• 대한바이러스학회지
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• 제29권2호
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• pp.129-136
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• 1999

Transient transfection assay has been done to evaluate whether the c-jun activation would be prerequisite to the induction of permissiveness against human cytomegalovirus using in vitro cell model in which U937 has been induced to express CD11b and CD14 to become potential monocyte/macrophage cells by TPA treatment. U937 cells were treated with $10\;{\mu}M$, $50\;{\mu}M$ or $100\;{\mu}M$ of TPA. The cell morphology change was observed and the expression of the CD11b and CD14 was confirmed by FACS. Differentiated cells were transfected with pJLuc reporter vector which contained the wild type murine c-jun promoter spanning the SP1, CTF, ATF/CREB and MEF-2 binding sites upstream of the firefly luciferase gene. After 48 hrs of transfection, the cells were infected with HCMV Towne strain and the luciferase activity was assessed at 1 hand 4 h pi. The transfection assay showed no activation of the c-jun promoter at 1 h pi, instead, it showed 2 times increase of the its activity at 4 h pi. There was no difference of the c-jun promoter activation between TPA treated and untreated U937 cells, implying that c-jun activation might not be prerequisite for allowing cells to be premissive to HCMV, although HCMV infection itself could activate c-jun promoter.

• Journal of Microbiology and Biotechnology
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• 제13권1호
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• pp.99-103
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• 2003

An Escherichia coli strain containing the recA promoter that fused to the luxCDABE operon originating from Photorhabdus luminescens was shown to respond sensitively to genotoxic stresses. Two different recombinant bacteria, one (DPDI 657) harboring a plasmid with the recA promoter that fused to the luxCDABE operon, and the other (DPD1710) containing a chromosomally-integrated recA promoter that fused with luxCDABE, were compared and it was found that the sensitivity of 'the two strains was significantly different in terms of their bioluminescent level, response time, and the minimum detectable concentration of a chemical causing DNA damaging stress. DPDI 710, with a chromosomally-integrated single copy, generally led to lower basal luminescence levels, faster responses, increased response ratios, and an enhanced sensitivity to mutagens, when compared to DPD 1657 with a multi-copy plasmid.

• 한국축산식품학회지
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• 제31권3호
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• pp.381-388
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• 2011

Dietary conjugated linoleic acid (CLA) play key roles in lipid metabolism. Here, we investigated the effect of CLA on the transcriptional activity of TR4, an orphan nuclear receptor that plays an important role in lipid homeostasis. CLA increased TR4 gene mRNA level in 3T3-L1 adipocytes, but inhibited TR4 transcriptional activity in a dose-dependent manner. TR4 induced perilipin expression in 3T3-L1 adipocytes by activating perilipin promoter activity. In a gel shift assay, TR4 bound direct to the putative TR4 response element in the perilipin promoter. Interestingly, CLA reduced the interaction between TR4 and consensus DR1, a well-known TR4 binding site. Additionally, CLA inhibited TR4-induced perilipin promoter activity in a dose-dependent manner. Together, our results suggest that CLA may play a role in lipid homeostasis in adipocytes by functionally regulating TR4.

• 대한화장품학회:학술대회논문집
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• 대한화장품학회 1996년도 4차 심포지움(Skin Biology Efficacy)
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• pp.10-14
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• 1996

최근 오존층의 파괴로 인한 지구 자외선량의 증가로 피부암 및 광노화의 발생가능성이 증가되고있다. 광노화의 주요한 현상중의 하나는 탄력섬유상 물질이 축적되는 일광탄력 섬유증이다. 자외선은 탄력소 유전자의 전사과정증가를 유도하며 결국 비정상적인 탄력소의 증가를 유발한다는 보고가 있다. 본 연구에서는 피부 섬유아세포 및 각질형성세포를 배양하여 자외선이 탄력소 유전자발현에 미치는 영향 및 전사조절인자에 미치는 영향을 알아보고자 하였다. 피부섬유아세포에 UVB를 30mJ-200J/m 조사하여 nothern blotting한 결과 UV양이 증가함에 따라 탄력소 전사물이 증가되었고, 1001/$m^2$ 에서 최대의 증가를 보였다. 탄력소 promoter와 CAT을 접합시킨 pEP62 vector을 섬유아세포에 형질전환 시키고 UV에 의한 Promoter 활성을 본 결과 UV조사량이 증가할수록 활성이 증가되었고 200J/$m^2$ 에서 대조군에 비해 5배의 활성 증가를 보였다. 이 system에 광노화 억제물질로 알려진 retinoid를 5 $\times$ $10^{-6}$M처리하였을 때 UV에 의한 탄력소 promoter활성을 약 1/3로 감소시켰다. 각질 형성세포에서 UVB에 의한 transcription factor의 활성을 mobility shift assay에 의해 조사한 결과 AP-1 및 NFkB가 활성화됨을 볼 수 있었다.

