• 한국미생물·생명공학회지
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• 제32권3호
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• pp.206-211
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• 2004

Pseudomonas aurantiaca 유래 levansucrase 유전자(lscA)를 GAL1 promoter 하류에 연결시킨 pYES-lscA와 CAL10 promoter와 Kluyveromyces marxianus exoinulinase의 분비 신호서열(INU1 ss)하류에 연결시킨 pYInu-lscA를 각각 구축하였다. 이들 plasmid를 invertase 결손 변이주(suc2-$\Delta$9)인 S. cerevisiae SEY2102에 형질전환시켜 고활성 형질전환주를 선발하였다. 효모 형질전환주를 galactose 함유 배지로 배양한 결과, pYES-lscA 함유 형질전환주인 경우 levansucrase의 총활성은 8.62 U/ml이고, pYInu-lscA 함유 형질전환주인 경우 5.43 U/ml에 도달하였다. 발현된 levansucrase의 약 80% 정도가 periplasmic space와 cytopla느에 존재하였고, INU1 ss에 의한 분비효율 증가는 관찰할 수 없었다. 또한, 효모에서 발현된 재조합 levansucrase는 과당쇄화된 형으로 생산되는 것으로 보여진다.

• 한국잠사곤충학회지
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• 제41권2호
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• pp.102-107
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• 1999

To analysis a promoter strength of Atographa californica nucler polyhedrosis virus (AcNPV) IE1 gene, an immediate viral gene, ${\beta}$-glactosidase gene as a reporter gene was introduced under the control of the IE1 promoter. The restriction fragment containing IE1 promoter and ${\beta}$-galctosidase gene from pAcIE1-gal were inserter into pBacPAK9 to yield transfer vector pAcNPV-IE1-gal. The pAcNPV-IE1-gal was cotransfected with AcNPV genomic DNA BacPAK6 into Sf9 cells to produce recombinant baculovirus AcNPV-IE1-gal. In addition, recombinant bacvulovirus AcNPV-gal, which express ${\beta}$-galac-tosidase under the control of the polyhedrin promoter, was constrer, was constructed to compared with AcNPV-IE1-gal. The recombinant viruses were respectively infected into Sf9 cells and characterized by the virus titer and expression of ${\beta}$-galactoxidase in Sf9 cells. The promoter strength of IE1 and polyhedrin promoters was determined by the amount of ${\beta}$-galactosidase secreted into medium by viral infection. The titer of AcNPV-IE1-Gal determined by plaque assays in Sf9 cells was similar to that of AcNPV-gal. However, expression level of ${\beta}$-galactosidase by AcNPV-IE1-gal was significantly lower than that by AcNPV-gal. In conclusion, promoter strength of IE1 was approximately 25-fold lower than that of polyhedrin.

• Asian Pacific Journal of Cancer Prevention
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• 제16권14호
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• pp.5749-5754
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• 2015

Cervical carcinoma is the main cause of cancer-related mortality in women and is correlated with more than 15 risk cofactors, including infection of cervical cells with high-risk types of HPV (hrHPV). Indeed, both aberrant methylation of the RASSF1A promoter and hrHPV infection are often observed in cervical carcinomas. The purpose of our meta-analysis was to evaluate the role of RASSF1A promoter methylation and hrHPV infection in cervical cancer. Our meta-analysis involved 895 cervical cancer patients and 454 control patients from 15 studies. Our results suggested that RASSF1A promoter hypermethylation increased the risk of cervical cancer (OR=9.77, 95%CI=[3.06, 31.26], P=0.0001, $I^2=78%$). By grouping cases according to cancer subtypes, we found that HPV infection was higher in cervical squamous cell carcinomas (SCCs) than in cervical adenocarcinomas/adenosquamous cancers (ACs/ASCs) (OR=4.00, 95%CI=[1.41, 11.30], P=0.009, $I^2=55%$). Interestingly, HPV infection tended to occur in cervical cancers with relatively low levels of RASSF1A promoter methylation (OR=0.59, 95%CI=[0.36, 0.99], P=0.05, I2=0%). Our study provides evidence of a possible interaction between HPV infection and RASSF1A promoter methylation in the development of cervical cancers.

