• 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.

• Journal of Internet Computing and Services
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• v.6 no.1
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• pp.85-93
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• 2005

This paper describes how to distribute high multi-dimensional facial expression data of vast quantity over a suitable space and produce facial expression animations by selecting expressions while animator navigates this space in real-time. We have constructed facial spaces by using about 2400 facial expression frames on this paper. These facial spaces are created by calculating of the shortest distance between two random expressions. The distance between two points In the space of expression, which is manifold space, is described approximately as following; When the linear distance of them is shorter than a decided value, if the two expressions are adjacent after defining the expression state vector of facial status using distance matrix expressing distance between two markers, this will be considered as the shortest distance (manifold distance) of the two expressions. Once the distance of those adjacent expressions was decided, We have taken a Floyd algorithm connecting these adjacent distances to yield the shortest distance of the two expressions. We have used CCA(Curvilinear Component Analysis) technique to visualize multi-dimensional spaces, the form of expressing space, into two dimensions. While the animators navigate this two dimensional spaces, they produce a facial animation by using user interface in real-time.

• Journal of Life Science
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• v.17 no.1 s.81
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• pp.39-44
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• 2007

The Rqh1 gene is essential for vegetative growth in fission Yeast. The rqh1 mutant showed that sensitivity of DNA damaging agent, a wild range of phenotype including abnormal gene expression and cell elongation. This result showed that the rqhl-overexpression cell was sensitivity to DNA damaging agent like rqhl mutant. When Rqh1 have an over-expression by $nmt1^+$ promoter of pREP vector, rqh1 mutant DNA damaging agent sensitivity could be compensated. We isolated two strong mutant containing complementation gene, rqh156 and rqh172, respectively. This result observed that the DNA damaging agent sensitivity of rqhl mutant was complemented by the expression of rqh156 and rqh172. They induced mRNA expression in a dose-dependent manner HU, MMS and UV. The HU sensitivity of the rqhl was complemented by the expression of rqh156 and rqh172. The mRNA expression of rqh156 decreased on HU dose dependent but the mRNA expression of rqh172 did not decrease on HU dose dependent. The MMS and W sensitivity of the rqhl was complemented by the expression of rqh156 and rqh172. These results indicate that the isolated rqhl gene may play an important role in DNA metabolism.

• Horticultural Science & Technology
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• v.32 no.1
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• pp.91-99
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• 2014

The objective of this study is to identify cold-tolerance genes in Brassica rapa. In order to acheive this goal, we analyzed a KBGP-24K oligo chip data [BrEMD (B. rapa EST and Microarray Database)] using B. rapa ssp. pekinensis inbred line 'Chiifu' under cold stress condition ($4^{\circ}C$). Among 23,929 unigenes of B. rapa, 417 genes (1.7%) were primarily identified as cold responsive genes that were expressed over 5-fold higher than those of wild type control, and then a gene which has unknown function and has full length sequence was selected. It was named BrCSR (B. rapa Cold Stress Resistance). BrCSR was transformed using expression vector pSL101 to confirm whether BrCSR can enhance cold tolerance in tobacco plants. $T_1$ transgenic tobacco plants expressing BrCSR were selected by PCR and Southern hybridization analyses, and the function of BrCSR was characterized by expression level analysis and phenotype observation under cold stress condition. The expression level of BrCSR in transgenic tobacco plants increased up to about two folds in quantitative real-time RT-PCR assay and this was very similar to Northern blot hybridization analysis. Analysis of phenotypic characteristics clearly elucidated that transgenic tobaccos expressing BrCSR were more cold tolerant than wild type control under $4^{\circ}C$ treatment. Based on these results, we conclude that the over-expression of BrCSR might be closely related to the enhancement of cold tolerance.

• Reproductive and Developmental Biology
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• v.32 no.1
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• pp.21-25
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• 2008

Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal and differentiation into a variety of cell types. They represent an attractive source of cells for gene and cell therapy. The purpose of this study is to direct the specific expression of the DsRed reporter gene in $Sca-1^+$ BMMSCs differentiated into a cardiomyogenic lineage. We constructed the prMLC-2v-DsRed vector expressing DsRed under the control of the 309 tp fragment of the rat MLC-2v 5'-flanking region. The specific expression of the DsRed reporter gene under the transcriptional control of the 309 bp fragment of the rat MLC-2v promoter was tested in 5-azacytidine healed-$Sca-1^+$ BMMSCs over 2 weeks after the prMLC-2v-DsRed transfection. The prMLC-2v-DsRed was specifically expressed in the $Sca-1^+$ BMMSCs with cardiomyogenic lineage differentiation and it demonstrates that the 309 bp sequences of the rat MLC-2v 5'-flanking region is sufficient to confer cardiac specific expression on a DsRed reporter gene. The cardiac-specific promoter-driven reporter vector provides an important tool for the study of stem cell differentiation and cell replacement therapy in ischemic cardiomyopathy.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.3
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• pp.1077-1082
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• 2015