• Clinical and Experimental Pediatrics
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• 제48권5호
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• pp.495-499
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• 2005

목 적 : IL-4는 Th2 면역 반응의 중요한 매개체로 IL4 유전자의 promoter 일배체형(haplotype)은 한국인 소아에서 RSV에 의한 심한 모세기관지염과 연관된다고 알려져 있다. 본 연구는 IL4 유전자의 promoter 다형성에 따른 IL-4 단백의 기능적인 변화를 분석하여 심한 RSV 하기도 감염증에 기여하는 IL4 유전자의 발병 기전의 연관성을 연구하고자 시행하였다. 방 법 : 면역 기능이 정상인 소아 20명을 대상으로 전혈을 채취한 후 genomic DNA를 추출하여 IL4 유전자 promoter 약 1.2 kb 부위를 증폭시켰다. 염기서열 분석을 통하여 IL4 유전자 promoter의 유전형을 결정하고, PHASE 분석으로 일배체형을 결정하였다. 각 일배체형별로 $5{\mu}g$의 DNA를 Jurkat T 세포에 핵형질변환 시켜서 정상 Jurkat T 세포와 PMA(50 ng/mL)로 자극한 세포에서의 luciferase 활성도를 분석하여 IL4 유전자 promoter의 전사능을 결정하였다. 결 과 : 한국인 소아의 일배체형은 3가지 유형 GCC(7%), TCC(17%), 및 TTT(76%)로 분포하였다. Jurkat T 세포의 절대 luciferase 활성도는 GCC형이 가장 낮았고 TTT형이 가장 높았다. GCC 일배체형을 기준으로 하여 나타낸 Jurkat T 세포의 상대 luciferase 활성도는 TCC형이 4.2배, TTT형이 5.3배로 증가되었다. PMA로 자극한 후에 측정한 각 일배체형의 luciferase 활성도 역시 GCC형에 비하여 TCC형이 3.0배, TTT형이 4.1배로 증가되어 자극하지 않은 세포에서와 유사한 활성도의 차이를 보였다. 결 론 : 소아의 심한 RSV 하기도 감염증과 연관된 IL4 유전자의 promoter 일배체형 TTT는 IL4 유전자의 promoter의 전사능을 증가시킴으로써 영아 및 소아의 RSV 하기도 감염증의 병인에 중요한 역할을 할 것으로 생각된다.

• IMMUNE NETWORK
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• 제12권5호
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• pp.207-212
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• 2012

T cell immunoglobulin mucin domain (TIM)-3 is an immunomodulatory molecule and upregulated in T cells by several cytokines. TIM-3 also influences mast cell function but its transcriptional regulation in mast cells has not been clarified. Therefore, we examined the transcript level and the promoter activity of TIM-3 in mast cells. The TIM-3 transcript level was assessed by real-time RT-PCR and promoter activity by luciferase reporter assay. TIM-3 mRNA levels were increased in HMC-1, a human mast cell line by TGF-${\beta}1$ stimulation but not by stimulation with interferon (IFN)-${\alpha}$, IFN-${\lambda}$, TNF-${\alpha}$, or IL-10. TIM-3 promoter -349~+144 bp region relative to the transcription start site was crucial for the basal and TGF-${\beta}1$-induced TIM-3 promoter activities in HMC-1 cells. TIM-3 promoter activity was increased by over-expression of Smad2 and Smad4, downstream molecules of TGF-${\beta}1$ signaling. Our results localize TIM-3 promoter activity to the region spanning -349 to ＋144 bp in resting and TGF-${\beta}1$ stimulated mast cells.

• International Journal of Industrial Entomology and Biomaterials
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• 제8권2호
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• pp.169-174
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• 2004

Bombyx mori nucleopolyhedrovirus(BmNPV) ecdysteroid UDP-glucosyltransferase gene (egt) promoter fragments of different lengths were amplified from BmNPV ZJ-8 genomic DNA by PCR. Reporter plasmids pBmegt542-luc, pBmegt309-luc and pBmegtl59-luc with luciferase (lue) driven by egt promoters were constructed. Both in vitro and in vivo expressions showed that BmNPV egt promoter activity requires the transactivation of viral factor(s), and expression of luc was detected earliest at 24 hrs post infection (pi). BmNPV ZJ-8 homologous region 3 (hr3) increased the expression of luc by over 1,600-fold. Molting hormone of 1.0 - 2.0 $\mu\textrm{g}$/$m\ell$ can dramatically down regulate expression of luc. Juvenile hormone analogue of 0.5-2.0 ${\mu}g$/$m\ell$ increased expression of luc by 145.8% to 75.7%. Deletion assay revealed that the promoter fragment of 159 bp contains the basal promoter structure; Promoter fragments of 309 bp and 542 bp showed similar but much higher transcriptional activities than that of 159 bp, suggesting that nucleotide from -159 to -309 nt upstream the translation initiation site harbors the main cis-acting elements.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.537-543
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• 2003

