• Title/Summary/Keyword: aP2 promoter

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Construction of Bioluminescent Escherichia coli from lux Operon and Heat Shock Promoter for the Detection of Toxic Substances (lux Operon과 Heat Shock Promoter 유전자 재조합을 통한 독성물질 탐지용 대장균의 개발)

  • 유승오;이은관;김현숙;정계훈;전억한
    • Microbiology and Biotechnology Letters
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    • v.27 no.4
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    • pp.278-285
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    • 1999
  • In order to use heat shock promoter for the detection of toxic substances, dnaK promoter was amplified from E. coli genomic DNA by using a polymerase chain reaction(PCR) followed by sequencing and sub-cloning into the multi-cloning site of the plasmid, pUCD615. The pUCD615 is a broad-host-range vector containing promoterless lux operon originated from V.fischeri. The recombinant plasmid was transfered to E. coli DH5$\alpha$ through electroporation. The recombinant E. coli showed several patterns of bioluminescent responses to ethanol stress. The bioluminescent E. coli also showed responses to other toxic substances including FeK3(CN)6, CdCl2, p-nitrophenol and HgCl2. The increases of RLU(Relative Light Unit) were observed at 100ppm of FeK3(CN)6, 10ppm and 100ppm and 100ppm of CdCl2, 1ppm of 10ppm of p-nitrophenol and at 1ppm of HgCl2.

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Deletion Analysis of Pichia PGK1 Promoter and Construction of an Episomal Vector for Heterologous Protein Expression in P. pastoris (Pichia PGK1프로모터의 분석과 P. pastoris에 있어 외래단백질발현을 위한 Episomal벡터의 제조)

  • Lee, Sung-Jae;Hong, In-Pyo;Baek, Seon-Yeol;Choi, Shin-Geon
    • Microbiology and Biotechnology Letters
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    • v.35 no.3
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    • pp.184-190
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    • 2007
  • Approximately 2.0 kb of the promoter region of the Pichia pastoris phosphoglycerate kinase gene (PGK1) was reduced to a 266 bp fragment and this minimized portion was used for construction of a new episomal constitutive expression vector in P. pastoris. As an approach to developing a constitutive expression vector in P. pastoris, the GAP promoter region of the Pichia expression vector pGAPZB was replaced with sequentially deleted PGK1 promoter fragments fused to a beta-galactosidase gene. When a lacZ gene was used as a reporter gene, PGK1 promoter strength was lower than that of the constitutive GAP promoter but it was higher than TEF1. We report here the development of the pPGKZ-E vector as a new episomal expression vector for heterologous gene expression by removing non-essential regions of the PGK1 promoter. This broadens the choice of episomal expression vectors for controlled constitutive expression in P. pastoris.

Organ Specific Expression of the nos-NPT II Gene in Transgenic Hybrid Poplar (형질 전환된 포플러에 대한 nos-NPT II 유전자의 기관별 발현 특성)

  • Chun, Young Woo;Klopfenstein, Ned B.
    • Journal of Korean Society of Forest Science
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    • v.84 no.1
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    • pp.77-86
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    • 1995
  • To effectively modify tree function with genetic engineering, transgenes must be expressed at the proper level in the appropriate tissues at suitable developmental stages. Toward understanding the spatial and temporal expression of transgenes in woody plants, transgene expression was evaluated in three greenhouse-grown, transgenic lines of Populus alba ${\times}$ P. grandidentata hybrid clone 'Hansen'. All transgenic poplar lines possess constructs containing the bacterial nopaline synthase(nos) promoter linked to a neomycin phosphotransferase II(NPT II) selectable marker gene. In addition, each transgenic poplar line contains one of the following gene constructs : 1) a wound-inducible potato proteinase inhibitor II (pin2) promoter linked to a chloramphenicol acetyltransferase(CAT) reporter gene. 2) a nos promoter linked to a PIN2 structural gene : or 3) a Cauliflower Mosaic Virus 35s promoter linked to a PIN2 structural gene. Polymerase chain reaction(PCR) was used to verify the presence of foreign genes in the poplar genome. Enzyme-linked immunosorbent assays(ELISAs) were used to evaluate organ specific expression of the nos-NPT II construct. NPT II expression was detected in leaves, petioles, stems, and roots of transgenic poplar, thereby indicating that the nos promoter is potentially effective for general constitutive expression of transgenes. NPT expression varied among transgenic poplar lines and among organs for one transgenic line, Tr15. With Tr15, NPT II levels were highest in older leaves and petioles. These results indicate that screening of several transgenic lines may be required to identify lines with optimal transgene expression.

