• Tuberculosis and Respiratory Diseases
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• v.45 no.3
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• pp.529-535
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• 1998

Background: Interleukin-4 plays an important role in pathogenesis of asthma, especially in developing atopy by means of switching B lymphocytes to produce IgE. It has been shown that there is polymorphism in the Interleukin-4 promoter region, transversion of cytosine to thymine at-598 from translation initiation site of IL-4 gene. There has also been quite a few works to reveal the role of the polymorphism of IL-4 gene in patients with asthma. We performed this investigation to determine the role of the polymorphism in the severity of symptoms of patients with asthma. We also examined the frequency and the type of the polymorphism in asthmatics compared with non-asthmatics as well. Method: The subjects enrolled in this study were 49 asthmatics and 33 non-asthmatics. All the asthmatics were classified as mild and moderate to severe by the NHLBI/WHO Workshop. DNA from both asthmatics and non-asthmatics was extracted, then performed ARMS(Amplification Refractory Mutation System) as well as RFLP using BsmFl restriction enzyme in order to confirm the polymorphism of Il-4 gene. Results: There was no significant difference in the occurrence of polymorphism of the IL-4 promoter sequence between asthm and non-asthma groups(P=0.7). Among those with polymorphisms, the number of C/C type was slightly more than C/T type in both asthmatics and non-asthmatics, 26 vs 21 in asthmatics and 18 vs 15 in non-asthmatics, which was, however, insignificant statistically. No significant relationship between the severity of asthma and the polymorphism was found(P=0.7). Conclusion: There was no significant difference between the severity of asthma and the IL-4 promoter polymorphism(P=0.709). Interestingly, the frequency of the polymorphism in both asthmatics as well as non-asthmatics was found to be even higher than that occurred in Caucasians. However, no significant difference in the frequency of the polymorphism was found in both groups.

• Microbiology and Biotechnology Letters
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• v.17 no.6
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• pp.600-605
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• 1989

For the purpose of isolation of the Zymomonas mobilis DNA fragments showing promoter activity in Escherichia coli, a promoter screening vector, PCMT215 was constructed by transferring a promoterless chloramphenicol acetyltransferase (CAT) gene of pYEJ001 into pMT21 which contains $\beta$-lactamase gene and multiple cloning sites. A library of Z, mobilis Sau3AI DNA fragments was constructed in E. coli using the newly constructed pCMT215. Fourteen clones showing resistance to chloramphenicol ranging in concentration from 30 to 750 $\mu$g/$m\ell$ were selected. From five clones of them, the Z. mobilis DNA fragments expressing CAT gene of the recombinant plasmids were sequenced and then sites of transcriptional initiation were identified. The nucleotide sequences of the cloned DNA shared AT rich regions, poly A's or T's stretches and palindromic regions. The positions of transcriptional initiation for CAT gene occurred at more than one site spaced over by 4 to 190 base pairs on the cloned fragments in E. coli.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.109-113
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• 1998

In this study we tried to transform an antimicrobial peptide gene (Shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) into tobacco and potato plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified ie nucleotide sequence to increase antimicrobial activity. Transgenic tobacco plants were regenerated and their seeds were tested on the media containing kanamycin (500 mg/L). The results of PCR amplification and genomic Southern blot hybridization confirmed the integration of construct (tPAL5 promoter-Shiva-NOS-GUS-NOS) into chromosome. We observed that one of the transgenic tobacco plants showed chromosome rearrangement when integrated. In case of potato transformation, the efficiency of regeneration was maximized at the medium containing Zeatin 2mg/L, NAA 0.01mg/L, GA$_3$ 0.1mg/L. We also observed the high expression of GUS (${\beta}$-glucuronidase) enzyme which was located next to the terminator sequence of nopaline synthase gene (NOS) in the vascular tissue of stem, leaves of transgenic potatoes. This result suggested that a short sequence of Shiva gene (120 bp) and NOS terminator sequence might be served as a leader sequence of transcript when translated.

• BMB Reports
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• v.52 no.9
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• pp.548-553
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• 2019

Ets-1 is a prototype of the ETS protein family. Members of the ETS protein family contain a unique ETS domain. Ets-1 is associated with cancer progression and metastasis in many types of cancer. Many studies have shown a link between elevated expression of Ets-1 in cancer biopsies and poor survival. CCR7 is a chemokine that binds to specific ligand CCL21/CCL19. CCR7 expression is associated with tumor metastasis and infiltration into lymph nodes. The objective of this study was to test whether Ets-1 could regulate CCR7 expression and enhance tumor metastasis. Our data showed that CCR7 expression was downregulated in Ets-1-deficient T cells upon T-cell stimulation. Overexpression of Ets-1 increased CCR7 expression in breast cancer cell lines. In contrast, knockdown of Ets-1 reduced CCR7 expression. Ets-1 could directly bind to CCR7 promoter and mediate CCR7 expression in luciferase reporter assays and chromatin immunoprecipitation assays. Transactivation activity of Ets-1 was independent of the Pointed domain of Ets-1. Ets-1 could also enhance $NF-{\kappa}B$ and CBP transactivation of CCR7 promoter. Our results also showed that Ets-1 could modulate cancer cell transmigration by altering CCR7 expression in transwell assay and wound healing assay. Taken together, our data suggest that Ets-1 can enhance CCR7 expression and contribute to tumor cell migration.

