• International Journal of Industrial Entomology and Biomaterials
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• v.39 no.1
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• pp.22-28
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• 2019

We previously isolated 9 clones that show stronger signal compared to Bombyx mori cytoplasmic actin gene (BmA3) by using a dot blot hybridization. In this study, we focused on one clone among these clones which has high amino acid similarity with ${\beta}$-tubulin gene of B. mori. This clone was ubiquitously expressed in all tissues and developmental stage of B. mori. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-750/-1) in the 5'-flanking region of ${\beta}$-tubulin gene, which has about 47 fold more intensive promoter activity than BmA3 promoter. Moreover, the ${\beta}$-tubulin promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that ${\beta}$-tubulin promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

• Food Science and Preservation
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• v.17 no.5
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• pp.752-756
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• 2010

Lepidoptera (butterflies) extracts, traditionally employed as medicines, have various biological activities. Five species of Lepidoptera (Papilio maackii, Papilio xuthus, Pieris rapae, Eurema hecabe, and Sasakia charonda) were extracted with distilled water (DW), dimethyl sulfoxide (DMSO), ethanol (EtOH), and methanol (MeOH). Each extract was analyzed for anti-oxidant and anti-inflammatory activities using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay method, the ferric reducing ability of plasma (FRAP) test, and a cyclooxygenase-2 (COX-2) promoter assay. The results suggest that Lepidoptera extracts have valuable anti-oxidant and anti-inflammatory properties, supporting the idea that the extracts may serve as a food biomaterial(s) preventing oxidative processes and inflammatory damage.

• Journal of Life Science
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• v.21 no.10
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• pp.1466-1472
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• 2011

We characterized embryo early gene (EEG)-704 promoter of the silkworm Bombyx mori, which is specifically regulated in the development stages. To determine core promoter region, 10 different partial mutant clones were tested by luciferase assay in Sf9 cells. About 1.5 kb promoter shows higher luciferase activity than constitutive promoter (BmA3). Interestingly, EEG-704 shares the same DNA sequences with BmHsp20.8 by the result of BLAST analysis; its expression is also increased under heat shock condition. Development of such promoter inducible, directly or indirectly in the developmental-stage, is very useful in making recombinant proteins in transgenic silkworms.

• International Journal of Industrial Entomology and Biomaterials
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• v.20 no.2
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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.

• Mycobiology
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• v.46 no.4
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• pp.429-439
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• 2018

To develop a convenient promoter analysis system for fungi, a null-pigment mutant (NPG) of Aspergillus nidulans was used with the 4'-phosphopantetheinyl transferase (PPTase) gene, npgA, which restores the normal pigmentation in A. nidulans, as a new reporter gene. The functional organization of serially deleted promoter regions of the A. nidulans trpC gene and the Cryphonectria parasitica crp gene in filamentous fungi was representatively investigated to establish a novel fungal promoter assay system that depends on color complementation of the NPG mutant with the PPTase npgA gene. Several promoter regions of the trpC and crp genes were fused to the npgA gene containing the 1,034-bp open reading frame and the 966-bp 3' downstream region from the TAA, and the constructed fusions were introduced into the NPG mutant in A. nidulans to evaluate color recovery due to the transcriptional activity of the sequence elements. Serial deletion of the trpC and crp promoter regions in this PPTase reporter assay system reaffirmed results in previous reports by using the fungal transformation step without a laborious verification process. This approach suggests a more rapid and convenient system than conventional analyses for fungal gene expression studies.

• Korean Journal of Microbiology
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• v.27 no.1
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• pp.10-15
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• 1989

The plasmid pKOneo, containing SV40 transcriptional promoter, has been used in the mouse tumorigenicity assay for oncogene studies. This assay employs a cotransfection of NIG3T3 fibroblast cells with the desired DNA and the plasmid pKOneo. This oncogene assay, however, has been speculated due to the SV40 transcriptional promoter in the plasmid pKOneo. This research was designed to investigate if the plasmid pKOneo alone is capable of transforming NiH3T3 fibroblast cells. The NIH3T3 subclones were established after the NIH3T3 cells were transfected with the plasmid pKOneo alone. The estabilished NIH3T3 subclones, containing the exogeneous plasmid pKOneo in their chromosomes, were examined for their expression of transformation-associated parameters. The results indicate that this plasmid pKOneo alone has positive effects on transformation of NIH3T3 cells after integration into cellular chromosomes.

• BMB Reports
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• v.40 no.6
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• pp.936-943
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• 2007

Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cellwall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region ($P_g$) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the $P_g$-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that $P_g$ in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced $P_g$ efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals.

• International Journal of Industrial Entomology and Biomaterials
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• v.24 no.2
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• pp.49-55
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• 2012

We previously isolated 9 clones that show stronger signal compared to B. mori cytoplasmic actin gene (BmA3) by using a dot blot hybridization. In this study, we focused on one clone among these clones which has high amino acid homology with elongation factor ${\alpha}$ gene of B. mori. This clone, named $bEF1{\alpha}$ (B. mori elongation factor ${\alpha}$) was ubiquitously expressed in all tissues and developmental stage of B. mori. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-702/+38) in the 5'-flanking region of $bEF1{\alpha}$ gene, which has about 20 fold more intensive promoter activity than BmA3 promoter. Moreover, the $bEF1{\alpha}$ promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that $bEF1{\alpha}$ promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

• Journal of Life Science
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• v.19 no.9
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• pp.1289-1293
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• 2009

Proto-oncogene human cervical cancer oncogene (HCCR) functions as a negative regulator of p53 and contributes to tumorigenesis in various human tissues. However, it is unknown how HCCR contributes to the cellular and biochemical mechanisms of human tumorigenesis. In this study, we showed how the expression of HCCR is modulated. The luciferase activity assay indicated that the HCCR 5'-flanking region at positions -370 to -406 plays an important role in the promoter activity. Computational analysis of this region identified one consensus sequence for the TG-interacting factor (TGIF) located at -390 to -366 (TG). Mobility shift assays (EMSA) revealed that nuclear proteins from K562 bind to the TG site, but not to the mutated TG site. The reporter activity assay with promoter constructs carrying mutated TGIF sequences pGL3-mTGIF significantly increased reporter activities compared to wild type constructs pGL3-$406{\sim}+30$. In this study, we characterized the HCCR promoter and found that HCCR expression was partially regulated by the transcription repressor TGIF, which bound the promoter at positions -390 to -366.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.207-212
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• 2012

The shortage of human organs for transplantation has induced the research on the possibility of using animal as porcine. However, pig to human transplantation as known as xeno-transplantation has major problem as immunorejection. Recently, the solutions of pig to human xenotransplantation are commonly mentioned as having a genetically modification which include alpha 1, 3 galatosyl transferase knockout (GTKO) and immune-suppressing gene transgenic model. Unfortunately, the expression level of transgenic gene is very low activity. Therefore, development of gene overexpression system is the most urgent issue. Also, the tissue specific overexpression system is very important. Because most blood vessels are endothelial cells, establishment of the endothelial-specific promoter is attractive candidates for the introduction of suppressing immunorejection. In this study, we focus the ICAM2 promoter which has endothelial-specific regulatory region. To detect the regulatory region of ICAM2 promoter, we cloned 3.7 kb size mini-pig ICAM2 promoter. We conduct serial deletion of 5' flanking region of mini-pig ICAM2 promoter then selected promoter size as 1 kb, 1.5 kb, 2 kb, 2.5 kb, and 3 kb. To analyze promoter activity, luciferase assay system was conducted among these vectors and compare endothelial activity with epithelial cells. The reporter gene assay revealed that ICAM2 promoter has critical activity in endothelial cells (CPAE) and 1 kb size of ICAM2 promoter activity was significantly increased. Taken together, our studies suggest that mini-pig ICMA2 promoter is endothelial cell specific overexpression promoter and among above all size of promoters, 1 kb size promoter is optimal candidate to overcome the vascular immunorejection in pig to human xenotransplantation.