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Transcriptional Regulation of the Glial Cell-Specific JC Virus by p53

  • Kim, Hee-Sun;Woo, Moom-Sook
    • Archives of Pharmacal Research
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    • v.25 no.2
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    • pp.208-213
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    • 2002
  • The human polyomavirus JC virus is the etiologic agent of progressive multifocal leukoencephalopathy (PML). As the JC virus early promoter directs cell-specific expression of the viral replication factor large T antigen, transcriptional regulation constitutes a major mechanism of glial tropism in PML. It has been demonstrated that SV4O or JC virus large T antigen interacts with p53 protein and regulates many viral and cellular genes. In this study we founts that p53 represses the JC virus early promoter in both glial and nonglial cells To identify the cis-regulatory elements responsible for p53-mediated repression, deletional and site-directed mutational analyses were performed . Deletion of the enhancer region diminished p53-mediated transcriptional repression. However, point mutations of several transcription factor binding sites in the basal promoter region did not produce any significant changes. In support of this observation, when the enhancer was fused to a heterologous promoter, p53 red reduced the promoter activity about three fold. These results indicate that the enhancer region is important for tole repression of JC virus transcription by p53. Furthermore, coexpression of JC virus T antigen with a p53 protein abolished p53-mediated repression of the JC virus early promoter in non-glial cells, but not in glial cells. This finding suggests that T antigen interacts with p53 and regulates JC virus transcription in a cell-specific manner.

Mechanism of Regulation of the pts Promoter Transcription Initiation by Carbon Sources in Escherichia coli (Carbon Source의 변화에 의한 대장균의 pts Promoter 전사 조절 기작)

  • Kim, Soon-Young;Kwon, Hyuk-Ran;Shin, Dong-Woo;Ryu, Sang-Ryeol
    • Applied Biological Chemistry
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    • v.42 no.4
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    • pp.293-297
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    • 1999
  • The pts operon, which encodes several factors in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) of Escherichia coli, has multiple promoters which respond to different signals to facilitate quick adaptation to changes in growth conditions. The influence of an 1 kbp DNA region upstream of the pts P0 promoter on pts expression was studied in vitro by employing the DNA templates containing both P0 and P1 promoter with or without the 1 kbp upstream DNA region for in vitro transcription assay. The 1 kbp DNA region upstream of the pts P0 promoter, however, had no effect on pts transcription in vitro. The intracellular concentration of cAMP was measured when cells were grown in the presence of glucose, mannose, or mannitol. The transcription of P0 was increased maximally in the presence of glucose even though the concentration of cAMP in the condition was lowest while the transcription from the P1b was highest when cells were grown in the presence of mannose or mannitol even though the intracellular concentration of cAMP was lower than cells grown in the absence of the sugar. These results suggest the possibility of the existence of a glucose inducible repressor specific for the P0 promoter and a second repressor that is inducible by glucose, mannose and mannitol specific for the P1 promoter.

