• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2003.10a
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• pp.178-178
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• 2003

Cytochrome P-450 3A4 (CYP3A4) is major enzyme in human liver, the role of this Is detoxification and metabolizing more than 50% clinical drugs in use. Expression of CYP3A4 is transciptionally regulated by the Pregnenolone X receptor (PXR), of which human form is Steroid and Xenobiotics receptor (SXR). SXR is activated by wide range of endogenous and exogenous compounds, and then induces CYP3A4 gene expression. In the previous study, it has been known that proximal promoter (-864 to ＋64) does not response to chemical inducers such as pregnenolone 16a-carbonitrile (PCN), Rifampicin, Estrogen in terms of transcription of CYP 3A4 in cultured cells. Here, we developed luciferase reporter gene assay system to detect SXR-based CYP 3A4 transcriptional activity. We have used CYP3A4-Luc plasmid that contains proximal promoter of human CYP3A4 gene upstream of the luciferase gene. We did transient transfection of 3A4-luciferase gene and SXR. In the HepG2 cells transfected with CYP3A4-Luc, when rifampicin treatment was combined with histone deacetylase inhibitor (HDAC Inhibitor), such as Trichostatin A, Hc-toxin and IN 2001 of the luciferase activity was induced 10-20 fold over control.

• Proceedings of the Botanical Society of Korea Conference
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• 1996.07a
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• pp.26-38
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• 1996

To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode different types of variants. Both genes had an intron in the coding region. In the promoters, some characteristics sequences, such as Oct and Nona motifs, which are conserved among plant histone genes were also found, and they were located in a short region (about 120 bp) upstream from the putative TATA box. Analyses of promoter activity with H2A-GUS fusion genes in the transient system using tobacco protoplasts revealed novel types of positive cis-acting sequences in the TH254 promoter: a direct repeat of a 13-bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). A quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUG chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were both active in developing seedlings and flower organs but regulated in different manner.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.277-283
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• 2009

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for $4{\times}14$ days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter-bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with $4mg\;1^{-1}$ phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the $R_1$ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.

• Molecules and Cells
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• v.27 no.4
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• pp.475-480
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• 2009

The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.

• The Journal of Korean Medicine
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• v.29 no.3
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• pp.88-99
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• 2008

Objectives: In order to identify the anti-inflammatory and analgesic properties of Salvia miltiorrhiza (Dan-Sam), widely used in Korean traditional medicine, an in vitro screening system was designed using pGL3, a luciferase reporter vector, and the tumor necrosis factor (TNF)-${\alpha}$ and cyclooxygenase (COX)-2 as target genes. Methods: The promoter regions of each gene were generated by PCR using the human chromosome as template DNA, and inserted into pGL3 vector with Kpn I and Hind III. The final construct was transfected into human myelomonocytic leukemia cells (U-937) that could be differentiated and activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). Using this system, the anti-inflammatory and analgesic effects of several herbal extracts regarded to have the medicinal effects of diminishing body heat and complementing Qi were tested. The chemicals PD98059 and berberine chloride were used as controls of the transcriptional inhibitors of TNF-${\alpha}$ and COX-2, respectively. Results: Salvia miltiorrhiza (Dan-Sam) demonstrated significant decrease of TNF-${\alpha}$ and COX-2 mRNA in the in vitro assay system. In MTT assay, Salvia miltiorrhiza (Dan-Sam) did not significantly inhibit the survival and proliferation of human myelomonocytic leukemia cells (U-937). Conclusions: Salvia miltiorrhiza (Dan-Sam) was found to exhibit the significant medicinal properties of anti-inflammatory and analgesic effects.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.236-241
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• 2000

A defensin is an inducible antibacterial peptide from a fleshfly and contains 40 residues basic peptide with six cysteines. For the consiruction of recombinant S cerevisiae expressing defensin, the structural gene coding for active defensin was chemically synthesized and fused in fiam to GAP promoter, MFul preprosequence and the GAL7 transcription terminator, generating a recombinant plasnlid pGMD18. S. ce~evisine 2805 Gells were transror~ned to uracil prototroph by the pGMDl8 arid the transformed cells showing antibacterial activity against 111. luteus TAM1056 were selected by growth inhibition zone assay. The optimal culture conditions for the unprovement of the defensin production of a selected tmdonnant were investigated. The optirmzed medium containing 0.4% yeast extract, 2% corn steep liquor, 2.5% glucose and 0.05% $C_2CO_3$, could be determined and the optimum lemperature. and initial pH could be detennnied as $28^{\circ}C$ and pH 3, ~mpectively. The optimized conditioiis revealed the trvofold Increase in the cell growth and the fourfold in the antibaclerial activity. coinpar-ed with tllc Yl'D medium.

