• Journal of Plant Biotechnology
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• 제2권3호
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• pp.135-142
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• 2000

The activity of promoter sequences was evaluated in garlic cells using the $\beta$-glucuronidase (GUS) gene as a reporter. Histochemical GUS assay indicated transient GUS activity in leaf, callus and root cells 48 hours after particle bombardment transformation. Quantitative fluorometric assays in extracts of transformed leaves demonstrated that the CsVMV promoter induced the highest level of gene expression, which was, on average, ten fold the level induced by CaMV35S and by the Arabidopsis Act2 promoters and two fold the level expression observed with a construct containing a double CaMV35S plus the untranslated leader sequence from AMV. No activity or very low levels were observed when cells were transformed with plasmids rontaining the typical monocot promoters, Actl, from rice or the Ubi-1, from maize. The green fluorescent protein (GFP) was also tested as a marker gene for garlic transformation. Intense fluorescence was observed in leaf, callus and root cells transformed with a construct containing the gfp gene under control of the CaMV35 Promoter. No fluorescence was detected when the gfp was under control of the Ubi-1 promoter.

• Molecules and Cells
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• 제19권2호
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• pp.279-282
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• 2005

In mammals, 7-dehydrocholesterol reductase (Dhcr7) is the terminal enzyme in cholesterol biosynthesis. We previously reported that the Dhcr7 proximal promoter (-179 to +1), which contains CpG islands, is responsible for sterol-mediated expression of the rat gene. In the present study, we examined whether methylation of this region affects the transcriptional activity of the Dhcr7 gene. In vitro DNA methylation of the Dhcr7 promoter and luciferase-reporter assays showed that DNA methylation of the CpG islands suppressed transcription. Furthermore, treatment of the methylated Dhcr7 promoter with the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-CdR), reversed the suppression of promoter activity. These results indicate that methylation of the CpG islands is an important transcriptional regulatory mechanism in the Dhcr7 promoter.

• Genomics & Informatics
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• 제1권2호
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• pp.113-118
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• 2003

Among a number of antigens characterized in M leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M leprae. We have previously determined a sequence specific element in the 18 kDa gene of M leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M bovis BCG or M smegmatis in front of LacZ gene resulted in normal $\beta$­galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, $\beta$-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the $\beta$-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.

• 한국발생생물학회지:발생과생식
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• 제14권2호
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• pp.123-129
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• 2010

배아 줄기세포는 미분화상태에서 자가 재생을 유지할 수 있다. 자가 재생은 OCT4, SOX2와 NANOG와 같은 많은 인자들이 작용한다. 생쥐 배아 줄기세포에서 OCT4와 SOX2가 Nanog 프로모터에 결합하여 Nanog 유전자의 발현을 촉진한다는 사실은 생쥐 promoter에 관한 정밀분석으로 알려져 있다. 본 연구에서는 인간 Nanog promoter를 정밀 분석하기 위해 연속적인 결손 돌연변이를 가진 promoter-reporter construct를 제조하였다. Promoter의 최대 활성은 0.6 kb(-253/+365) promoter-reporter construct에서 발견되었으며, 이 construct에는 OCT4 및 SOX2의 결합부위가 포함된다. OCT4와 SOX2의 기여도를 확인하기 위하여 OCT4 및 SOX2의 결합부위에 자리 특이적 돌연변이를 유도하고 promoter 활성에 미치는 영향을 조사한 결과, OCT4나 SOX2 어느 한 군데라도 돌연변이가 존재하면 promoter 활성이 현저히 저해되었다. 본 연구 결과를 통해 인간 Nanog 유전자 발현에 있어 OCT4 및 SOX2가 필수적임을 직접적으로 확인할 수 있었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제25권1호
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• pp.111-114
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• 2012

In previous studies we have shown that a sw17255 gene was expressed in hemocyte-specific tissues of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). It was verified that the sw17255 core promoter region contains elements that regulate the expression of this gene in hemocyte tissue; the selected promoter region spans nucleotides -1 to -2,112 upstream of the start codon. Each of the luciferase reporter gene expression vectors under the control of 4 different kinds of promoter candidates, (-2,112/-1), (-1,640/-1), (-1,169/-1) and (-579/-1), and the control reporter plasmid DNA, were introduced into B. mori larval coelom by direct injection using a syringe. The promoter candidate [E] (-579/-1) showed more than 1.67 fold transcriptional activity compared to control promoter activity. Higher productivity of an expressed gene in the transgenic silkworm by this promoter combination could be achieved in the near future. The foreign recombinant protein could be easily harvested from the blood of the transgenic silkworm.

• Journal of Plant Biology
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• 제37권1호
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• pp.17-25
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• 1994

Two potato (Solanum tuberosum L.) cultivars were transformed by Agrobacterium tumefaciens harboring cauliflower mosaic virus (CaMV) 35S promoter and $\beta$-glucuronidase (GUS) gene. Expression patterns of the CaMV 35S promoter according to tissue types and developmental stages, and genetic stability of GUS gene were investigated in the clonal progenies of transgenic potatoes. Kanamycin-resistant shoot emerged from tuber disc after 4 weeks of culture, and root was induced 6 weeks after culture on the selection medium. Shooting frequency of cvs. Superior and Dejima were 43% and 27%, respectively. Mature transformants and their clonal progenies showed no phenotypical abnormality. GUS activity was expressed primarily at parenchymatous cells of phloem tissue around the vascular cambium in the stem and root, and higher activity was found at the apical meristem of shoot, root and adventious shoot bud. GUS activity was higher at tubers of young explants than at stored tubers. These facts indicate that expression level of the CaMV 35S promoter differed according to tissue types and developmental stages of the organs. The GUS gene was stably inherited to each clonal progeny and normally expressed.

