• Biotechnology and Bioprocess Engineering:BBE
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• 제5권4호
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• pp.247-252
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• 2000

The strength and regulatory characteristics of the heat-inducible HSA1, HSA2 and TPS1 promoters were compared with those of the well-established, carbon source-regulated FMD promoter in a Hansenula polymorpha-based host system in vivo. In addition, the Saccharomyces cerevisiae-derived ADH1 promoter was analysed. While ADH1 promoter showed to be of poor activity in the foreign host, the strength of the heat shock TPS1 promoter was found to exceed that of the FMD promoter, which at present is considered to be the strongest promoter for driving heterologous gene expression in H. polymorpha.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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• pp.639-647
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• 2008

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the ${\lambda}O_L1$ and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the $O_L1$ from the ${\lambda}P_L$ promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one ${\lambda}O_L1$, and CJ1OX2, which has two successive ${\lambda}O_L1$, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature-sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

• Journal of Microbiology
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• 제34권2호
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• pp.137-143
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• 1996

We isolated a promoter inducible by paraquat, a superoxide-generating agent, from Escherichia coli using a promoter-probing plasmid pRS415. From sequence analysis we found out the promoter is for fpr ENCODING nadph : ferredoxin oxidoreductase. We constructed on operon fusion of lacZ gene with fpr promoter to monitor the expression of the gene in the single-copy state. LacZ expression generators, menadione and plumbagin, also induced the expression of .betha.-galactosidase in the fusion strain. On the other hand, no significant induction was observed by treatment with hydrogen peroxide, ethanol, and heat shock. Induction of .betha.-galactosidase was significantly reduced by introducing a .DELTA. sox 8 :: cat of soxS3 :: Tn10 mutation into the fusion strain, indicating that fpr gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis. Possible roles of fpr induction in superoxide stress were discussed.

• BMB Reports
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• 제40권6호
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• pp.936-943
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• 2007

Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cellwall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region ($P_g$) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the $P_g$-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that $P_g$ in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced $P_g$ efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals.

• 생명과학회지
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• 제21권10호
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• pp.1466-1472
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• 2011

본 연구에서는 누에의 초기 배아시기에 유전자 발현 조절이 가능한 EEG-704 promoter를 개발하고자 하였다. Promoter의 핵심 영역을 결정하기 위하여, 10개의 서로 다른 partial mutant clone들을 만들고 이를 Sf9 곤충세포주에 도입하여 luciferase assay 방법을 사용하여 각각의 clone의 활성을 분석하였다. Constitutive promoter인 BmA3 promoter에 의한 활성과 비교하였을 때, 약 1.5 kb의 promoter 염기서열을 포함하는 clone이 가장 높은 luciferase 발현율을 나타내었다. 특히 EEG-704 유전자의 경우 BLAST를 이용한 유전자 비교 분석의 결과 누에의 열충격 단백질20.8 (BmHsp20.8) 과 동일한 것으로 밝혀졌으며, 정상 온도조건과 비교하였을 때 열충격을 가한 조건하에서 발현율이 증가하는 현상을 나타내었다. 특이적으로 발생단계에서 직 간접적으로 발현 조절이 가능한 이러한 promoter는 여러 유용 재조합 단백질 생산을 위한 형질전환 누에 개발 시 매우 유용할 것으로 생각된다.

• Journal of Microbiology
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• 제38권3호
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• pp.145-149
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• 2000

GroEL is a typical molecular chaperone. GroEL synthesis patterns at various culture temperatures in Escherichia coli were investigated in this study. No significant differences in the amount of GroEL produced from the chromosome were found at 30 and 37$^{\circ}C$. However, GroEL production increased 3.4-fold at 42$^{\circ}C$. GroEL synthesis was not transient but continuous at 42$^{\circ}C$, although most heat shock gene expression is known to be transient. To understand the role of the groEL structural gene, a groE promoter-lacZ fusion was constructed. Interestingly , while transcriptional fusion is not thermally inducible, it is inducible by ethanol, suggesting that the secondary structure of the groEL transcript is involved in thermal regulation of the groEL gene. Secondary structures of groE mRNA at 37 and 42$^{\circ}C$ were compared using the computer program RNAdraw. Distinct structures at the two temperatures were found, and these structures may be related to a high level of GroEL expression at 42$^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제12권6호
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• pp.986-988
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• 2002