• Journal of Life Science
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• v.12 no.4
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• pp.477-482
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• 2002

As a biological model system for the production of an active antibacterial peptide, we have attempted the expression and secretion of insect defensin in Saccharomyces cerevisiae. Nucleotide sequences encoding mature defensin composed of 40 amino acids were fused in frame with promoter and signal sequence of Saccharomyces diastaticus glucoamylase, and mating factor $\alpha$ l[MF $\alpha$1] prosequence. The host strain, S. cerevisiae 2805 was transformed with the resulting plasmid, pSMFll The secretion of functional defensin was confirmed by growth inhibition zone assay using Micrococcus luteus as a test organism. Insect defensin was secreted to the culture supernatant in biologically active form by glucoamylase signal sequence and mating factor $\alpha$1 prosequence. Most of antibacterial activity was detected in the culture supernatant. Defensin was also active against Staphylococcus aureus and Listeria monocytogenes.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.196-205
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• 2015

A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

• International Journal of Industrial Entomology and Biomaterials
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• v.9 no.2
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• pp.235-239
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• 2004

Drosophila melanogaster heat shock protein 70 gene promoter (Dhsp70p) is widely used in transgenic insect to drive exogenous gene, and the homologous region 3 from Bombyx mori nucleopolyhedrovirus (BmNPVhr3) functions as an enhancer for several promoters. To test whether BmNPVhr3 can enhance the Dhsp70ps transcriptional activity, the reporter plasmids, which contain the Dhsp70p, the reporter $\beta$-galactosidase gene with SV40 terminator and BmNPVhr3 fragment, are constructed and transfected into the insect cell lines (Bm-N cells and Sf-21 cells) by lipofectin-mediated method. The results from the transient expression assay show that BmNPVhr3 significantly increases transcriptional activity of Dhsp70p both under the normal condition and under the heat-shock treatment, although the effects are significantly different between in Bm-N cells and in sf-21 cells. The enhancing behavior of BmNPVhr3 on the Dhsp70p is in an orientation-independent manner. Meanwhile, the effects of heat-shock treatment on Dhsp70p alone or Dhsp70p/BmNPVhr3 combination present no significant difference, indicating that BmNPVhr3 only enhances the transcriptional activity of Dhsp70p, but cant alter its characteristic of the response to the heat-shock stress. The above results suggest that the Dhsp70p/BmNPVhr3 combination is more effective one to drive exogenous gene for transgene or stable cell expression system in insects.

• Molecules and Cells
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• v.44 no.10
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• pp.723-735
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• 2021

Spemann organizer is a center of dorsal mesoderm and itself retains the mesoderm character, but it has a stimulatory role for neighboring ectoderm cells in becoming neuroectoderm in gastrula embryos. Goosecoid (Gsc) overexpression in ventral region promotes secondary axis formation including neural tissues, but the role of gsc in neural specification could be indirect. We examined the neural inhibitory and stimulatory roles of gsc in the same cell and neighboring cells contexts. In the animal cap explant system, Gsc overexpression inhibited expression of neural specific genes including foxd4l1.1, zic3, ncam, and neurod. Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) and promoter analysis of early neural genes of foxd4l1.1 and zic3 were performed to show that the neural inhibitory mode of gsc was direct. Site-directed mutagenesis and serially deleted construct studies of foxd4l1.1 promoter revealed that Gsc directly binds within the foxd4l1.1 promoter to repress its expression. Conjugation assay of animal cap explants was also performed to demonstrate an indirect neural stimulatory role for gsc. The genes for secretory molecules, Chordin and Noggin, were up-regulated in gsc injected cells with the neural fate only achieved in gsc uninjected neighboring cells. These experiments suggested that gsc regulates neuroectoderm formation negatively when expressed in the same cell and positively in neighboring cells via soluble factors. One is a direct suppressive circuit of neural genes in gsc expressing mesoderm cells and the other is an indirect stimulatory circuit for neurogenesis in neighboring ectoderm cells via secreted BMP antagonizers.