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Selection of Constitutive Promoter for Exoinulinase Production in Fed-Batch Culture of Recombinant Yeast (재조합 효모의 유가배양에서 Exoinulinase생산을 위한 Promoter의 선별)

  • 김이경;고지현;김연희;김성구;남수완
    • Microbiology and Biotechnology Letters
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    • v.29 no.4
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    • pp.206-211
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    • 2001
  • In order to overexpress constitutively the Kluyveromyces marxianus exoinulinase gene (INUI) in Saccharomyces cerevisiae, four episomal expression systems employing GAPDH, ADHI, PGK and ENOI promoters were constructed as p YIGP aADHI -INU, pPGK-INU, and pENOI- INU plasmids respectively, When S cereviais transformants harboring each plasmid were batchwisely cultivated in the fermentor containing 5% glucose medium no significant differences in the cell growth are observed How- ever the experession level of exoinulinase and plasmid stability showed a strong dependency on the promoter employed. The expression levels of exoinulinase were about 1.70 unit/ml for GAPDH promoter 1.67 unit/ml for PGK promoter, 1.29 unit /ml for ADH1 promoter, and 0.80 unit/ml for ENOl promoter. The plasmid stabilites were maintaines above 80% in all experession systems. except the GAPDH promoter system of 55%, Based on the plas- mid stability and expression level of exoinulinase the ADHl and PGK promoter system were selected for the fed - batch culture to overproduce exoinulinase By the intermittent feeding of yeast extract and glucose, both promoter systems gave the cell concentration of about 30 g-dry cell weight/1 byt the maximal exoinulinase activity of 3.70 unit/ml and plasmid stability of 96% in the ADH1 promoter were higher than those (2.70 unit/ml, 80%) of PGK sys- tem Taking into account the plasmid stability and extended culture time the ADH1 promoter systems would be the most feasible expression systems for the constitutive overproduction of exoinulinase through high cell-density fed- batch cultures using non-selective rich medium.

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Development of E. coli Expression System to Overproduce a Harmful Protein, Carboxypeptidase Taq.

  • Lee, Sang-Hyeon
    • Journal of Life Science
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    • v.11 no.2
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    • pp.108-110
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    • 2001
  • The E. coli expression system to overproduce a harmful protein, carboxypeptidase Taq was developed. Since expression plasmid pCK305N containing the colicin promoter already has the initiation codon on the restriction site, the initiation codon of the CPase Taq gene was removed. Expression plasmid pCP4-col includes the entire CPase Taq gene, which is directed by the colicin promoter. E. coli cells harboring pCP-col produced a high amount of the enzyme when they were cultured in the present of mitomycin C (0.4 ${\mu}g$/ml). An amount of purified enzyme produced by pCP4-col directed by the colicin promoter was 10.5 mg. This result indicated that the novel E. coli expression system controlled by the colicin promoter could produce almost twice amounts of CPase Taq than the conventional system controlled by the tart promoter.

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Optimal Expression System for Production of Recombinant Neoagarobiose Hydrolyase in Saccharomyces cerevisiae (출아효모에서 재조합 neoagarobiose hydrolyase의 생산을 위한 최적 발현시스템)

  • Jung, Hye-Won;Kim, Yeon-Hee
    • Microbiology and Biotechnology Letters
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    • v.47 no.4
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    • pp.662-666
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    • 2019
  • In this study, the NABH558 gene expression system was constructed to efficiently produce neoagarobiose hydrolase (NABH) in Saccharomyces cerevisiae strain. The ADH1 and GAL10 promoters of the pAMFα-NABH and pGMFα-NABH plasmids were examined to determine the suitable promoter for the NABH558 gene expression, respectively. The effect of promoter and carbon sources on NABH558 gene expression was investigated by transforming each plasmid into the S. cerevisiae 2805 strain. The NABH activity in the 2805/pAMFα-NABH strain was 0.069 unit/ml/DCW in YPD medium, whereas that in the 2805/pGMFα-NABH strain was similar (0.02-0.027 unit/ml/DCW) irrespective of the medium composition. The higher NABH activity in the YPD medium was due to the increased NABH558 gene transcription. NABH produced in the recombinant strains could degrade agarose to galactose and AHG. This indicated that ADH1 promoter was a more optimal promoter for the expression of NABH558 gene than the GAL10 promoter. The NABH activity induced by the ADH1 promoter was about 3-fold higher than that induced by the GAL10 promoter.