• 한국미생물·생명공학회지
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• 제26권6호
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• pp.497-506
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• 1998

Bacillus thurintensis subsp. kurstaki HD1 살충성 단백질 ICP 유전자가 있는 NdeI 단편 3.856 kb를 클로닝하여 제조한 pHLN2-80(-) 클론이 pHLN1-80(-)에 비해서 대장균에서 ICP발현량이 과다발현되는 현상을 규명하고자 하였다. 본 연구에서는 상기의 pHLN2-80(+) 클론의 발현량을 조절하는 원인을 규명하기 위하여 ICP의 아미노산 서열은 변화되지 않는 범위 내에서 pHLN1-80(+) 클론에 있는 Plac프로모터와 ICP유전자 프로모터의 일부인 -80 bp프로모터의 염기서열, 전사 개시점과 종결부위의 변이가 ICP유전자발현에 미치는 영향을 조사하였다. pHLN1-80(+)에 5'-말단에 존재하는 -80 bp 프로모터만을 보유한 pHLNK-80 클론은 ICP 생산은 매우 저조하였다. Plac프로모터와 -80 bp 프로모터의 구조 골격을 일부 변이 시킨 pHLNF1-80클론의 ICP생산량은 pHLN2-80(-)가 생산한 양보다는 낮아서 과다발현이 안되었다. Plac프로모터 상류를 약 350bp을 제거하여 만든 클론 pHLND2-80의 ICP 생산량은 모클론인 pHLN2-80(-) 보다 매우 높게 과다발현 되었다. ICP 유전자의 과다발현 현상에 대한 전사 개시점과 전사종결 부위의 역할을 알아보기 위해서 -72bp ICP유전자프로모터를 갖는 클론 pHLD1-72는 재조합 클론 pHLN2-80(-)가 생산한 양보다 적은 양의 ICP을 생성하였고, 클론 pHLD2-72는 재조합 클론 pHLN2-80(-)보다 적은 ICP을 발현하여 과다 발현되었으며, 클론 pHLN2-72는 모클론인 pHLN2-80(-)보다 약간 높은 ICP 생산량을 보여 과다발현되었다. 클론 pHLN2-72를증식하여 파쇄액을 만든 후에 Bombyx mori유충에 대한 살충력 검사에서 클론 pHLN2-72이 생산한 ICP는 pHLN1-80(+)이 생산한 ICP보다 약 90배의 살충력을 보였다. SDS-PAGE와 Western blot 분석에서도 클론 pHLN2-72는 재조합 클론 pHLN2-80(-)보다 약간 높게 ICP가 생성이 되었었다. 이상의 결과는 과다발현에 Plac프로모터와 종결부위가 반드시 필요하며, -72 bp ICP 프로모터가 -80 bp 프로모터보다 과다발현률이 높았으며, ICP 유전자는 반드시 Plac프로모터의 전사 방향에 역방향으로 삽입이 되어야 하는 것으로 나타났다.

• 농업과학연구
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• 제48권2호
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• pp.353-365
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• 2021

Previously, we reported that tissue factor (Tf) was included in the list of differentially expressed genes as an upregulated gene in a rejected porcine heart after xenotransplantation into monkey. In this study, we analyzed that expression of Tf in aortic endothelial cells (pAEC) isolated from alpha 1,3-galactosyltransferase knockout pig in response to allogeneic porcine serum and xenogeneic human serum. The consequence was significant upregulation of Tf expression by responding to human serum compared with porcine serum. To analyze the function of Tf gene as a promoter, we constructed reporter vectors for expression of luciferase linked to 1,246 and 787 base pairs of porcine Tf (pTF1246 and pTF787), and 535 base pairs of human TF (hTF535) sequences including putative promoter regions and AP-1 biding site at the 5' end. The reporter vectors were transfected into pAEC including cytomegalovirus enhancer/chicken β-actin (CAG)-luciferase vector as a control. Luciferase assay showed that all of the promoters were insufficient to express luciferase compared with CAG promoter in basic culture conditions. Notably, pTF1246, pTF787, and hTF535 led to a significant increase of luciferase expression in response to human serum compared with porcine serum while no change of CAG. pTF1246 and pTF787 showed higher expression than hTF535. Taken together, our findings suggest that pTF1246 and pTF787 promoters could mediate target gene expression specifically at xenogeneic stress condition.

• Journal of Plant Biology
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• 제38권1호
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• pp.107-113
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• 1995

pRLYS1 containing intact rbcL gene of maize (Zea mays L. cv Golden X Bantam T-51; Zm-A) was digested with several restriction enzymes to construct subcones carrying promoter region of rbcL. The DNA fragments of 0.20, 0.19, 0.92 and 1.55 kb among the EcoRI digests, the EcoRI-DdeI digests, the AvaI digests and the EcoRI-BamHI digests of pRLYS1 were subcloned into pBluscriptSK＋and named pRLPS2, pRLPS3, pRLPS14 and pRLPS35, respectively. Four subclones contain the 1.92 kb portion from 136 nucleotide downstream to 1780 nucleotide upstream from the ATG initiation codon of rbcL gene. pRLPS2 (-29 to -229) and pRLPS3 (-239 to -420 from the ATG) were sequenced. When nucleotide sequence of Zm-A was compared with sequence of rbcL promoter region of a different cultivar of maize (Zea mays L. cv WFG TMS X BS7; Zm-B), the difference rate between two cultivars was 4.3%. The mean of sequence divergence between Zm-A and three grass species in the same tribe, Andropogoneae, in the upstream region from 29 to 420 of ATG was 1.8%, whereas between Zm-B and above-mentioned three species was 5.4%. Therefore, Zm-A seems to evolutionarily closer to three other species in Andropogoneae tribe than Zm-B is.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.178-178
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• 1996

