• BMB Reports
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• v.36 no.5
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• pp.514-519
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• 2003

The ribonucleotide reductase (RR) 2 gene of the HSV-2 strain G was cloned, sequenced, and expressed in an E. coli cell. The RR2 gene was located on the PstI 2.4 kb fragment, which was cloned and sequenced. The ORF of the gene was 1,011 bp and its termination codon was TAG; also, the CATATAA sequence was present in the promoter of the RR2 gene. A Poly A signal sequence (AATAAA) was found in the 3'-noncoding region. The RR2 proteins that were produced in the E. coli and Vero cells were confirmed using a Western blot analysis. SDS-PAGE revealed that the molecular weights of the fusion-RR2 that was produced in the E. coli cells were approximately 24 kDa and 38 kDa in the Vero cells. The RR2 proteins were soluble. The differences in the molecular weights might be due to modifications in the Vero cells.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.187-192
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• 2010

In order to monitor the possibility of horizontal gene transfer between transgenic rice and microorganisms in a paddy rice field, the gene flow from a bifunctional fusion (TPSP) rice containing trehalose-6-phosphate synthase and phosphatase to microorganisms in soils was investigated. The soil samples collected from the paddy rice field during June 2004 to March 2006 were investigated by multiplex PCR, Southern hybridization, and amplified fragment length polymorphism (AFLP). The TPSP gene from soil genomic DNAs was not detected by PCR. Soil genomic DNAs did not show homologies on the Southern blotting data, indicating that gene transfer did not occur during the last two years in the paddy rice field. In addition, the AFLP band patterns produced by soil genomic DNAs from both transgenic and non-transgenic rice fields appeared similar to each other when analyzed by the NTSYSpc program. Thus, these data suggest that transgenic rice does not give a significant impact on the communities of soil microorganisms, although long-term observation may be needed.

• Applied Biological Chemistry
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• v.39 no.3
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• pp.195-199
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• 1996

We have cloned several E. coli sfs genes which stimulate mal gene expression with $crp^{{\ast}1}$). One the genes (pPVC2) was sequenced and potential CRP binding site is located in the upstream of the putative promoter in the regulatory region. In order to investigate the regulation of the sfs1 gene by the cAMP-CRP complex, we have constructed the sfs-lacZ fusion gene in this research. The overall transcriptional stimulations of sfs1 gene in the presence cAMP were confirmed by ${\beta}-galactosidase$ activity and Western blot analysis of sfs1-lacZ fusion gene. Transcriptional regulation by cAMP-CRP was also confirmed by Northern blot analysis. End-labelled DNA of the DNA fragment in sfs1 regulation region were used for gel retardation assay to examine the CRP-DNA complex in the presence of cAMP. Results here indicate that CRP binding site in the regulatory region of sfs1 gene is positive regulator for the expression of sfs1 gene.

• BMB Reports
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• v.42 no.11
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• pp.731-736
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• 2009

The generation of functional recombinant antibodies from hybridomas is necessary for antibody engineering. However, this is not easily accomplished due to high levels of aberrant heavy and light chain mRNAs, which require a highly selective technology that has proven complicated and difficult to operate. Herein, we attempt to use an alkaline phosphate (AP)-fused form of single-chain variable fragment (scFv) for the simple identification of a hybridoma-derived, functional recombinant antibody. As a representative example, we cloned the scFv gene from a hybridoma-producing mouse IgG against branched-chain keto acid dehydrogenase complex-E2 (BCKD-E2) into an expression vector containing an in-frame phoA gene. Functional recombinant antibodies were easily identified by conventional enzyme-linked immunosorbent assay (ELISA) by employing scFv-AP fusion protein, which also readily serves as a valuable immuno-detective reagent.

• Microbiology and Biotechnology Letters
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• v.31 no.3
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• pp.307-310
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• 2003

A gene encoding streptodornase(sdc) from Streptococcus equisimilis H46A was expressed in S. equisimilis H64H sdc under the control of the streptokinase gene promoter. Secretion of the streptodornase was directed by the signal sequences of streptokinase or streptodornase. The expressed streptodornase activity from S. equisimilis H46A sdc transformant with streptokinase promoter - streptodornase coding sequence fusion vector was 2.3 fold higher than that from wild type. Construct of signal sequence region replaced by streptokinase ones was similarly expressed as a wild type. But constructs of skc or lrp core regions of streptokinase promoter streptodornase fusion were similarly expressed as in sdc mutant. In conclusion, improved expression of streptodornase by use of streptokinase promoter required the full length of promoter.