Recent studies have suggested that the RAS protein activator like-1 (RASAL1) functions as a tumor suppressor in vitro and may play an important role in the development of gastric cancer. However, whether or not RASAL1 suppresses tumor growth in vivo remains to be determined. In the present study, we investigated the role of RASAL1 in gastric carcinogenesis using an in vivo xenograft model. A lentiviral RASAL1 expression vector was constructed and utilized to transfect the human poorly differentiated gastric adenocarcinoma cell line, BGC-823. RASAL1 expression levels were verified by quantitative real-time RT-PCR and Western blotting analysis. Then, we established the nude mice xenograft model using BGC-823 cells either over-expressing RASAL1 or normal. After three weeks, the results showed that the over-expression of RASAL1 led to a significant reduction in both tumor volume and weight compared with the other two control groups. Furthermore, in xenograft tissues the increased expression of RASAL1 in BGC-823 cells caused decreased expression of p-ERK1/2, a downstream moleculein the RAS/RAF/MEK/ERK signal pathway. These findings demonstrated that the over-expression of RASAL1 could inhibit the growth of gastric cancer by inactivation of the RAS/RAF/MEK/ERK pathway in vivo. This study indicates that RASAL1 may attenuate gastric carcinogenesis.

• Horticultural Science & Technology
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• v.32 no.6
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• pp.845-852
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• 2014

Among various abiotic stress factors, soil salinity decreases the photosynthetic rate, growth, and yield of plants. Recently, many genes have been reported to enhance salt tolerance. The objective of this study was to characterize the Brassica rapa Salt Stress Resistance (BrSSR) gene, of which the function was unclear, although the full-length sequence was known. To characterize the role of BrSSR, a B. rapa Chinese cabbage inbred line ('CT001') was transformed with pSL94 vector containing the full length BrSSR cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the expression of BrSSR in the transgenic line was 2.59-fold higher than that in the wild type. Analysis of phenotypic characteristics showed that plants overexpressing BrSSR were resistant to salinity stress and showed normal growth. Microarray analysis of BrSSR over-expressing plants confirmed that BrSSR was strongly associated with ERD15 (AT2G41430), a gene encoding a protein containing a PAM2 motif (AT4G14270), and GABA-T (AT3G22200), all of which have been associated with salt tolerance, in the co-expression network of genes related to salt stress. The results of this study indicate that BrSSR plays an important role in plant growth and tolerance to salinity.

• Microbiology and Biotechnology Letters
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• v.36 no.4
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• pp.260-267
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• 2008

The choice of expression vector is very important for industrial production of proteins. Therefore, the systematic mining of promoters over a wider range of genetic resource and/or host is required. We previously reported a novel bidirectional reporting system (pBGR) for the isolation of promoters from metagenome and screened useful promoters that functioned constitutively in E. coli under general culture conditions. Among them, three promoter sequences including each upstream region were amplified by PCR and used to construct new expression vectors. To facilitate subcloning, a multi-cloning site was incorporated into the downstream region of the revere primer sequence. At these sites, GFP, esterase and $\beta$-glucosidase were subcloned and analyzed the constitutive expression ability of new promoter in terms of protein solubility and expression level. As a result, these vectors expressed the proteins constitutively to a level of $2{\sim}3%$ of the total cell protein in soluble fraction (>80 %). This study suggested that excavation of metagenomic promoters for construction of expression vector in a certain strain could provide a way for the development of the expression systems.

• Fisheries and Aquatic Sciences
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• v.6 no.1
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• pp.20-26
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• 2003

A bacterial strain was isolated from seawater to screen for phytase activities. A colony had the highest activity and was identified as an Escherichia coli strain. Using primers derived from E. coli acid phosphatase appA sequence, we cloned a 1,495 bp DNA fragment connected with the pGEM-T vector. It was over-expressed under lac promoter combined with its native promoter in E. coli $DH5\alpha$. The expression of the phytase gene occurred during late exponential growth and the intracellular phytase production was 16.9 units/ml. The yield of recombinant phytate was 412-fold higher than that of wild type E. coli K13.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.789-795
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• 2004

A promoter-probe shuttle vector pSK1Cat was constructed for the isolation of transcriptional signal sequences from Corynebacterium glutamicum. Besides conferring resistance to kanamycin in Escherichia coli and C. glutamicum, the vector carried a promoterless cat gene to confer resistance to chloramphenicol upon insertion of the appropriate transcriptional signals in the multiple cloning site. By utilizing the vector, a series of transcriptionally active fragments were isolated from the genome of C. glutamicum. The clones, ranging from 200 bp to 1 kb in size, were grouped into 3 classes of strong, medium, and weak, based on the chloramphenicol acetyltransferase (CAT) activity and sensitivity to the chloramphenicol of the clone-carrying C. glutamicum cells. C. glutamicum cells carrying the $P_{19}$ clone, a representative in the strong class, were able to grow on minimal agar plates containing over $40 mg/mell$ chloramphenicol, and showed CAT activity of 10 m㏖/mgㆍmin, performing slightly better than the cells carrying $P_{tac}$ , a strong E. coli promoter. Subcloning analysis of the $P_{19}$ clone identified a 180 bp intergenic fragment ($P_{180}$), which was located upstream of a gene encoding a hypothetical membrane protein. The expression conferred by $P_{180}$ was not affected by either the kinds of carbon sources or changes in temperature. These properties make the $P_{180}$ clone useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation.