• Journal of Plant Biology
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• 제37권3호
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• pp.371-380
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• 1994

To determine the patterns and the levels of expression of the cauliflower mosaic virus (CaMV 35S) promoter and the rice actin 1 (Act1) promoter in rice, transgenic rice plants containing CaMV 35S-$\beta$-glucuronidase (GUS) and Act1-GUS constructs were generated and examined by fluorometric and histochemical analyses. The fluorometric analysis of stably transformed calluses showed that the activity of the rice Act1 promoter was stronger than that of the CaMV 35S promoter in rice cells. In a histochemcial study of the transgenic rices, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in parenchymal cells of vascular tissues of leaves and roots and mesophyll cells of leaves. These results are similar to those of potato, a dicot plant. In contrast, rice plant transformed with Act1-GUS fusion construct revealed strong GUS activity in parenchymal cells of vascular tissue, mesophyll cells, epidermal cells, bulliform cells, guard subsidiary cells of leaves and most cells of the root, suggesting that the rice Act1 promoter is more constitutive than the CaMV 35S promoter. It was also confirmed that in both types of transgenic rice little or no staining was localized in metaxylen tracheary elements of vascular tissue from leaves or roots. These results indicate that the rice Act1 promoter can be utilized more successfully for expression of a variety of foreign gene in rice than the CaMV 35S promoter.

• Journal of Applied Biological Chemistry
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• 제66권
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• pp.128-137
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• 2023

Transcriptome analysis revealed that the sinR gene encoding a transition-state regulator of Bacillus pumilus, genetically close to B. subtilis, was expressed at high levels during growth. The sinR gene is the second gene of the sinIR operon consisting of three promoters and two structural genes in B. subtilis. This study used the sinIR promoter of B. subtilis DB104 to construct a recombinant protein expression system. First, the expression ability depending on the number of sinIR promoter was investigated using enhanced green fluorescent protein (eGFP). The expression level of eGFP was slightly higher when using two promoters (Psin2) than using original promoters. The Psin2 promoter was further engineered by modifying the repressor binding site and -35 and -10 regions. Shine-Dalgarno (SD) sequence of the sinI gene was modified to the consensus sequence. Finally, combining the engineered Psin2 promoter with the modified SD sequence increased the expression level of eGFP by about 13.4-fold over the original promoter. Our results suggest that the optimized sinIR promoter could be used as a novel tool for recombinant protein expression in B. subtilis.

• KSBB Journal
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• 제10권1호
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• pp.38-45
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• 1995

Aspergillus oryzae에서 A. nidulans의 glyceral d dehyde-3-phosphate dehydrogenase (gpdA)와 trpC, prmoter의 발현 능력 을 E. coli lacZ gene fusion을 이용하여 비교.분석하였다. A. oryzae 내에서 발현된 E. coli $\beta$galactosidase의 specific activIty를 조사하여 본 결과, gpdA promoter를 가지는 transformant들 에서는 2,000unit/ug of protem 정도의 activity를 보이는 반면, trpC, promater를 가지고 있는 transformant들에서는 10.5~52.3unit/ug of protein 정도의 activity를 보였다. 이 결과로부터 A. oryzae 내에서 A. nidulans의 gpdA promoter가 trpC, promoter에 비해 70 배 정도 더 강한 발현 능력을 보이고 있음을 알 수 있다. Western blot 분석에서도 gpdA promoter를 가지고 있는 transf ormant에서 더 많은 E. coli $\beta$-galactosidase가 발현된 것 을 보여 주고 있다. 또한 southern blot 분석에서는 이러한 강한 발현이 transform된 plasmid의 copy number와 상관 없음을 보여주고 있다.

