• 대한골관절종양학회지
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• 제15권1호
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• pp.13-25
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• 2009

서론: $O^6$-methylguanine-DNA methyltransferase (MGMT)는 DNA 염기 손상에 의해 형성된 $O^6$-methylguanine에서 알킬기를 제거하여 DNA 염기 손상을 복구하는 역할을 한다. MGMT의 후생유전적 불활성화가 인체 종양에서 보고되고 있으며, 항암 치료에 대한 저항성에 영향을 주는 요소로 알려져 있다. 본 연구에서는 연부조직육종에서 이러한 MGMT 불활성화가 미치는 영향에 대해 살펴보고자 하였다. 재료 및 방법: 총 62예의 연부조직육종조직에서 메틸화 특이 중합효소연쇄반응을 이용하여 MGMT 유전자의 촉진자 부위 메틸화 정도를 알아보고, 면역조직화학염색을 통하여 MGMT 단백 발현의 소실 양상을 살펴보았다. 결과: MGMT 단백 발현 소실은 진행성 병기(p=0.000), 조직학적 고등급(p=0.005), 종양의 재발 또는 전이(p=0.011), 그리고, 낮은 생존률(p=0.017)과 통계학적 유의성을 보였다. MGMT 유전자 촉진자 부위 메틸화는 조직학적 고등급, 종양의 재발 또는 전이, 그리고, 낮은 생존률과 관련성을 보였다. 또한, MGMT 단백 발현의 소실은 MGMT 유전자 촉진자 부위 과메틸화와 높은 상관성을 나타내었다(p=0.000). 결론: 본 연구는 MGMT 단백 발현의 소실과 MGMT 유전자 촉진자 부위 과메틸화가 연부조직 육종에서 흔히 발생하며 종양의 공격적인 양상 및 나쁜 예후와 관련성이 있다는 것을 보여준다. 또한 MGMT 단백 발현의 소실은 대개 MGMT 유전자 촉진자 부위 과메틸화로 인해 발생한다는 사실을 보여주고 있다.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1362-1368
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• 2006

A new constitutive episomal expression vector, pGAPZ-E, was constructed and used for initial screening of eukaryotic target gene expression in Pichia pastoris. Two reporter genes such as beta-galactosidase gene and GFPuv gene were overexpressed in P. pastoris. The expression level of the episomal pGAPZ-E strain was higher than that of the integrated form when the beta-galactosidase gene was used as the reporter gene in P. pastoris X33. The avoiding of both the integration procedure and an induction step simplified the overall screening process for eukaryotic target gene expression in P. pastoris. Nine human protein targets from the Core 50, family of Northeast Structural Genomics Consortium (http://www.nesg.org), which were intractable when expressed in E. coli, were subjected to rapid screening for soluble expression in P. pastoris. HR547, HR919, and HR1697 human proteins, which had previously been found to express poorly or to be insoluble in E. coli, expressed in soluble form in P. pastoris. Therefore, the new episomal GAP promoter vector provides a convenient and alternative system for high-throughput screening of eukaryotic protein expression in P. pastoris.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.321-326
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• 2000

To examine the feasibility of the long-term production of the human granulocyte colony stimulating factor (hG-CSF) using the $_{L}-arabinose$ promoter system of Escherichia coli, flask relay culture and cyclic fed-batch culture were performed. In the flask relay culture, it was found that the pismid was maintained stably up to about 170 generations in an uninduced condition, whereby the cells could also maintain the capability of expressing hG-CSF expression were maintained stably up to at least 100 generations. In contrast, in the cyclid fed-batch culture, segregational plasmid instability was observed within about 4 generations after induction, even though the cell growth and hG-CSF production reached their maximum balues, 78.0 g/l of dry cell weight and 7.0 g/l of hG-CSF, respectively. It would appear that, when compared to the flask relay culture, the high-cell density and high-level expression of hG-CSF in the cyclic fed-batch cultrure led to the segregational plasmid instability; in other words, a severe metabolic burden existe on the cells due to the high-level expression of hG-CSF. Accordingly, based on these long-term cultures, the segregational and structural plasmid instability was observed and a strategy to overcome such problems could be designed.

• BMB Reports
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• 제30권5호
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• pp.320-325
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• 1997

Thioredoxin is a multi-functional protein which is ubiquitous in microorganisms, animals and plants. Previously, expression of the E. coli thioredoxin gene was found to be negatively regulated by cAMP. In the present study, the effect of cAMP on two separate promoters of the E. coli thioredoxin gene was investigated. Cyclic AMP had a repressible effect on P1 and P1P2 promoter activity of the constructs. This effect was also observed in the cya strain. The P2 promoter construct gave very high -galactosidase activity, and its expression was not affected by exogenous cAMP. It was assumed that a cis-acting negative element, probably the cAMP-CRP binding site, might have been deleted in the P1 promoter construct. Repression of the thioredoxin gene expression by cAMP appeared to be independent of ppGpp.

• Reproductive and Developmental Biology
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• 제33권1호
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• pp.63-69
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• 2009

In this study we tried to construct a more efficient tetracycline-inducible gene expression system by replacing previous key elements with more advance ones. At the beginning, we substituted PGK (phophoglycerate kinase) promoter for CMV (cytomegalovirus) promoter to control "$rtTA2^sM2$" which has been known for high induction efficiency in response to tetracycline. With this modification, expression of the EGFP marker gene under the induction condition was significantly increased. Next, we replaced "TRE" fragment with a modified version named "TRE-tighf" which has been reported to have higher affinity and specificity to the transactivator by minor base change of the "TRE" DNA fragment sequence. Use of "TRE-tighf" instead of "TRE" resulted in more than 10 fold increment in terms of induction efficiency and significant decrement of background expression in non-inducible condition. By combining PGK promoter and "TRE-tight" fragment, we could upgrade previous tetracycline-inducible system to show more stringent turn on/off gene switch ability and stronger expression of the gene of our interest. Use of this newly developed system must be very helpful to the studies of gene expression, especially to the transgenic animal study in which non-controllable constitutive expression of the transgene has been one of the urgent problems to be solved.

