• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.1-11
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• 2001

Lactic acid bacteria (LAB) are widely used for various food fermentation. With the recent advances in modern biotechnology, a variety of bio-products with the high economic values have been produced using microorganisms. For molecular cloning and expression studies on the gene of interest, E. coli has been widely used mainly because vector systems are fully developed. Most plasmid vectors currently used for E, coli carry antibiotic-resistant markers. As it is generally believed that the antibiotic resistance markers are potentially transferred to other bacteria, application of the plasmid vectors carrying antibiotic resistance genes as selection markers should be avoided, especially for human consump-tion. By contrast, as LAB have some desirable traits such that the they are GRAS(generally recognized as safe), able to secrete gene products out of cell, and their low protease activities, they are regarded as an ideal organism for the genetic manipulation, including cloning and expression of homologous and heterologous genes. However, the vec-tor systems established for LAB are stil insufficient to over-produce gene products, stably, limiting the use of these organisms for industrial applications. For a past decade, the two popular plasmid vectors, pAM$\beta$1 of Streptococcus faecalis and pGK12 theB. subtilis-E. coli shuttle vector derived from pWV01 of Lactococcus lactis ssp. cremoris wg 2, were most widely used to construct efficient chimeric vectors to be stably maintained in many industrial strains of LAB. Currently, non-antibiotic markers such as nisin resistance($Nis^{r}$ ) are explored for selecting recombi-nant clone. In addition, a gene encoding S-layer protein, slp/A, on bacterial cell wall was successfully recombined with the proper LAB vectors LAB vectors for excretion of the heterologous gene product from LAB Many food-grade host vec-tor systems were successfully developed, which allowed stable integration of multiple plasmid copies in the vec-mosome of LAB. More recently, an integration vector system based on the site-specific integration apparatus of temperate lactococcal bacteriophage, containing the integrase gene(int) and phage attachment site(attP), was pub-lished. In conclusion, when various vector system, which are maintain stably and expressed strongly in LAB, are developed, lost of such food products as enzymes, pharmaceuticals, bioactive food ingredients for human consump-tion would be produced at a full scale in LAB.

• IMMUNE NETWORK
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• v.5 no.3
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• pp.157-162
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• 2005

Background: 1-8D gene is a member of human 1-8 interferon inducible gene family and was shown to be overexpressed in fresh colon cancer tissues. Three peptides 1-6, 3-5 and 3-7 derived from human 1-8D gene were shown to have immunogenicity against colon cancer. Methods: To study tumor immunotherapy, of three peptides we established an active immunization model using HHD mice. $D^{b-/-}{\times}{\beta}2$ microglobulin $({\beta}2m)$ null mice transgenic for a chimeric HLA-$A2.1/D^{b-}\;{\beta}2m$ single chain (HHD mice) were challenged with B16/HHD/1-8D tumor cells and were immunized with irradiated peptide-loaded RMA- S/HHD/B7.1 transfectants. In therapy model tumor growth was retarded in HHD mice that were injected with 3-5 peptide-loaded RMA-S/HHD/B7.1. In survival test vaccination with 1-8D-derived peptide protects HHD mice from tumor progression after tumor challenge. Results: These studies show that peptide 3-5 derived from 1-8D gene can be the most effective candidate for the vaccine of immunotherapy against colon cancer and highlight 1-8D gene as putative colon carcinoma associated antigens. Conclusion: We demonstrated that RMA-S/HHD/ B7.1 loaded with 1-8D peptides, especially 3-5, immunization generates potent antitumor immunity against tumor cells in HHD mice and designed active immunization as proper immunotherapeutic protocols.

• Journal of Plant Biology
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• v.37 no.1
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• pp.77-83
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• 1994

DNA uptake in dry embryos of rice by DNA imbibition was detected by monitoring the expression of chimeric vectors. The selective markers of expression vectors used were ${\beta}-glucuronidase$ ronidase (GUS) and hygromycin phosphotransferase (HPT) genes under the control of CaMV35 S promoter. Frequency of transient expression of the foreign gene was generally 30-50% varying according to the types of vectors and rice cultivars. Dot blot analysis and DNA sequence analysis of inverse polymerase chain reaction products showed that selected rice in hygromycin B (HmB) medium had HPT gene and CaMV35S promoter DNA sequence in genomic DNA of rice. To investigate what ratio of rice having two marker genes simultaneously as rice embryos imbibed the vector DNA having two HPT and GUS gene, transform ants selected in lImB medium were subjected to PCR for GUS gene. It was shown that about 90 percentage of surviving ones in HmB medium had GUS gene.S gene.

