• Korean Journal of Plant Tissue Culture
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• v.25 no.5
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• pp.341-346
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• 1998

In this paper we describe the cloning and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol 4-reductase (DFR) in Matthiola incana R. Br. A heterologous cDNA probe from Zea mays was used to isolate full-size DFR cDNA clone from a corolla-specific cDNA library. Comparison of the coding region of this DFR cDNA sequence including the sequences of Zea mays, Anthirrinum majus, Petunia hybrida, Callistephus chinensis, Dianthus caryophyllus and Rosa hybrida reveals a identity higher than 61% at the nucleotide level. The DFR transcript is G/C rich in monocotyledonous plants show a strong codon bias preferring codons with a G or C in the third position. The function of this nucleotide sequences were verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRNA from wild type and mutant plants and by in vitro expression yielding an enzymatically active reductase. Genomic southern blot analysis showed the presence of one gene for DFR in Matthiola incana. Northern blot analysis of the DFR wild type and mutant lines showed that the lack of DFR activity in the stable acyanic mutant k17b is clearly by a transcriptional block of the DFR gene.

• Korean Journal of Veterinary Research
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• v.36 no.1
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• pp.109-118
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• 1996

The Salmonella rfb gene encoding for the biosynthesis of the oligosaccharide-repeating units of the O-antigenic determinants was cloned and sequenced. A set of nucleotide primers(a forward and reverse) was selected to target a defined region of the guanosine diphospho-mannose(GDP-Man) pyrophosphorylase synthase gene : rfbM of Salmonella C serogroup. The primer set was used to develop a PCR-based rapid and specific detection system for Salmonella C1 serogroup. Amplification bands of predicted size(1,422bp) were generated from 11 different Salmonella C1 isolates. The bands were verified to be specific for the C1 serogroup by Southern blot analysis using reference homologous DNA specificity was further confirmed by the lack of reactivity with heterologous DNA derived from non-salmonella members of the family enterobacteriaeceae. A specificity of 100% was deduced along with a very high sensitivity shown by a detection limit of 1fg of a purified DNA template. The isolated DNA sequence was found to be 99.8% homologous to S montevideo but the related primers amplified with the predicted band sizes with all the Salmonella C1 serogroups tested. It is concluded that the PCR protocol based on the rfbM gene from S cholerasuis is optimal fast and specific for the detection of Salmonella C1 serogroup and also the corresponding probe is suitable for rapid detection of all Salmonella C1 serogroup DNA tested. This technology should facilitate the identification of contaminated pig products and for any other products contaminated with the Salmonalla C1 serogroup. The immediate impact of this developed method will be in the area of food safety of pig products with the potential prospect for adaptation to other food inspection technologies.

• Journal of Microbiology
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• v.44 no.3
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• pp.301-310
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• 2006

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr =166,208). The EMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.

• Journal of Plant Biotechnology
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• v.33 no.2
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• pp.85-91
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• 2006

Flavanone 3$\beta$-hydroxylase (FHT) is an enzyme acting in the central part of the flavonoid biosynthesis pathway. FHT catalyses the hydroxylation of flavanone to dihydroflavonols in the anthocyanin pathway. In this paper we describe the cloning and expression of the genes encoding the flavonoid-biosynthetic enzyme FHT in Gypsophila paniculata L. A heterologous cDHA probe from Dianthus cavophyllus was used to isolate FHT-encoding cDHA clones from Gypsophila paniculata L.. Inspection of the 1471 bp long sequence revealed an open reading frame 1047 bp, including a 190 bp 5' leader region and 288 bp 3' untranslated region. Comparison of the coding region of this FHT cDHA sequence including the sequences of Arabidopsis thaliana, Citrus sinensis, Dianthus caryophyllus, Ipomoea batatas, Matthiola incana, Nierembergia sp, Petunia hybrida, Solanum tuberosum, Vitis vinifera reveals a identity higher than 69% at the nucleotide level. The function of this nucleotide sequences were verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRHA from wild type and mutant plants, by in vitro expression yielding and enzymatically active hydroxylase, as indicated by the small dihydrokaempferol peak. Genomic southern blot analysis showed the presence of only one gene for FHT in Gypsophila paniculata.

• Journal of Life Science
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• v.19 no.7
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• pp.845-851
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• 2009

A vector harboring double cassettes; a heterologous gene expression cassette of pHCE-InaN-antigen and a ghost formation cassette of pAPR-cI-E lysis 37 SDM was constructed and introduced to E. coli DH5a. For the production of a bacterial ghost vaccine, bacterial ghosts from E. coli / Streptococcus iniae with four different types of antigens - enolase, GAPDH, sagA and piaA - were produced by the optimization of fermentation parameters such as a glucose concentration of 1 g/l, agitation of 300 rpm and aeration of 1 vvm. Efficiency of ghost bacteria formation was evaluated with cultures of OD$_{600}$=1.0, 2.0 and 3.0. The efficiency of the ghost bacteria formation was 99.54, 99.67, 99.99 and 99.99% with inductions at OD$_{600}$=3.0, 1.0, 2.0 and 1.0 for E. coli/S. iniae antigens enolase, piaA, GAPDH and sagA, respectively. Ghost bacteria as a vaccine was harvested by centrifugation. The antigen protein expressions were analyzed by SDS-PAGE and western blot analysis, and the molecular weights of the enolase, piaA, GAPDH and sagA were 78, 26, 67 and 26 kDa, respectively. The molecular weights of the expressed antigens were consistent with theoretical sizes obtained from the amino acid sequences.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.271-279
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• 1994