• International Journal of Industrial Entomology and Biomaterials
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• 제20권2호
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• pp.107-114
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• 2010

For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (85%) with hypothetical protein 32 gene of Lonomia obliqua. This clone, named bHp32 (B. mori hypothetical protein 32) was ubiquitously expressed in all tissues and developmental stage of fifth instar B. mori larvae. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1,200/+220) in the 5'-flanking region of bHp32 gene, which has 42-fold more intensive promoter activity than BmA3 promoter. Moreover, the bHp32 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHp32 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

• 원예과학기술지
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• 제18권5호
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• pp.594-598
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• 2000

본 실험은 형질전환 담배내에서의 배유 특이 promoter에 의한 gus 유전자의 조직 특이적 발현 여부와 전이유전자의 후대로의 유전 양상을 확인하고자 수행하였다. 배유 특이 promoter에 의해 유도되는 gus 유전자(Z4pro-gus)와 kanamycin 저항성유전자를 운반하는 BV3 construct를 A. tumerfaciens을 이용하여 담배 형질전환체를 유기시켰다. 형질전환체 중에서 8개체를 선발하여 nptII primer를 이용하여 PCR을 실시한 결과 8개체 모두에서 700bp의 PCR 산물을 얻을 수 있었다. Promoter에 따른 유전자의 발현양상을 알아보기 위하여 Z4pro-gus가 전이된 형질전환체의 잎과 CaMV35S와 gus 유전자(35Spro-gus)로 구성된 pBI121 construct를 전이시킨 형질전환체의 잎으로부터의 발색정도를 비교하였다. 그 결과 Z4pro-gus가 전이된 형질전환체의 경우 잎에서 매우 부분적으로 극소량 발색되었으나 35Spro-gus가 전이된 형질전환체의 잎에서는 상대적으로 많은 양의 발색정도를 보여 promoter에 따른 발현정도의 차이를 보였다. 보다 명확한 Z4 promoter의 조직 특이 발현 양상을 확인하기 위하여 Z4pro-gus로 형질전환시킨 $T_0$ 식물체를 자가수분하여 얻은 $R_1$ 종자와 35Spro-gus를 형질전환시켜 같은 방법으로 얻은 $R_1$ 종자를 histochemical assay하였다. 그 결과 35Spro-gus로 형질전환된 담배 종자는 절단면 전체에서 gus 유전자가 발현되어 배유뿐만 아니라 종자 내 다른 조직에서도 발색되는 양상을 나타내었으나 Z4pro-gus를 형질전환 시켜 얻은 종자의 경우는 배유 부분만이 조직 특이적으로 파랗게 발색되었고 배 또는 그 이외의 조직에서는 gus 발색이 전혀 관찰되지 않았다. Kanamycin 저항성검정을 실시한 결과 모든 계통에서 전이유전자가 후대로 안정적으로 전이됨을 확인할 수 있었다.

• Journal of Crop Science and Biotechnology
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• 제10권1호
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• pp.50-56
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• 2007

Barley S-adenosylmethionine synthetase1 gene, which was differentially expressed in seed development of extra early barley, was regulated by the phytohormones and abiotic stresses. In order to identify the regulation regions which were involved in transcriptional control of the phytohormones and abiotic stresses, we isolated 1459 bp fragment of HvSAMS1 gene promoter using genome walking strategy and deletion series were constructed. Deleted upstream fragments(-1459, -1223, -999, -766, -545, -301 bp) were fused to the GUS reporter gene and evaluated via Agrobacterium-mediated transient expression assay. Increased GUS activity of HvSMAS1 promoter -301/GUS construct under each of NaCl, $GA_3$, ABA and ethylene application was found. However, GUS activity was negligible in the leaves transformed with the HvSMAS1 promoter(-1459, -1223, -999, -766 and -545)/GUS constructs. No significant induction of GUS activity was observed for the ethionine and spermidine treatments. In order to locate promoter sequence of the HvSAMS1 gene that was critical for the activation of gene expression, deletion and addition promoter derivatives(+, includes 43 bp of 5' ORF) of the HvSAMS1 gene fused to the GUS reporter gene were applied. The tobacco leaves which harbored the additional HvSAMS1 promoter(-1459+, -1459 to -546, -545+ and -301+)/GUS construct did not significantly induce GUS activity as compared to the HvSAMS1 promoter(-1459, -545 and -301)/GUS constructs under each of NaCl, ABA and $GA_3$ treatment. However, the GUS activity was high in the tobacco leaves which harboring the -211 to -141 regions of the HvSAMS1 promoter. This result suggested that HvSAMS1 gene expression might be regulated by this region(from -211 to -141).

• 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.