• Preventive Nutrition and Food Science
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• v.8 no.1
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• pp.61-65
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• 2003

This study was investigated to identify the regulatory mechanism of ACC gene expression by hormones and nutrition. The fragment of ACC promoter I (PI) -220 bp region was recombined to pGL3-Basic vector with luciferase as a reporter gene. The primary hepatocyte from the rat was used to investigate the regulation of ACC PI activity. ACC PI (-220 bp)/luciferase chimeric plasmid was transfected into primary rat hepatocyte by using lipofectin. ACC PI activity was shown by measuring luciferase activity. The addition of insulin, dexamethasone, and triiodothyronine to the culture medium increased the activity of ACC PI by 2.5-, 2.3- and 1.8-fold, respectively. In the presence of 1 $\mu$M dexamethasone, the effects of insulin was amplified about 1.2-fold showing the additional effects of dexamethasone. Moreover the activity of luciferase was increased by insulin, dexamethasone, and triiodothyronine treatment approximately 4-fold. These results indicated that insulin, dexamethasone and thyroid hormone coordinately regulate ACC gene expression via regulation of promoter I activity. On the -220 to ＋21 region of ACC PI, the addition of the glucose to the culture medium increased the activity of ACC PI. With 25 mM glucose, luciferase activity increased by 7-fold. On the other hand, on the -220 bp region, ACC PI activity was not changed by polyunsaturated fatty acids. Therefore, it can be postulated that there are response elements for insulin, triiodothyronine, dexamethasone, and glucose, but not PUFAs on the -220 bp region of ACC PI.

• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.147-152
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• 1998

AL1-gene, necessary for the replication of the genome of a gemini virus TGMV, was inserted in the opposite direction to the promoter CaMV35S resulting in the construction of a plant transformation binary vector pAR35-2. The vector pAR35-2 contains the chimeric gene cassette involving the duplicated promoter CaMV35S, opposite direction of AL1-gene fusioned with hygromycin resistant gene, and the gene cassette of the neomycin phosphotransferase II gene. The plasmid was transferred to tobacco and tomato plants by leaf disk infection via Agrobacterium. The transgenic plants were selected and grown on the MS-agar medium containing kanamycin and hygromycin. The shoots induced from the calli were regenerated to the whole transgenic plants. The antisense AL1-gene was detected in the genomic DNA isolated from the leaves by using the PCR mediated Southern blot analysis. The expression of the antisense AL1-gene was also observed using the RT-PCR mediated Southern blot analysis. The observation of chloroplasts in guard cell pair indicated that the transgenic tomato plants were diploid.

• The Plant Pathology Journal
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• v.21 no.3
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• pp.288-292
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• 2005

Pathogenesis-related protein-1a (PR-1a) is strongly induced in tobacco plants by pathogen attack, exogenous salicylic acid (SA) application and by other developmental processes. In order to develop a rapid screening system for the selection of plant defense-inducing compounds originated from various sources, we have transformed tobacco Samsun NN plants with a chimeric construct consisting of GUS $(\beta-glucuronidase)$. In the $T_1$ generation, three transgenic lines having stable GUS expression were selected for further promoter analysis. Using GUS histochemical assay, we observed strong GUS induction driven by PR-1a promoter in PR1a-GUS transgenic tobacco leaves in response to the exogenous application of SA or benzol (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH), a SA­derivative compound. In addition, GUS expression was maintained locally or systemically in PR1a-GUS transgenic line $\#5\;T_2$ generation) until after 3 days when they were treated with same chemicals. Our results suggested that the PR1a-GUS reporter gene system in tobacco plants may be applicable for the large-scale screening of defense-inducing substances.

• BMB Reports
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• v.37 no.5
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• pp.556-564
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• 2004

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

• Molecules and Cells
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• v.19 no.1
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• pp.131-136
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• 2005

The ETV6-NTRK3 gene fusion, first identified in the chromosomal translocation in congenital fibrosarcoma, encodes a chimeric protein tyrosine kinase with potent transforming activity. ETV6-NTRK3-dependent transformation involves the joint action of NTRK3 signaling pathways, and aberrant cell cycle progression resulting from activation of Mek1 and Akt. The level of glutathione (GSH) was found to be markedly increased in ETV6-NTRK3-transformed NIH3T3 cells. The activities of the two GSH biosynthetic enzymes as well as of glutathione peroxidase, together with their mRNAs, were also higher in the transformed cells. The transformed cells were able to grow in the presence of GSH-depleting agents, whereas the control cells were not. L-Buthionine-(S,R)-sulfoximine (BSO) inhibited activation of Mek1 and Akt in the transformed NIH3T3 cells. These observations imply that up-regulation of GSH biosynthesis plays a central role in ETV6-NTRK3-induced transformation.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.323-327
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• 2004

Leuconostoc citreum CBUE isolated from kimchi proved to harbor a small cryptic plasmid, pNS75. The complete nucleotide sequence of pNS75 was 1,821 bp and had a low G+C content of 39.2%. Computer analysis using DNASIS revealed one open reading frame (ORF), having ATG as putatitive start condon and potentially encoding proteins with molecular mass of 38 kDa. The chimeric plasmid pLeuCM was first constructed wih pNS75, pUC19 and chroamphenicol acetyltransferase (CAT) from Staphylococcus sp.. pLeuCM replicated and expressed chroamphenicol acetyltransferase in Leuconostoc citerum CBNF after transformation. To test the availability of shuttle vector as cloning vehicle of foreign gene, $\alpha$-amylase gene of Streptococcus bovis was cloned and all transformants secreated the $\alpha$-amylase successfully. The result indicates that pLeuCM is a potential shuttle vector for Leuconostoc spp. and lactic acid bacteria.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.