류를 시행 받았으며 산화기로는 막형 또는 기포형 산화기가 사용되었다. 두군의 환자들은 연령, 성별, 체표면적, 심폐바이패스 시간, 재가온 시간 등에서 유의한 차이가 없었다. 관찰 결과 두군의 환자들에서 모두 회복실에 도착한후 16시간후 까지 측정한 직장 온도에서 후하강은 없었으며 두군간 시간대별 직장 체온의 차이도 관찰되지 않았다. 두군간 심박수와 수축기 및 확장기 혈압에서도 유의한 차이는 발견되지 않았다. 그리고 재가온 종료시 식도 온도는 직장 온도에 비해 높게 측정되었으며 중환자실에서 측정한 액와 온도는 직장 온도보다 항상 낮게 측정되었다. 전율은 전례에서 관찰되지 않았다. 결론적으로 본 실험 결과는 소아에서는 직장 온도를 기준으로 적어도 섭씨 35.5도 이상에서 재가온을 종료하면 후하강이 일어나기 어렵다는 것을 의미하고 있다. 8례(11%),술후 일과성 신경학적 합병증이 7례 (9%),술후 일과성 정신과적 합병증이 6례 (8%), 급성 신부전이 3례 (4%),술후 출혈 및 폐렴이 각각 2례 (3%), 창상 감염과 십이지장 궤양 천공이 각각 1례 (1 %)이었다. 역행성심정 지액을 이용한 심근 보호법은 고위험도의 허혈성 심질환 환자에서의 관상동맥 우회술에서 수술 위험도를 최소화할 수 있는 유용한 심근 보호법이라고 생각된다.처리구를 무작위로 할당하여 배양하였으며, 또한 740개의 정상수정란도 같은 반복수로 배양을 실시하였다. 미세주입한 수정란은 8일 후 11.6%(26/255) 및 5.2% (14/267)가 대조구 및 G418 처리구에서 배반포기까지 발달하였으며 정상수정란은 27.2% (151/740)가 배반포기 배까지 발달하였다. 미세주입후 대조구에서는 23.1$\pm$2.6/70.7$\pm$4.7 (32.7%)의 할구가 $\beta$-Gal 활력을 보였고, 반면에 100$\mu\textrm{g}$/ml G418 처리구에서는 40.3$\pm$4.1/48.8$\pm$7.5 (82.6%)가 $\beta$-Gal 활력을 보였다. 비록 mosaic 형태로 외래유전자가 발현되었지만 대조구에서 87.0% (26/30개) 배반포기가 $\beta$-Gal 활력을 보인 반면, G418 처리구에서는 모든 배반포기가 $\beta$-Gal 활력을 보였다 (P<0.05). 그러나 대조구 및 G418 처리구의 ICM colony에서는 영양배엽과 내배엽을 제외한 epiblast에서는 확인되지 않았다. 그러나 이 결과로부터 $\beta$-actin promoter/lacZ gene이

• 생명과학회지
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• 제21권7호
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• pp.932-938
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• 2011

Ginsenoside Rg1은 인삼에서 분리한 약물학적인 활성을 가지는 물질이다. 본 연구는 Rg1이 제2형 당뇨병 모델 동물에서 혈당과 지질대사에 유익한 효과를 가지는지를 확인하기 위한 목적으로 수행되었다. 10주령의 db/db 마우스에 Rg1을 10 mg/kg 농도로 15일간 경구투여한 결과 공복혈당이 감소하였고, 포도당 내성이 개선되었다. 특히 혈중 중성지방과 유리지방산이 유의적으로 감소하였고 혈중 HDL-콜레스테롤이 증가되었다. 또한 chimeric GAL4-PPAR${\alpha}$ receptor 활성 프로모터를 활성화시켰고 PPAR${\alpha}$ gene인 CPT-1 (carnitine palmitoyltransferase-1)과 ACO (acyl-CoA oxidase)의 발현을 증가시켰는데 이것으로 Rg1의 지질대사 개선이 PPAR${\alpha}$ 활성에 의한 지방산 산화에 의한 것임을 확인할 수 있었다. 모든 결과를 종합해 볼 때, Rg1은 제2형 당뇨병과 관련된 고혈당증과 고지혈증에 유용한 효과를 가짐을 확인하였다.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2010년도 정기총회 및 추계학술발표회
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• pp.14-14
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• 2010