Analysis of Transcriptional Activity and Estrogen Responsiveness of Regulatory Elements in Chicken Ovalbumin Promoter (닭 오브알부민 프로모터의 길이에 따른 유전자 발현 활성 및 에스트로겐 반응성 분석)

  • Yang, Hyeon;Kim, Kyung-Woon;Kim, Jeom Sun;Woo, Jae-Seok;Lee, Hwi-Cheul;Choi, Hoonsung;Jung, Sun Keun;Sureshkumar, Shanmugam;Lee, Haesun;Oh, Keon Bong;Byun, Sung June
    • Korean Journal of Poultry Science
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    • v.46 no.1
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    • pp.17-24
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    • 2019
  • Chickens have been considered as well-defined animal bioreactor. The optimized ovalbumin promoter is essential for recombinant protein production in transgenic chicken. Here we try to compare the activity and identify the effect of estrogen on ovalbumin promoter according to each promoter length with estrogen response element (ERE) existence. We cloned two (2.8 and 5.5 kb) ovalbumin promoters that the 5.5 kb contained the ERE but the 2.8 kb did not, and these two promoters were cloned to pGL4.11 vector. Additionally, we constructed another pGL4.11 vector containing of the 4.4 kb (with ERE) ovalbumin promoter deleted with 1 kb between ERE region and the 2.8 kb promoter. For reporter assay, HeLa, MES-SA, LMH/2A, and cEF cells were transfected with all the pGL4.11 vectors. The comparative analysis showed that the mutated 4.4 kb promoter has more potent activity than the 2.8 and 5.5 kb promoters in HeLa, MES-SA, and LMH/2A cells. However, there is no significant difference in cEFs. Also, these cells transfected with the mutated 4.4 kb promoter were treated with the $17{\beta}$-estradiol (0~3,000 nM) and HeLa, MES-SA, and LMH/2A cells showed estrogen responsibilities, but cEFs did not. Besides, the mutated 4.4 kb promoter has still higher activity than the 2.8 and 5.5 kb promoter, and there is no transcriptional induction effect in 2.8 kb promoter at 500 nM estrogen that is blood concentration of laying hens. Hence our study strongly suggested that the mutated 4.4 kb promoter is considered as one of the most efficient length for generating transgenic chicken.

Expression and Secretion of Recombinant Inulinase under the Control of GAL or GAP Promoter in Sacharomyces cerevisiae (Sacharomyces cerevisiae에서 GAL또는 GAP 프로모터 조절에 의한 재조합 Inulinase의 발현 및 분비)

  • 남수완;임현정정봉현장용근
    • KSBB Journal
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    • v.11 no.4
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    • pp.445-452
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    • 1996
  • To investigate the promoter effect on heterologous gene expression in S. cerevisiae, the recombinant plasmids pYI11, pYI12, pYI10-2, and pYIGP were constructed to contain the inulinase gene (INUI) as a reporter under the control of GAL10, GAL7, GAL1, and GAP promoters, respectively. When the yeasts transformants were cultivated on galactose-containing rich media, the cell growth reached to 36-39 OD600 at 72 hours of cultivation. The specific growth rates of the cells harboring the four different plasmids decreased similarly : they dropped from $0.24 h^{-1}$ during the glucose-consuming period to 0.04 -$0.10 h^{-1}$ during the galactose-consuming period (gene expression phase for GAL promoter system). After the depletion of glucose, the expression of inulinase gene was started and reached to maximal levels of 4.3(GAL1 promoter), 4.0(GAL10 promoter), 3.8(GAL7 promoter), and 1.6(GAP promoter) unit/mL at 72 hours of cultivation. Based on the maximal expression level and activity staining on the plate, the promoter strength was in the order of GAL1, GAL10, GAL7 and GAP promoter. While the GAL-promoter systems showed a high plasmid stabilities of more than 78%, the GAP-promoter plasmid revealed a lower plasmid stability of 55%. Most of inulinase activity (98%) was found in the extracellular medium, indicating that the secretion efficiency of inulinase is independent on the type of promoter.

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