A plasmid expression vector of pEStxl encoding a mature form of the B chain of the Shiga toxin was constructed without a signal peptide under the control of an inducible n promoter. The encoded protein was purified to 90% by simple heat treatment, and then further purified to 95% by Phenyl-Sepharose and DEAE-Sepharose chromatographies, all in a single day. Accordingly, this expression system and heat treatment could facilitate the rapid purification of gram-scale amounts of the Shiga toxin B subunit from recombinant Escherichia coli cells.

• The Korean Journal of Physiology and Pharmacology
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• 제18권5호
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• pp.403-409
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• 2014

The Bis protein is known to be involved in a variety of cellular processes including apoptosis, migration, autophagy as well as protein quality control. Bis expression is induced in response to a number of types of stress, such as heat shock or a proteasome inhibitor via the activation of heat shock factor (HSF)1. We report herein that Bis expression is increased at the transcriptional level in HK-2 kidney tubular cells and A172 glioma cells by exposure to oxidative stress such as $H_2O_2$ treatment and oxygen-glucose deprivation, respectively. The pretreatment of HK-2 cells with N-acetyl cysteine, suppressed Bis induction. Furthermore, HSF1 silencing attenuated Bis expression that was induced by $H_2O_2$, accompanied by increase in reactive oxygen species (ROS) accumulation. Using a series of deletion constructs of the bis gene promoter, two putative heat shock elements located in the proximal region of the bis gene promoter were found to be essential for the constitutive expression is as well as the inducible expression of Bis. Taken together, our results indicate that oxidative stress induces Bis expression at the transcriptional levels via activation of HSF1, which might confer an expansion of antioxidant capacity against pro-oxidant milieu. However, the possible role of the other cis-element in the induction of Bis remains to be determined.

• The Korean Journal of Physiology and Pharmacology
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• 제22권4호
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• pp.457-465
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• 2018

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

• Fisheries and Aquatic Sciences
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• 제20권7호
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• pp.9.1-9.13
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• 2017

Background: Metal-responsive transcription factor-1 (MTF-1) is a key transcriptional regulator playing crucial roles in metal homeostasis and cellular adaptation to diverse oxidative stresses. In order to understand cellular pathways associated with metal regulation and stress responses in Pacific abalone (Haliotis discus hannai), this study was aimed to isolate the genetic determinant of abalone MTF-1 and to examine its expression characteristics under basal and experimentally stimulated conditions. Results: The abalone MTF-1 shared conserved features in zinc-finger DNA binding domain with its orthologs; however, it represented a non-conservative shape in presumed transactivation domain region with the lack of typical motifs for nuclear export signal (NES) and Cys-cluster. Abalone MTF-1 promoter exhibited various transcription factor binding motifs that would be potentially related with metal regulation, stress responses, and development. The highest messenger RNA (mRNA) expression level of MTF-1 was observed in the testes, and MTF-1 transcripts were detected during the entire period of embryonic and early ontogenic developments. Abalone MTF-1 was found to be Cd inducible and highly modulated by heat shock treatment. Conclusion: Abalone MTF-1 possesses a non-consensus structure of activation domains and represents distinct features for its activation mechanism in response to metal overload and heat stress. The activation mechanism of abalone MTF-1 might include both indirect zinc sensing and direct de novo synthesis of transcripts. Taken together, results from this study could be a useful basis for future researches on stress physiology of this abalone species, particularly with regard to heavy metal detoxification and thermal adaptation.