• Journal of Microbiology and Biotechnology
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• 제31권6호
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• pp.833-839
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• 2021

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (P_cry3A, P₁₀, P_SG1, P_srfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P₁₀ promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

• IMMUNE NETWORK
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• 제8권4호
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• pp.137-142
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• 2008

Background: CD11c, also known as integrin alpha x, is one of the optimum markers of dendritic cells. However, the regulation of the CD11c expression in mouse has not been identified yet. In this study, in order to analyze the regulation of CD11c expression, the promoter of CD11c was cloned and characterized. Methods: To identify the promoter portion, various sizes of what are considered to be CD11c promoter fragments was amplified by polymerase chain reaction (PCR), using mouse genomic DNA as a template. After sequence was obtained, these fragments were transfected into various cell lines including mouse dendritic cell lines such as JAWSII and DC2.4 and L929 as control cell line.. The promoter activity of three promoter fragments was measured and compared by luciferase activity in the transfected cells. Results: Three clones with size of 1kb, 3kb and 6kb were obtained from mouse genomic DNA. Flow cytometry analysis of JAWSII cells revealed that 52% of the cells expressed CD11c, which was confirmed by RT-PCR analysis. On the contrary, L929 and DC 2.4 cells did not express CD11c. The CD11c+ JAWSII cells were enriched from 52% to 90% with cell sorter. The comparative luciferase activity analyisis demonstrated that the region responsible for tissue specific expression was contained within -3 kb and the clone with size of 3 kb particularly showed higher luciferase activity than 6 kb and 1 kb clones. Conclusion: The CD11c promoter region containing the region responsible for tissue specificity was successfully cloned and -3 kb region showed the highest activity.

• 식물조직배양학회지
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• 제28권5호
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• pp.255-261
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• 2001

VVTL1은 포도 과육에서 특이적으로 다량 발현되는, PR5 계열의 thaumatin과 높은 상동성을 나타내는 단백질로서, 품종에 따라 염기서열의 차이를 나타낸다고 알려져 있다. 현재까지 포도의 VVTL1에 대해서 몇몇 연구가 진행되었지만, 우리나라에서 가장 많이 재배되는 품종인 캠벨에서는 전혀 이루어지지 않았다. 본 연구에서는 캠벨 품종으로부터 VVTL1-homolog 유전자의 게놈 DNA를 분리하여, 염기서열을 분석하였다. VVTL1-homolog 유전자는 일반적으로 PR5 유전자의 구조와 동일한 구조인, intron이 없는 하나의 exon으로만 구성되어 있었다. 염기서열로부터 추론된 VVTL1-homolog 단백질의 아미노산 서열은 VVTL1을 비롯한 다른 품종의 포도에서 분리된 TLP와는 달리 염기성의 등전점을 가지고 있었다. Primer extension 분석을 통해 전사개시 부위를 결정하였고, promoter영역을 포함하는 5'upstream 지역에 전사에 중요한 TATA box (4개)와 CAAT box (1개)가 존재하였으나, 이들의 위치와 수는 다른 PR5 유전자의 promoter와는 다랐다. 이러한 연구결과는 VVTL1-homolog 유전자의 발현이 과육 성숙과정동안 abscisic acid와 스트레스 또는 자극에 의해 발현이 유도되고 있음을 제시해준다. 포도 과육특이발현 promoter인 VVTL1-homolog 유전자의 promoter 분리는, 유전자의 도입에 의해 유용형질을 과육에 나타내는 포도품종을 개발하고자 할 때 효율적으로 사용될 수 있을 것으로 사료된다.

• 한국미생물·생명공학회지
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• 제31권2호
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• pp.111-116
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• 2003

Xylan 분해 균주인 Bacillus stearothermophilus No. 236 분리균의 $\beta$-xylosidase 생산 유전자(xylA)의 염기 서열 및 transcription start site를 결정한 이전 연구 결과에 의하면 xylA 유전자는 매우 특이하게 UUG codon에서 translation이 시작되며 initiation codon 15dp 윗쪽에는 promoter로 추정되는 염기 서열을 가지고 있는 것으로 분석되었다. 이와 같은 xylA 유전자 promoter region의 구조는 E. coli에 클로닝된 xalA 유전자를 이용한 실험 결과로도 확인되었다. xalA promoter의 -10 element는 CATAAT로서 6개의 염기 중 5개가 그리고 -35 element의 경우는 TTGTTA로서 6개의 염기 중 4개가 consensus sequence와 일치되었으나 두 hexamer 사이의 거리가 최적 거리에서 크게 벗어난 12 bp인 것으로 분석되었다. 본 연구에서는 $\beta$-xylosidase의 대량 생산을 위한 연구의 일환으로 xalA promoter sequence의 체계적 구조 변화에 의한 promoter strength에 미치는 효과를 E. coli와 B. subtilis두 숙주 세포에서 조사 분석해 본 결과, 첫째로 두 promoter elements사이의 거리를 최적거리인 17 bp로 바꾸었을 때 xalA의 발현율은 E. coli에서는 1.6배, B. subtilis에서는 2.5배 정도 증가함을 보여주었다. 그리고 -35 element는 consensus sequence와 같이 5'쪽에서 네번째 위치에 있는 T$\longrightarrow$A로 변이 시켰을 때 E. coli경우 2.3배, 특히 B. subtilis에서는 35배나 되는 가장 높은 promoter 활성의 증가를 보였다. 그러나 -10 sequence의 경우 consensus sequence와 같이 5' 쪽에서 첫번째 위치에 있는 C$\longrightarrow$T로 transition시켰을 때 예상외로 오히려 발현율이 5~15배까지 낮아지는 특이한 결과를 얻었다. 따라서 본 연구 결과 xalA promoter의 경우 -10 sequence인 CATAAT의 C와 -35 element의 두 염기가 promoter활성에 있어 가장 중요한 염기임을 알 수 있었다.