• Fisheries and Aquatic Sciences
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• 제2권1호
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• pp.93-97
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• 1999

The promoter region of alginate lyase gene (aly) from Pseudomonas sp. W7 was used for the high expression of gilthead seabream (Sparus aurata) growth hormone (GH) gene in Esherichia coli. PCR product encoding the premature segment of the growth hormone. was cloned to the downstream of aly promoter. GH was overexpressed With 46 ammo acid of alginate lyase as fusion protein. GH was immunoreactive and production of GH was repressed with supplementation of $0.4\%$ glucose into culture media.

• 미생물학회지
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• 제27권4호
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• pp.317-322
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• 1989

A gene can be selectively overexpressed in E. coli by utilizing the phage T7 RNA polymerase's stringent recognition and active transcription of the T7 promoter. The T7 expression system was constructed such that the T7 RNA polymerase gene is under the control of lacUV5 promoter in one plasmid, and that the target gene, the promoterless chloramphenicol acetyltransferase (CAT) gene with E. coli ribosome binding site is under the control of T7 promoter in the other plasmid. Only the E. coli cells containing both plasmids show high resistance to chloramphenicol. When the copy number of the runaway plasmid containing the polymerase gene was varied by a temperature shift, amounts of the CAT protein synthesized upon induction was correspondingly changed as shown in SDS gel electrophoresis.

• 한국미생물·생명공학회지
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• 제19권6호
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• pp.568-574
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• 1991

효모에서 대랑의 물질생산계를 구축하기 위하여 먼저 여러 종류의 베터의 이용이 가능한 다양한 영양요구성 marker를 지니며 형질전환율이 향상된 효모숙주를 선별 개량하였다. 벡터의 제작에 사용되는 프로모터로는, 효모의 여러 유전자 중에서 그 활성이 매우 높은 해당계의 효소 GAP-DH의 구조 유전자 GAP를 이용하기로 하여, 효모염색체 DNA중에서 GAP 프로모터를 분리하여 이용하기 쉽게 변형하였다. 분리된 GAT promoter의 기능을 검토하기 위하여, reporter로 APase의 구조유전자 PHO5'를 이용하여 세포내의 copy수가 상이한 발현 벡터를 제작하여 GAP 프로모터에 의한 APase의 활성 및 전사산물을 측정한 결과, 정상적인 전사가 이루어 졌으며, 효소활성도 높게 나타났으며, 벡터의 copy수에 의한 효소활성의 차이도 감지되었다.

• 한국미생물·생명공학회지
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• 제23권3호
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• pp.288-296
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• 1995

To investigate high-level expression of Serratia marcescens metalloprotease (SMP) in Escherichia coli and S. marcescens, we constructed various recombinant plasmids: pSP2, containing SMP gene and lac promoter; pKSP2, containing SMP gene and tac promoter; pTSP2, containing SMP gene, trc99a promoter, and lacI$^{q}$. The recombinant E. coli (pKSP2) strain expressed SMP to a high-level, about 36% of total cellular proteins but accumulated inactive SMP precursors intracellularly, which indicated that E. coli does not have activation and secretion system for SMP. To overproduce active SMP, we transformed S. marcescens with the recombinant plasmids by a modified CaCl$_{2}$ method. The recombinant S. marcescens ATCC27117 (pSP2) containing lac promoter for SMP transcription produced 530 U/ml of active SMP on LB broth, which is about 5.1 times of the SMP yield, 105 U/ml of a control strain, S. marcescens ATCC27117 (pUC19). However, S. marcescens ATCC27117 (pKSP2) containing tac promoter for SMP transcription did not grow healthy and hardly produced SMP. To overcome a harmful effect of the strong tac promoter, we constructed a regulatory plasmid pTSP2 containing a strong trc99a promoter and its repressor gene lacI$^{q}$. When S. marcescens ATCC27117 (pTSP2) was induced with 1.0 mM IPTG after 9 hr cultivation, 2,200 U/ml of SMP was obtained in LB broth, which is about 21 times of that of a control strain.

• Asian Pacific Journal of Cancer Prevention
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• 제16권13호
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• pp.5181-5185
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• 2015

Background: CCAT1 has been reported to be linked with pathogenesis of malignancies including colon cancer and gastric cancer. However, the regulatory effect of CCAT1 in hepatocellular carcinoma (HCC) remains unclear. The purpose of this research was to identify any role of CCAT1 in the progression of HCC. Materials and Methods: Real time-PCR was performed to test the relative expression of CCAT1 in HCC tissues. A computation screen of CCAT1 promoter was conducted to search for transcription-factor-binding sites. The association of c-Myc with CCAT1 promoter in vivo was tested by Pearson correlation analysis and chromatin immunoprecipitation assay. Additionally, Kaplan-Meier analysis and Cox proportional hazards analyses were performed. Results: c-Myc directly binds to the E-box element in the promoter region of CCAT, and when ectopically expressed increases promoter activity and expression of CCAT1. Moreover, Kaplan-Meier analysis demonstrated that the patients with low expression of CCAT1 demonstrated better overall and relapse-free survival compared with the high expression group. Cox proportional hazards analyses showed that CCAT1 expression was an independent prognostic factor for HCC patients. Conclusions: The findings demonstrated CCAT1, acting as a potential biomarker in predicting the prognosis of HCC, is regulated by c-Myc.