To develop a yeast strain which stably secretes both $\alpha$-amylase and glucoamylase and therefore is able to convert starch directly to ethanol, a mouse salivary $\alpha$-amylase cDNA gene with a yeast alcohol dehydrogenase I promoter has been introduced into the cell of a Saccharomyces diactaticus hybrid strain secreting only glucoamylase. To secrete both enzymes more stably without loss of the $\alpha$-amylase gene during a cell-multiplication, an integrating plasmid vector containing $\alpha$-amylase gene was constructed and introduced into the yeast cell. The results showed that the linearized form of the integrating vector was superior in the transformation efficiency and the rate of the expression of the $\alpha$-amylase gene than the circular type of the vector. The yeast transformant having a linearized plasmid vector exhibited higher mitotic stability than the yeast transformant habouring episomat plasmid vector. The transformant containing the linearized vector producing both $\alpha$-amylase and glucoamylase exhibited 2-3 times more amylolytic activity than the original untransformed strain secreting only glucoamylase.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.73-79
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• 2017

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous ${\alpha}1,3$-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig ($GalT^{-MCP/+}$), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig ($GalT^{-MCP/-MCP}$) by crossbreeding $GalT^{-MCP/+}$ pigs. Two female founders gave birth to six of $GalT^{-MCP/-MCP}$, and seven $GalT^{-MCP/+}$ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the $GalT^{-MCP/-MCP}$ pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the $GalT^{-MCP/-MCP}$ pig is a useful candidate to apply xenotransplantation study.

• Microbiology and Biotechnology Letters
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• v.42 no.2
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• pp.190-193
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• 2014

CMP-Neu5Ac synthetase is a key enzyme for the synthesis of CMP-Neu5Ac, which is an essential precursor of sialylated glycoconjugates. For the soluble expression of the CMP-Neu5Ac synthetase gene (neuA) from Escherichia coli K1, various heat shock proteins were co-expressed in E. coli BL21 (DE3) Star. In order to do this, a pG-KJE8 plasmid, encoding genes for GroEL-ES and DnaK-DnaJ-GrpE, was co-transformed with neuA and was expressed at $20^{\circ}C$ by the addition of 0.01 mM IPTG and 0.005 mg/ml L-arabinose. The co-expression of a variety of heat shock proteins resulted in the remarkably improved production of soluble CMP-Neu5Ac synthetase in E. coli.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1326-1334
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• 2008

The prime-boost vaccination with DNA vaccine and recombinant viral vector has emerged as an effective prophylactic strategy to control infectious diseases. Here, we compared the protective immunities induced by multiple alternating immunizations with DNA vaccine (pCIgB) and replication-incompetent adenovirus (Ad-gB) expressing glycoprotein gB of pseudorabies virus (PrV). The platform of pCIgB-prime and Ad-gB-boost induced the most effective immune responses and provided protection against virulent PrV infection. However, priming with pCIgB prior to vaccinating animals by the DNA vaccine-prime and Ad-boost protocol provided neither effective immune responses nor protection against PrV. Similarly, boosting with Ad-gB following immunization with DNA vaccine-prime and Ad-boost showed no significant responses. Moreover, whereas the administration of Ad-gB for primary immunization induced Th2-type-biased immunity, priming with pCIgB induced Th1-type-biased immunity, as judged by the production of PrV-specific IgG isotypes and cytokine IFN-$\gamma$. These results indicate that the order and injection frequency of vaccine vehicles used for heterologous prime-boost vaccination affect the magnitude and nature of the immunity. Therefore, our demonstration implies that the prime-boost protocol should be carefully considered and selected to induce the desired immune responses.

• Journal of Dairy Science and Biotechnology
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• v.21 no.2
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• pp.97-104
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• 2003

It is extremely important to destroy the antigenicity of milk proteins for dietetic treatment of infants with milk allergy. Enzymatic digestion of milk protein is not only effective for destroying antigenicity, but it also is less liable to alter the nutritive value. Bovine casein was hydrolyzed with eight different commercial proteases derived from bacterias or fungi, either individually or in combination to eliminate protein allergenicity. The average molecular weight of casein hyrdolysates determined by size exclusion chromatography is about 550${\sim}$2,300 dalton range. Antigenicity of the casein hyrdolysates was not detected by heterologous passive cutaneous anaphylaxis in guinea pig-rabbit antiserum system. The inhibition test on the enzyme-linked immunosorbent assay(ELISA) showed that the antigenicity of casein hydrolysates is lowed up to 1/8,000 than that of intact bovine casein. As the enzyme reaction was carried out by the combination of bacterial and fungal protease, casein hydrolysates showed much lower bitterness and antigenicity. It suggests that these hydrolysates will be applied to many kinds of foods including the development of hypo-allergenic infant formula.