• 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.

• Journal of Animal Science and Technology
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• 제45권2호
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• pp.169-182
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• 2003

ADRP 유전자가 24개월령 한우 등심조직에서 발현량이 급격히 증가하여 30개월령 등심조직에서는 발현량이 다소 감소하는 발현양상 분석결과로부터 이전 연구에서는 ADRP 유전자를 한우 성장단계 특이발현 유전자로 선정하였다. 본 연구에서는 ADRP 유전자의 발현조절 기작을 분석하기 위하여 promoter 영역을 포함하는 ADRP 유전자 전체영역을 cloning하였으며, 구조를 분석하고 promoter의 특성을 조사하였다. 한우 ADRP cDNA 단편을 probe로 합성하여 Southern blot 분석을 실시한 결과로부터 ADRP 유전자가 한우 genome 상에서 single copy로 존재하고 크기는 대략 12 kb에 해당하는 것을 확인하였다. Genomic DNA library screening을 실시하여 promoter 영역을 포함하는 ADRP 전체 유전자에 해당하는 clone을 확보하고 HwADRPg-1으로 명명한 후, 염기서열을 결정하고 분석하였다. 한우 ADRP 유전자, HwADRPg-1은 8개의 exon과 7개의 intron으로 구성되어 있으며 모든 exon-intron 경계는 GT/AG 원칙을 따르고 있었고, coding 영역은 7,633 bp로서 6개의 intron에 의해 7개의 exon으로 나누어져 있었다. HwADRPg-1의 promoter 영역에서는 TATAA box는 발견되지 않았으며, -70 위치에 근육 특이적 transcription activator인 Myo G 서열이 존재하였고, -629 위치에는 지방세포의 분화를 유도하는 것으로 알려진 C/EBP (CCAAT/enhancer binding protein) 서열이 존재하였다. HwADRPg-1의 조절영역에 있는 Myo G factor가 근육조직에서 ADRP 유전자가 발현될 수 있도록 하며, 근육의 발달정도를 신호로써 감지하여 근육조직에서 성장단계에 따른 ADRP 유전자의 발현량을 조절할 것으로 추정되고, 다른 종류의 지방세포 특이적인 전사인자 및 지방세포의 분화정도를 신호로 인식하는 전사단계 조절인자를 조사하기 위하여 promoter 영역의 추가분석이 이루어져야 할 것으로 사료된다.

• Plant Biotechnology Reports
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• 제4권1호
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• pp.29-35
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• 2010

NPR1 is a positive regulator of systemic acquired resistance in Arabidopsis and rice. Expression of the rice gene OsNPR1 is induced by salicylic acid (SA). To identify the region of the OsNPR1 promoter involved in response to SA, we carried out deletion mutagenesis of the region 1005 bp upstream of the OsNPR1 start codon. Ciselement analysis revealed that the OsNPR1 promoter contains W-boxes and ASF1 motifs, both of which are known to be functional cis-elements of the WRKY and bZIP proteins, respectively. The deletion constructs 1005:LUC and 752:LUC, were induced by up to 4.3- and 3.8-fold, respectively, following SA treatment, suggesting that W-boxes and ASF1 motifs may play an important role in the strong induction of these constructs by SA. Using mutation analysis, we also showed that both the W-box and ASF1 motif are necessary for SA-induced expression of OsNPR1.