Vitamin C (ascorbic acid) is an essential component for collagen biosynthesis and also for the proper functioning of the cardiovascular system in humans. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. In this study, we have developed two different kinds of transgenic potato plants (Solanumtuberosum L. cv. Taedong Valley) overexpressing strawberry GalUR and mouse GLoase gene under the control of CaMV 35S promoter with increased ascorbic acid levels. Integration of the these genes in the plant genome was confirmed by PCR and Southern blotting. Ascorbic acid(AsA) levels in transgenic tubers were determined by high-performance liquid chromatography(HPLC). The over-expression of these genes resulted in 2-4 folds increase in AsA intransgenic potato and the levels of AsA were positively correlated with increased geneactivity. The transgenic lines with enhanced vitamin C content showed enhanced tolerance to abiotic stresses induced by methyl viologen(MV), NaCl or mannitol as compared to untransformed control plants. The leaf disc senescence assay showed better tolerance in transgenic lines by retaining higher chlorophyll as compared to the untransformed control plants. Present study demonstrated that the over-expression of these gene enhanced the level of AsA in potato tubers and these transgenics performed better under different abiotic stresses as compared to untransformed control. We have also investigated the mechanism of the abiotic stress tolerance upon enhancing the level of the ascorbate in transgenic potato. The transgenic potato plants overexpressing GalUR gene with enhanced accumulation of ascorbate were investigated to analyze the antioxidants activity of enzymes involved in the ascorbate-glutathione cycle and their tolerance mechanism against different abiotic stresses under invitro conditions. Transformed potato tubers subjected to various abiotic stresses induced by methyl viologen, sodium chloride and zinc chloride showed significant increase in the activities of superoxide dismutase(SOD, EC 1.15.1.1), catalase, enzymes of ascorbate-glutathione cycle enzymes such as ascorbate peroxidase(APX, EC 1.11.1.11), dehydroascorbate reductase(DHAR, EC 1.8.5.1), and glutathione reductase(GR, EC 1.8.1.7) as well as the levels of ascorbate, GSH and proline when compared to the untransformed tubers. The increased enzyme activities correlated with their mRNA transcript accumulation in the stressed transgenic tubers. Pronounced differences in redox status were also observed in stressed transgenic potato tubers that showed more tolerance to abiotic stresses when compared to untransformed tubers. From the present study, it is evident that improved to lerance against abiotic stresses in transgenic tubers is due to the increased activity of enzymes involved in the antioxidant system together with enhanced ascorbate accumulated in transformed tubers when compared to untransformed tubers. At moment we also investigating the role of enhanced reduced glutathione level for the maintenance of the methylglyoxal level as it is evident that methylglyoxal is a potent cytotoxic compound produced under the abiotic stress and the maintenance of the methylglyoxal level is important to survive the plant under stress conditions.

• 생명과학회지
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• 제15권1호
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• pp.118-123
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• 2005

B. stearothermophilus 유래의 CGTase 유전자(cgtS)를 보유하고 있는 재조합 plasmid pCGTS (4.8 kb)을 효모 표면 발현용 vector인 pYDl (GAL1 promoter)에 subcloning 하였다. 구축된 재조합 plasmid, pYDCGT (7.2 kb)는 S. cerevisiae EBY100에 형질전환하였고, tryptophan이 결여된 SD 배지에서 1차 선별된 형질전환체들을 YPGS배지에서 배양 후 활성 염색을 통하여 CD가 생 성 됨을 확인하였다. 배양시간과 효소반응시간에 따른 반응 산물을 TLC로 분석 한 결과, 배양 12시간째부터 효소활성이 나타났고, 반응 10분 이후부터 CD가 생성되어 시간이 지남에 따라 CD 생성양이 증가하는 것을 확인하였다. 회분 배양한 결과 $25^{\circ}C$와 $30^{\circ}C$에서 CGTase의 최대 활성이 각각 21.3 unit/1 와 16.5 unit/1로 나타났고, plasmid 안정성은 각각 $86\%$와 $82\%$로 나타나 배양온도에 상관없이 plasmid는 비교적 안정하게 유지되었다.

• Journal of Microbiology
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• 제40권4호
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• pp.313-318
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• 2002

To perform the prophylactic study of a vaccine derived from human papillomavirus (HPV) using Balb/c mice, we produced virus like particles consisting of HPV capsid protein L1 which has been reported to induce significant humoral and cellular immunity using various animal model systems. In order to produce HPV16 VLPs, the cDNA of L1 capsid protein in HPV type 16, obtained by polymerase chain reaction, was inserted into yeast expression vector, YEG$\alpha$-HIR525 under the control of GAL10 promoter. The transformation of YEG$\alpha$-HPV16 L1 was performed into the yeast Saccharomyces cerevisiae Y2805 by the lithium acetate method and the yeast clone expressing the highest level of L1 capsid protein of human papillomavirus type 16 was selected by Western blot analysis using anti-HPV16 L1 antibody. The purification of HPV16 VLP has been performed by the ultracentrifugation and gel-filtration methods. To validate the vaccine efficacy of the purified HPV16 VLPs and investigate the properties of HPV16 VLPs to induce humoral immunity, ELISA assay was performed. A significantly increased production of anti-HPV16 VLP antibodies was observed in sera from immunized mice. The neutralization activity of antibodies in the sera from the vaccinated mice was demonstrated by a rapid and simple assay to detect hemagglutihation inhibition activity.

• Journal of Microbiology and Biotechnology
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• 제13권3호
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• pp.451-456
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• 2003

An expression vector system was developed for the secretory production of recombinant Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae. The mature L1 lipase gene was fused to ${\alpha}-amylase$ signal sequence from Aspergillus oryzae for the effective secretion into the culture broth and the expression was controlled under GAL10 (the gene coding UDP-galactose epimerase of S. cerevisiae) promoter. S. cerevisiae harboring the resulting plasmid successfully secreted L1 lipase into the culture broth. To examine an optimum condition for L1 lipase expression in the fed-batch culture, L1 lipase expression was induced at three different growth phases (early, mid, and late-exponential growth phases). Maximum product on of L1 lipase (1,254,000 U/l, corresponding to 0.65/1) was found when the culture was induced at an early growth phase. Secreted recombinant L1 lipase was purified only through CM-Sepharose chromatography, and the purified enzyme showed 1,963 U/mg of specific activity and thermoalkalophilic properties similar to those reported for the enzyme expressed in Escherichia coli.