• Food Science and Biotechnology
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• v.16 no.5
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• pp.778-782
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• 2007

This study assessed the effects of the antimicrobial substances sulforaphane, grapefruit seed extracts (GSE), and reuterin on the expression of Salmonella HilA and InvF virulence gene using a LacZY assay (${\beta}$-galactosidase assay) with hilA:lacZY and invF:lacZY fusion strains of Salmonella typhimurium SL1344. Salmonella was grown for 8 hr at $37^{\circ}C$ in the presence of diluted antimicrobial substances ($2\;{\mu}g/mL$ sulforaphane, $20\{\mu}g/mL$ GSE, and 0.26 mM reuterin) at concentrations that did not inhibit the cellular growth of Salmonella. Sulforaphane inhibited the expression of HilA and InvF by 50-90 and 20-80%, respectively. GSE also inhibited the expression of both genes, but to a lesser degree. Among the 3 antimicrobial substances, reuterin showed the least inhibition, which was abolished after 3-4 hr. None of the antimicrobial substances inhibited the ${\beta}$-galactosidase enzyme activity of S. typhimurium. The assay used in this study represents a very sensitive method for screening bioactive substances that inhibit the expression of virulence genes in Salmonella.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.12-19
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• 1994

KEM1 is known to control the spindle pole body or microtubule function, probably in response to the cellular nutritional conditions in Saccharomyces cerevisiae. Transposon insertions were performed in the cloned KEM1 gene using mini-Tn10-LUK element carrying E. coli ${\beta}$-galactosidase structural gene. A collection of ranfom Tn10-LUK insertions defined an approximately 3.5 kb region required for the KEM1 function. From this collection functional KEM1::lacZ protein fusions were identified. Indirect immunofluorescence using anti-${\beta}$-galacatosidase antibodies localized the KEM1::lacZ fusion protein to the periphery of the nucleus.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.48-54
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• 2005

A comparison of promoter fusions with the luxCDABE genes from Vibrio fischeri and Photorhabdus luminescens was made using promoters from several genes (katG, sodA, and pqi-5) of E. coli that are responsive to oxidative damage. The respective characteristics, such as the basal and maximum bioluminescence and the relative bioluminescence, were compared. E. coli strains carrying fusions of the promoters to P. luminescens lux showed higher basal and maximally induced bioluminescent levels than strains carrying the same promoter fused to the luxCDABE genes from V. fischeri. The sensitivities between the strains were similar, regardless of the luciferase used, but lower response ratios were seen from strains harboring the P. luminescens lux fusions. Furthermore, using the two katG::lux fusion strains, the bioluminescence from the P. luminescens lux fusion strain, DK1, was stable after reaching a maximum, while that of strain DPD2511 decreased very rapidly due to substrate limitation.

• Horticultural Science & Technology
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• v.32 no.3
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• pp.375-381
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• 2014

Transgenic potato was generated to express recombinant 5 repeated ${\beta}$-amyloid ($A{\beta}$) peptides, potential antigens to be applied as a preventive accine for Alzheimer's disease using Agrobacterium mediated transformation. The $A{\beta}$ peptides were fused to the human IgG Fc fragment enhancing protein and KDEL, which is the endoplasmic reticulum (ER) retention signal ($5A{\beta}$-FcK). The $5A{\beta}$-FcK, was expressed under the control of the duplicated 35S promoter. PCR analysis confirmed the presence of the transgene in several transgenic potato lines. Southern blot analysis showed only a single gene copy number in transgenic line 22, whereas multiple gene copy numbers were shown for transgenic lines 31 and 44. Northern blot analysis showed that line 22 had stronger mRNA levels when compared to lines 31 and 44. Immunoblot analysis confirmed that the $5A{\beta}$-FcK protein was expressed in the transgenic potato plant. These results indicate that $5A{\beta}$ fused to Fc can be expressed in potato plants.

• 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.