Hepatitis B virus (HBV) infection is one of the serious problems in Southeast Asia including Korea because it causes chronic hepatitis, which can easily be transformed In fatal conditions such as cirrhosis and hepatoma. Even though lots of informations on structural characteristics and gene expression mechanisms have been accumulated, the mechanism for HBV-induced hepatocellular injury which is believed to be the consequences of the immunological response is not well understood. In order tn perform immunopathological studies for prevention and treatment of HBV infection, we designed transgenic mice as a disease model which can mimic HBV infection, In this study, a promoter-HBV DNA fragment for the preparation of HBV transgenic mice has been constructed. To add a proper enzyme site on 5' end of HBV gene, total HBV (subtype adr) gene was inserted into BamHI site of pBluescript SK vector and reextracted by PstI-SacI treatment A liver-specific promoter, rat ${\alpha}$ 2u globulin gene promoter, was insrted to pBluescript SK vector and reextracted by BamHI-PstI treatment, Promoter-HBV DNA was constructed by ligation of two fragments using identical PstI sites. For large scale production of promoter-HBV DNA, it was inserted to BamHI-SacI site of pBluescript SK vector.

• Journal of Microbiology and Biotechnology
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• 제2권1호
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• pp.26-34
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• 1992

The effect of induction temperature on fermentation parameters has been investigated extensively using Escherichia coli M5248[pNKM21], a producer of recombinant human interleukin-2 (rhIL-2). In this recombinant microorganism, the gene expression of rhIL-2 is regulated by the cI857 repressor and $P_L$ promoter system. The recombinant fermentation parameters studied in this work include the cell growth, protein synthesis, cell viability, plasmid stability, $\beta$-lactamase activity, and rhIL-2 productivity. Interrelationships of such fermentation parameters have been analyzed through a quantitative assessment of the experimental data set obtained at eight different culture conditions. While the expression of rhIL-2 gene was repressed at culture temperatures below $34^\circ{C}$ with little effect on other fermentation parameters, under the conditions of rhIL-2 production $>(36~44^\circ{C})$ the cell growth, plasmid stability, and $\beta$-lactamase activity were, as induction temperature was increased, more profoundly reduced. Although the rhIL-2 content in the insoluble protein fraction was maximum at $40^\circ{C}$, total rhIL-2 production in the culture volume was found to be highest at the induction temperature of $36^\circ{C}$. This was in contrast to the previously known optimum induction temperature of the P$_{L}$ promoter system $>(40~42^\circ{C})$.Explanations for such a discrepancy have been proposed based on a product formation kinetics, and their implications have been discussed in detail.l.

• The Plant Pathology Journal
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• 제21권1호
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• pp.39-46
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• 2005

The promoter region flanking the 5’ CAZFP1 coding region was isolated from the genomic DNA of Capsicum annuum. To identify the upstream region of the CAZFP1 gene required for promoter activity, a series of CAZFP1 promoter deletion derivatives was created. Each deletion construct was analyzed by Agrobacterium-mediated transient transformation in tobacco leaves after infection by Pseudomonas syringae pv. tabaci, or treatment with methyl jasmonate (MeJA), ethylene, abscisic acid (ABA), salicylic acid (SA), cold and wounding. Promoter fragments of 685 bp or longer showed 7-fold or greater induction after P. s. pv. tabaci infection and MeJA treatment. The CAZFP1 full-length promoter (-999 bp) also showed 6-fold induction in response to ethylene. The transiently transformed tobacco leaves with the CAZFP1 full length promoter fused-GUS gene showed more than 5-fold induction in response to SA, ABA and cold. These results suggest that the CAZFP1 promoter contains responsive elements for pathogen, MeJA, ethylene, SA, ABA and cold.

• BMB Reports
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• 제32권1호
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• pp.45-50
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• 1999

We previously found three transcription factor-binding motifs in the rat p53 promoter. They are two recognition motifs of NF1-like protein (NF1-like element 1: -296 ~ -312, NF1-like element 2: -195 ~ -219) and a bHLH protein binding element (-142 ~ -146). In this study, we investigated the DNA-protein complex formation of the three elements with nuclear extracts from both normal and regenerating liver to find the element involved in the induced transcription of p53. The level of each DNA-protein complex on NF1-like and bHLH motifs was not changed. Instead, a new element located at -264 ~ -284 was detected in the DNase I footprinting assay with regenerating nuclear extract. This element has partial homology to the AP1 consensus motif. However, the competition studies with diverse oligonucleotides suggest that the binding protein is not AP1. An in vitro transcription assay shows that this element is important for the transcriptional activation of the rat p53 promoter. Therefore, for the induced transcription of the rat p53 promoter, the-264 ~ -284 region is required in addition to two NF1-like and one bHLH motif.