• Journal of Microbiology and Biotechnology
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• v.17 no.10
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• pp.1727-1732
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• 2007

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAA-CoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.

• Journal of Physiology & Pathology in Korean Medicine
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• v.26 no.4
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• pp.437-440
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• 2012

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined that Cryptotanshione(CT), a tanshinone from oriental traditional medicinal herb Danshen (Salvia miltiorrhiza Bunge), had the inhibitory effects on hypoxia-mediated activation of STAT3 in androgen independent human prostate cancer PC-3 cells. CT inhibited the protein expression of hypoxia-inducible factor-1alpha (HIF-$1{\alpha}$) under hypoxic condition. Consistently, CT blocked hypoxia-induced phosphorylation and nuclear accumulation of STAT3. In addition, CT reduced cellular of vascular endothelial growth factor (VEGF), a critical angiogenic factor and a target gene of STAT3 induced under hypoxia. Of note, chromatin immunoprecipitation (ChiP) assay revealed that CT inhibited binding of STAT3 to VEGF promoter. Taken together, our results suggest that CT has anti-angiogenic activity by disturbing the binding STAT3 to the VEGF promoter in PC-3 cells.

• Journal of Plant Biotechnology
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• v.6 no.3
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• pp.145-150
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• 2004

Cabbage plants were transformed with the potato proteinase inhibitor II (PINII) gene, bar gene, and hpt gene using Agrobacterium. The expression of the PINII gene was driven by its own promoter which was wound-inducible. Ten transgenic plants were obtained from medium containing hygromycin as a selection antibiotic. The integration and expression of PINII and bar genes were confirmed by Southern and Northern hybridization. Growth and development of diamondback moths (Plutella xylostella) and tobacco cutworm (Spodoptera litura) larvae were examined on $T_1$ plants. The weight of the larvae and pupae of these two insects grown on transgenic plants was not different compared to those grown on wild type plants. However, the pupation and emergence rate of diamondback moths and tobacco cutworms fed on some transgenic plants was lower than on wild type plants. These results suggest that the PINII transgene under the control of a wound-induced promoter may be used for control of insects in transgenic cabbage through reduction of insect progeny number.

• Journal of Microbiology and Biotechnology
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• v.5 no.4
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• pp.224-228
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• 1995

Fed-batch fermentations were conducted to produce human lipocortin-I (LC1), a potential anti-inflammatory agent, from recombinant Sacchromyces cerevisiae carrying a galactose-inducible expression system. The cell growth, expression level of LC1, and the plasmid stability were investigated under various LC1 induction modes performed by three different galactose feeding strategies. Galactoe was fed to induce the expression of LCl from the beginning (initial induction) of culture or when the cell concentration reached 120 OD (mid-phase induction) or 300 OD (late induction). Among the three galactose-induction modes tested, the initial induction mode yielded the best result with respect to a final expression level of LC1. Fedbatch fermentation with initial induction mode produced LC1 at a conentration of 220 mg/l, which corresponded to 1.38- and 1.53-fold increases over those produced by mid-phase and late induction modes.

• Journal of Microbiology
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• v.33 no.3
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• pp.201-207
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• 1995

We have previously reported that SPP2 gene product of yeast Saccharomyces cerevisiae is involved in the pre-mRNA splicing. To investigate the rol ein the splicing pathway of the Spp2p protein, the SPP2 gene was overexpressed in Escherichia coli and polyclonal antibodies to Spp2p were generated from rabbits. First, a DNA fragment containing the SPP2 GENE without its promoter was subcloned into an E. coli expression vector, pKK233-3. The resulting recombinant plasmid pBQ14 contained an IPTG inducible tac promoter and the SPP2 structural gene. Overexpression of the SPP2 gene was achieved by additionof 0.1 to 1.0 mM IPTG to a logarithmic culture of E. coli JM103(pBQ14) for 90 min at 37.deg.C. Sequence of N-terminal 15 amino acids of the overproduced protein was well matched to the deduced one from the SPP2 reading frame. Then, polyclonal antibodies were generated from rabbits immunized with gel-purified SppSp protein. These antibodies reacted specifically with the Spp2p protein extracted from yeast cells expressing the SPP2 gene to a great extent. The antibodies could also block the activity of yeast splicing extracts.

• Microbiology and Biotechnology Letters
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• v.35 no.4
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• pp.272-277
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• 2007

The physiological responses of microorganisms to specific nutrient limitation can be regulated at the transcriptional levels. In this study, in order to develop the Pichia pastoris-derived promoter inducible by nutrient-limited condition, we constructed cDNA libraries using RT-PCR of total RNA from P. pastoris in steady-states of phosphate-limited chemostat with different dilution rates. Various genes were detected from cDNA library. Among these genes, the gene encoding putative sodium/phosphate ($Na^+$/Pi) symporter (NPS), high affinity transporter of phosphate, was detected. It was observed that expression of NPS increased in a manner specific to phosphate-limited condition through Northern blot. Therefore, it is thought that the promoter from NPS gene may have the potential as auto-inducible promoter by phosphate-limited culture condition without inducer.