The mouse interferon gene (MuIFN-${\gamma}$) was cloned and then used to transform Saccharomyces cerevisiae. Expressed MuIFN-$\{gamma}$ protein (MuIFN-${\gamma}$) was successfully secreted into culture medium due to the presence oi the signal peptide of rice amylase 1A. Two different promoters fused to MuIFN-${\gamma}$ were tested: glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD (AG) promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. Using the hybrid promoter, the accumulation of MuIFN-${\gamma}$transcript was the highest after the 24 h cultivation, and then gradually decreased as the cultivation proceeded. However, both cell growth and recombinant MuIFN-${\gamma}$production reached their peaks after the 4-day cultivation. It was possible to produce 6.5 mg/l of MuIFN-${\gamma}$ without any changes in cell growth. Using GPD promoter, the MuIFN-${\gamma}$ transcript accumulation and the recombinant MuIFN-${\gamma}$ production followed the same pattern as the cell growth. However. compared to that of the hybrid promoter, the production of recombinant MuIFN-${\gamma}$ was 0.2 mg/l. The secreted MuIFN-${\gamma}$ had estimated molecular masses of 21 kDa and 23 kDa, which were larger than that of the encoded size due to glycosylation. The protection assay against the viral infection indicated that the recombinant MuIFN-${\gamma}$ was bioactive.

• Animal cells and systems
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• 제15권3호
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• pp.227-233
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• 2011

The Nramp1/Slc11a1 locus encodes a proton-coupled divalent cation transporter, expressed in late endosomes/lysosomes of macrophages, that constitutes a component of the innate immune response to combat intracellular pathogens and it was shown to play an important role in regulating inherent immunity. The previously identified Z-DNA forming polymorphic repeat(GT)n in the promoter region of the human Nramp1 gene does act as a functional polymorphism influencing gene expression. Research has shown that INF-${\gamma}$, TNF-${\alpha}$, IL-$1{\beta}$ and bacteria LPS increase the level of Nramp1 expression. However, the molecular mechanism for Nramp1 gene regulation is unclear. In this research, bovine Nramp1 5'-flanking region (-1748~+769) was cloned and analyzed by bioinformatics. Then to find the core promoter and the cis-acting elements, deletion analysis of promoter was performed using a set of luciferase reporter gene constructs containing successive deletions of the bovine Nramp1 5'-flanking regions. Promoter activity analysis by the dual luciferase reporter assay system showed that the core promoter of Nramp1 was located at +58~-89 bp. Some positive regulatory elements are located at -89~-205 bp and -278~-1495 bp. And the repressor elements were in region -205~-278 bp, intron1 and -1495~-1748 bp. LPS-responsive regions were located at -1495~-1748 bp and -278~-205 bp. The present study provides an initial effort to explore the molecular mechanism of transcriptional activation of the bovine Nramp1 gene and should facilitate further studies to decode the complex regulatory process and for molecular breeding for disease resistance in bovines.

• 한국동물학회지
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• 제38권3호
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• pp.433-441
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• 1995

염소 베타-락토글로불린 유전자 프로모터의 유선 조직 특이성은, 비 발현 세포에서 -470 에서 -205 부위에 의해서 매개되는 억제 조절에 의해서 보증된다. 이 음성 조절 기작과 억제 조절을 매개하는 인자를 확인하기 위하여 상류 염기 서열을 자세히 분석하였다. 상류 염기 서열은 어느 방향으로 위치하든 연결된 베타-락토글로불린 유전자 프로모터의 활성을 억제할 수 있었다. 이와 같이 규명된 염소 베타-락토글로불린 유전자의 잠정적 음성 조절 인자는 다른 유전자의 프로모터들에 대해서는 다양한 활성을 보였는데, herpes simplex 바이러스의 thimidine kinase 프로모터는 상류 염기서열의 방향에 따라 억제 또는 활성화되었으며, SV40 프로모터는 억제되기보다는 오히려 활성화되었다. 염소 베타-락토글로불린 유전자의 억제 조절 인자를 포함하는 조절 부위는 비 유선 세포인 HeLa 및 CV-1 세포에서 추출된 핵 추출물에 의해서 이동성이 강력하게 지연되는 반면, 유선세포인 HC11 세포의 핵 추출물에 의해서는 약하게 지연되었다. 이와 같은 활성 억제와 인자 결합간의 연관성은 foot-printing 분석에서 관찰된 결합 부위가 염소의 베타-락토글로불린 유전자의 조직특이적 억제에 작용할 음성 조절 인자일 가능성을 시사한다.