• Microbiology and Biotechnology Letters
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• v.44 no.2
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• pp.140-144
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• 2016

The gene encoding chondroitinase ABC from a genomic library of Bacteroides stercoris HJ-15, which was isolated from human feces, was cloned. The cloned gene consisted of 3,090 bp and was predicted to encode a 1,029−amino-acid protein. The B. stercoris chondroitinase ABC gene was not homologous to other chondroitinase ABC genes; however, its amino acid sequence showed 71% homology to that of Bacteroides thetaiotaomicron. The gene was cloned in the pET-26b+ expression vector and expressed under the T7 promoter in Escherichia coli BL21(DE3). The purified recombinant chondroitinase ABC degraded chondroitin sulfates A, B, and C.

• BMB Reports
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• v.34 no.6
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• pp.502-508
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• 2001

A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N-terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.

• Environmental Mutagens and Carcinogens
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• v.26 no.3
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• pp.69-76
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• 2006

2,3,7,8-Tetrachlorodibenzo-$\rho$-dioxin(TCDD) is a ubiquitous, persistent environmental contaminant and the most powerful carcinogen categorized by IARC. Although the mechanism of carcinogenesis by TCDD is poorly understood, several studies have shown that the skin is one of target organs far TCDD. In this study, we investigated the neoplastic transformation of human keratinocyte-derived cell line, HaCaT, by chemical transformation method using N-methyl-N'-nitro-N-nitrorsoguanidine(MNNG) and TCDD. We found that subsequent exposure to TCDD for 3 weeks after initial exposure to MNNG markedly induced transformed cells. It was suggested that TCDD can act as a potent promoter in HaCaT cells. Furthermore, these transformed cells showed morphological alternations in soft agar and increased telomerase activity. Therefore, the TCDD treatment of HaCaT cells by initiated with MNNG could promote neoplastic transformation without stimulation by exogenous growth factors. As a result, TCDD had a strong potency as a promoter in nontumorigenic immortalized human epidermal keratinocytes.

• The Korean Journal of Zoology
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• v.28 no.3
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• pp.166-178
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• 1985

In order to express hepatitis B surface antigen $(HB_sAg)$ containing pre-surface antigen region in mammalian calls, 2.7 kb DNA fragment containing pre-surface region-$HB_sAg$ gene poly(A) addition site of HBV genome was cloned into simian virus 40(SV 40) based chimeric vector pSVOB. 2.7 kb DNA fragment was derived from pHBVD 107 containing tandem copies of the HBV genome in a head-to-tail arrangement by Bgl II digestion. Construction of the vector pSVOE involved the incorporation of SV40 sequences spanning the viral origin of replication and 72 bp repeats (enhancer) into a pBR 322 derivative lacking sequences which inhibit replication in mammalian cells. Bam HI linker was inserted at the Pvu II site in the proximity of SV40 late promoter of pSVOE and named as pSVOB. To construct the recombinant plasmid pSVBS, pHBVD 107 was digested with Bgl II to isolate 2.7kb DNA fragment and the fragment was ligated into the Bam HI site of pSVOB by ligation. Preliminary result showed that the recombinant plasmid pSVBS produced $HB_sAg$ in the monkey cell producing large T antigen (COS cell).

• Journal of Life Science
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• v.14 no.3
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• pp.429-434
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• 2004

Levansucrase gene (levU) from Zymomonas mobilis was subcloned downstream of GALl promoter in pYES 2.0 and pYInu-AT [GALl0 promoter＋exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-levU and pYInu-levU, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Saccharomyces cerevisiae SEY2102, and then transformants showing high activity of levansucrase were selected. When each yeast transformants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 7.17 U/㎖ with the strain harboring pYES-levU and 6.61 U/㎖ with the strain harboring pYInu-levU. It was found that about 50% of levansucrase were detected in the medium and periplasmic space, and exoinulinase signal sequence didn't enhance the secretion efficiency. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.8
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• pp.477-484
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• 2019

Several types of scavenger receptors, including the Collectin-Placenta 1 (CL-P1) receptorthat is present in mammals, are molecules that are expressed on the surfaces of endothelial cells, macrophages and smooth muscle cells. These molecules are cell surface glycoproteins that can be conjugated to oxidized low density lipoprotein (oxLDL). Among these molecules, the effect of quercetin on CL-P1 activation has been confirmed. Quercetin is known as an antioxidant that stops oxidation because it acts to remove free radicals that are responsible for the oxidation reaction. In this study, fragments from the transcription start site of the mouse CL-P1 gene promoter to the -500th base were cloned using DNA polymerase. These fragments were then introduced into macrophage like RAW264.7 cells and fibroblast-like NIH3T3 cells to study the effect of quercetin on the CL-P1 gene expression. As a result, we found that bases ranging from -250 to -350 in the anterior part where gene expression starts are important for producing CL-P1 protein. Among them, the DNA mutation experiments we performed confirmed that the E2F binding sites are critical for producing the CL-P1 protein? In addition, when quercetin was added to the RAW264.7 culture medium, which was a culture of adherent cells, observedthe phenomenon of the cells falling off from the surface of the culture container.