• 한국환경성돌연변이발암원학회지
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• 제22권3호
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• pp.175-182
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• 2002

Endocrine disruptors (EDs) are the chemicals that affect endocrine systems through activation or inhibition of steroid hormone response. It is necessary to have a good system to evaluate rapidly and accurately endocrine-disrupting activities of suspected chemicals and their degradation products. The key targets of EDs are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We constructed a co-expression system of Gal4p DNA binding domain (DBD)- ligand binding domain of human estrogen receptor $\alpha$ or $\beta$, and Gal4p transactivation domain (TAD)-co-activator AIB-1, SRC-1 or TIF-2 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of CYC1 promoter and Gal4p binding site (GAL4 upstream activating sequence, GAL4$_{UAS}$). Expression of this reporter gene was dependent on the presence of estrogen or EDs in the culture medium. We found that the two-hybrid system with combination of the hER$\beta$ LBD and co-activator SRC-1 was most effective in the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by those chemicals correlated with their estrogenic activities measured by other assay systems, indicating that this assay system is efficient and reliable for measuring estrogenic activity. The data in this research demonstrated that the yeast detection system using steroid hormone receptor and co-activator is a useful tool for identifying chemicals that interact with steroid receptors.s.

• Molecules and Cells
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• 제41권4호
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• pp.342-350
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• 2018

Flowering time is determined by florigens. These genes include, Heading date 3a (Hd3a) and Rice FT 1 (RFT1) in rice, which are specifically expressed in the vascular tissues of leaves at the floral transition stage. To study the cis-regulatory elements present in the promoter region of Hd3a, we generated transgenic plants carrying the 1.75-kb promoter fragment of Hd3a that was fused to the ${\beta}$-glucuronidase (GUS) reporter gene. Plants expressing this construct conferred a vascular cell-specific expression pattern for the reporter gene. However, GUS was expressed in leaves at all developmental stages, including the early seedling stage when Hd3a was not detected. Furthermore, the reporter was expressed in roots at all stages. This suggests that the 1.75-kb region lackings cis-elements that regulate leaf-specific expression at the appropriate developmental stages. Deletion analyses of the promoter region indicated that regulatory elements determining vascular cell-specific expression are present in the 200-bp region between -245 bp and -45 bp from the transcription initiation site. By transforming the Hd3a-GUS construct to rice cultivar 'Taichung 65' which is defective in Ehd1, we observed that Ehd1 is the major regulatory element that controls Hd3a promoter activity.

• 한국환경성돌연변이발암원학회지
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• 제23권1호
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• pp.30-34
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• 2003

Recent industrial society has human widely exposed to PAHs (polynuclear aromatic hydrocarbons) that are comming from the incomplete combustion of organic material as wider spread environmental contaminants. Biological activities of PAHs are not known although PAHs are considered as carcinogens. Our laboratory have been studied the effect of PAHs in the mouse liver hepa 1 cells. In this study, we examined the mouse liver hepa-l cells as a new bioassay system to evaluate bioactivity of PAHs. We have selected 13 PAHs to examine bioassay using cyp1a1-luciferase reporter gene expression system where cyp1a1 1.6 Kb 5flanking region DNA was cloned in front of luciferase reporter gene and this plasmid was transfected into hepa 1 cells transiently. This cells then used for the study to observe the effect of PAHs. We demonstrated that PAHs induced the CYP1A1 promoter and 7-ethoxyresolufin O-deethylase (EROD) activities in a concentration-dependant manner. Some of PAHs showed stronger stimulatory effect on CYP1 gene expression than TCDD. Acenaphthene, anthracene, fluorine, naphthalene, pyrene, phenanthrene, carbazole were weak responders to cyp1a1 promoter activity stimulation and EROD induction in hepa 1 cells and these chemicals seemed to respond less to EROD than cyp1a1 promoter activity. Benz(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, chrysene, and dibenzo(a,h)anthracene showed strong response to cyp1a1 promoter activity stimulation and also EROD induction in hepa 1cells. Results of dose response study suggested that four strong responding PAHs, such as benzo(a)anthracene benzo(k)fluoranthene, chrysene, and dibenzo(a, h)anthracene might be mediated through arylhydrocarbon receptor system in hepa1 cells.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1249-1255
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• 2004

This study has developed Corynebacterium ammoniagenes (c. ammoniagenes) purine gene transcriptional reporters (purF-lacZ and purE-lacZ) that function in Escherichia coli (E. coli) DH5a. After transformation of a C. ammoniagenes gDNA library into E. coli cells harboring either purF-lacZ or purE-lacZ, C. ammoniagenes clones were obtained that repress purF-lacZ and purE-lacZ gene expression. The potential purE and purF regulatory genes are homologous to the genes encoding transcription regulators, the regulatory subunit of RNA polymerase, and genes for purine nucleotide biosynthesis of various bacteria. The C. ammoniagenes purE-lacZ and purF-lacZ reporters were repressed by adenine and guanine within E. coli, indicating similarity in the regulatory mechanism of purine biosynthesis in C. ammoniagenes and E. coli. Gene regulation of pur-lacZ by adenine and guanine was partly abolished in cells expressing potential purine regulatory genes, indicating functionality of the purine gene regulators in repression of purE-lacZ and purF-lacZ. The purE-lacZ and purF-lacZ reporters can be used for the screening of genes involved in the regulation of the de novo synthesis of the purine nucleotides.