• Reproductive and Developmental Biology
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• v.38 no.3
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• pp.99-105
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• 2014

In this study, to further understand the mechanism of animal growth and to develop a miniature transgenic animal model, we constructed and tested tetracycline-inducible RNAi system using shRNA targeting the mRNA of mouse insulin-like growth factor (mIGF-1) or mouse growth hormone receptor (mGHR) gene. Quantitative real-time PCR analysis of mouse liver cell (Hepa1c1c7) cells transfected with these vectors showed 85% or 90% of expression inhibition effect of IGF-1 or GHR, respectively. In ELISA analysis, the protein level of IGF-1 in the cells expressing the shRNA targeting IGF-1 mRNA was reduced to 26% of non-transformed control cells. Unexpectedly, in case of using shRNA targeting GHR, the IGF-1 protein level was decreased to 75% of control cells. Further experiments are needed to explain the lower interference effect of GHR shRNA in IGF-1 protein. Accumulated knowledge of this approach could be applicable to a variety of related biological area including gene function study, gene therapy, development of miniature animals, etc.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.12
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• pp.1689-1695
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• 2017

Objective: The cytokine inducible SH2-containing protein (CISH), which might play a role in porcine intestine immune responses, was one of the promising candidate genes for piglet anti-disease traits. An experiment was conducted to characterize the porcine CISH (pCISH) gene and to evaluate its genetic effects on pig anti-disease breeding. Methods: Both reverse transcription polymerase chain reaction (RT-PCR) and PCR were performed to obtain the sequence of pCISH gene. A pEGFP-C1-CISH vector was constructed and transfected into PK-15 cells to analysis the distribution of pCISH. The sequences of individuals were compared with each other to find the polymorphisms in pCISH gene. The association analysis was performed in Min pigs and Landrace pigs to evaluate the genetic effects on piglet diarrhea traits. Results: In the present research, the coding sequence and genomic sequence of pCISH gene was obtained. Porcine CISH was mainly localized in cytoplasm. TaqI and HaeIII PCR restriction fragment length polymorphism (RFLP) assays were established to detect single nucleotide polymorphisms (SNPs); A-1575G in promoter region and A2497C in Intron1, respectively. Association studies indicated that SNP A-1575G was significantly associated with diarrhea index of Min piglets (p<0.05) and SNP A2497C was significantly associated with the diarrhea trait of both Min pig and Landrace piglets (p<0.05). Conclusion: This study suggested that the pCISH gene might be a novel candidate gene for pig anti-disease traits, and further studies are needed to confirm the results of this preliminary research.

• Bulletin of the Korean Chemical Society
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• v.30 no.4
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• pp.897-901
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• 2009

Heme oxygenase-1 (HO-1) is an anti-inflammatory and anti-apoptotic protein and has been applied to various gene therapy researches. However, constitutive expression of HO-1 may induce deleterious side effects. In this research, hypoxia inducible HO-1 expression plasmid, pEpo-SV-HO-1, was constructed with the erythropoietin (epo) enhancer and simian virus 40 (SV40) promoter to avoid these unwanted side effects. Dexamethasone conjugated polyethylenimine (PEI-Dexa) was used as a gene carrier. It was previously reported that dexamethasone protected cardiomyocytes from apoptosis under hypoxia. In this research, PEI-Dexa reduced the caspase-3 level in hypoxic H9C2 cardiomyocytes as a derivative of dexamethasone, suggesting that PEI-Dexa is an anti-apoptotic reagent as well as a gene carrier. pEpo-SV-HO-1 was transfected to H9C2 cardiomyocytes using PEI-Dexa and the cells were incubated under normoxia or hypoxia. HO-1 expression was induced in the pEpo-SV-HO-1 transfected cells under hypoxia. In addition, cell viability under hypoxia was higher in the pEpo-SV-HO-1 transfected cells than the pEpo-SV-Luc transfected cells. Also, caspase-3 level was reduced in the pEpo-SV-HO-1 transfected cells under hypoxia. In addition to the anti-apoptotic effect of PEI-Dexa, hypoxia inducible HO-1 expression by pEpo-SVHO- 1 may be helpful to protect cardiomyocytes under hypoxia. Therefore, pEpo-SV-HO-1/PEI-Dexa complex may be useful for ischemic heart disease gene therapy.

• Journal of The Korean Society of Grassland and Forage Science
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• v.37 no.3
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• pp.223-230
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• 2017

Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) is an upstream signaling molecule that has been shown to induce stress tolerance in plants. In this study, the AtNDPK2 gene, under the control of a stress-inducible SWPA2 promoter, was introduced into the genome of tall fescue (Festuca arundinacea Schreb.) plants. The induction of the transgene expression mediated by methyl viologen (MV) and NaCl treatments were confirmed by RT-PCR and northern blot analysis, respectively. Under salt stress treatment, the transgenic tall fescue plants (SN) exhibited lower level of $H_2O_2$ and lipid peroxidation accumulations than the non-transgenic (NT) plants. The transgenic tall fescue plants also showed higher level of NDPK enzyme activity compared to NT plants. The SN plants were survived at 300 mM NaCl treatment, whereas the NT plants were severely affected. These results indicate that stress-inducible overexpression of AtNDPK2 might efficiently confer the salt stress tolerance in tall fescue plants.