• Journal of Microbiology and Biotechnology
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• 제16권11호
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• pp.1706-1712
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• 2006

Because of the need for new antimicrobial substances with novel mechanisms of action, we report here the use of an Acinetobacter reporter system for high-throughput screening of active antimicrobial agents. The bioreporter Acinetobacter strain DF4/PUTK2 carrying luciferase genes luxCDABE was chosen because of its ecological importance and it is widespread in nature. This bioreporter is genetically engineered to emit light constitutively that can be measured in real time by luminometry. Hence, this reporter system was employed to determine the bacteriostatic actions of spent-culture supernatants derived from twelve bacterial isolates. Out of the results, the strongest bioluminescence inhibitory effect of the supernatants was recorded with Bacillus cereus strain BAC (S5). Subsequently, ethyl acetate extracts of extracellular products of strain BAC (S5) were separated by a thin-layer chromatography (TLC). Based on the bioluminescence inhibitory assay, three fractions were found to have antimicrobial activity. One fraction (C) having the strongest antimicrobial activity was further purified using TLC and characterized by IR, $^1H$ NMR, mass spectrometry, SDS-PAGE, and amino acid composition analysis. The results predicted the presence of 2-pyrrolidone-S-carboxylic acid (PCA) and the octadeconic-acid-like fatty acid. Fraction C also demonstrated a broad inhibitory activity on several Gram-negative and Gram-positive bacteria. In conclusion, the Acinetobacter reporter system shows great potential to be a reliable, sensitive, and real-time indicator of the bacteriostatic actions of the antimicrobial agents.

• Genomics & Informatics
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• 제5권2호
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• pp.46-55
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• 2007

The convergence of molecular and genetic disciplines with non-invasive imaging technologies has provided an opportunity for earlier detection of disease processes which begin with molecular and cellular abnormalities. This emerging field, known as molecular imaging, is a relatively new discipline that has been rapidly developed over the past decade. It endeavors to construct a visual representation, characterization, and quantification of biological processes at the molecular and cellular level within living organisms. One of the goals of molecular imaging is to translate our expanding knowledge of molecular biology and genomic sciences into good patient care. The practice of molecular imaging is still largely experimental, and only limited clinical success has been achieved. However, it is anticipated that molecular imaging will move increasingly out of the research laboratory and into the clinic over the next decade. Non-invasive in vivo molecular imaging makes use of nuclear, magnetic resonance, and in vivo optical imaging systems. Recently, an interest in Positron Emission Tomography (PET) has been revived, and along with optical imaging systems PET is assuming new, important roles in molecular genetic imaging studies. Current PET molecular imaging strategies mostly rely on the detection of probe accumulation directly related to the physiology or the level of reporter gene expression. PET imaging of both endogenous and exogenous gene expression can be achieved in animals using reporter constructs and radio-labeled probes. As increasing numbers of genetic markers become available for imaging targets, it is anticipated that a better understanding of genomics will contribute to the advancement of the molecular genetic imaging field. In this report, the principles of non-invasive molecular genetic imaging, its applications and future directions are discussed.

• BMB Reports
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• 제40권6호
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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.

• 대한한의학회지
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• 제29권3호
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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.

• Toxicological Research
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• 제20권3호
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• pp.205-211
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• 2004

Concern that some chemicals in our environment may affect human health by disrupt-ing normal endocrine function has prompted a research on interactions of environmental contaminants with steroid hormone receptor. Toxaphene and chlordane are among the 12 persistent organic pollutants identified by the United Nations Environment Programme as requiring urgent attention. We compared the estrogenic activity of two organochlorine pesticides, toxaphene and chlordane, at estrogen receptor a (ER$\alpha$) and estrogen receptor $\beta$ (ER$\beta$). Human hepatoma cells (HepG2) were transiently transfected with rat ER$\alpha$ or ER$\beta$ plus an estrogen-responsive complement C3-luciferase (C3-Luc) reporter gene. After transfection, cells were treated with various concentrations of toxaphene and chlordane to investigate agonism or antagonism of these chemicals. Both toxaphene and chlordane were potent agonists in HepG2 cells for ER$\alpha$. In contrast, these chemicals had a minimal agonist activity with ER$\beta$ and almost abolished 17$\beta$-estradiol-induced ER$\beta$-mediated activity. Therefore, toxaphene and chlordane behaved as an ER$\alpha$ agonist and an ER$\beta$ antagonist with estrogen-responsive reporter plasmid C3-Luc, and exposure to these organochlorine pesticides could have a crictical effect on normal endocrine function.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2003년도 Annual Meeting of KSAP : International Symposium on Pharmaceutical and Biomedical Sciences on Obesity
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• pp.89-89
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• 2003

Exposure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a variety of biological and toxicology effects, most of which are mediated by aryl hydrocarbon receptor (AhR). The ligand-bound AhR as a heterodimer with AhR nuclear translocator (ARNT) binds to its specific DNA recognition site, the dioxin-responsive element (DRE), and it results in increased transcription of CYP1A1 gene. Retinoic acid (RA) regulates the transcription of various genes for several essential functions through binding to two classes of nuclear receptors, the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Constitutive androstane receptor (CAR) also regulates the transcription of gene. In this study, we have examined how RAR, RXR and CAR regulated CYP1A1 in rainbow trout hepatoma cell (RTH 149) using luciferase reporter gene assay system. We did transient transfection with CYP1A1 luciferase reporter gene and treated with TCDD, all-trans RA, 9-cis RA and phenobarbital. Treatment of all-trans RA, 9-cis RA or phenobarbital decreased the TCDD induced transcription of CYP1Al. When we did transient cotransfection with CYP1A1 luciferase reporter gene and RXR, as increase of RXR concentration, the TCDD induced transcription of CYP1A1 was decreased. Transfection with CAR also decreased the TCDD induced transcription of CYP1A1 in RTH 149 cells.