• Journal of Microbiology and Biotechnology
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• v.2 no.4
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• pp.237-242
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• 1992

In order to clone the gene coding for alkaline phosphatase in the yeast Kluyveromyces fragilis, a genomic library was constructed using the yeast-E. coli shuttle vector pHN114 as a cloning vector. From the genomic library, a clone carrying the gene was isolated and the plasmid was designated as pSKH101. A restriction enzyme map was made using this plasmid. Subcloning experiments and complementation studies showed that alkaline phosphatase was active only in the original 3.1 kb insert. Southern hybridization analysis confirmed that the cloned DNA fragment was derived from K. fragilis genomic DNA. Using a minicell experiment, the product of the cloned gene was identified as a protein with a molecular weight of 63 KDa. A 0.6 kb HindIII fragment, which showed promoter activity, was isolated using the E. coli promoter-probe vector pKO-1.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.568-574
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• 1991

The cloned glyceraldehyde-3-phosphate dehydrogenase (GAP) gene of Saccharomyces cereviszae (Holland et al., 1983) has been characterized. Based on the communication, we have also cloned 2.1 kb CAP DNA fragment and modified this fragment as a portable promoter. Two yeast expression vectors, one is YCp type vector being maintained at low copy number (1 or 2) and the other is YEp type vector at high copy number, have been constructed with the GAP promoter and the PH05' gene as a reporter. Our plasrnids were introduc,ed into S. cerevisiae HY-1, which has been improved. The $Trp^+$ transformants expressed APase activity efficiently and showed high level of PH05' transcripts.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.217-228
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• 2009

Mobile genetic segments, or transposons, are also referred to as jumping genes as they can shift from one position in the genome to another, thus inducing a chromosomal mutation. According to the target site-specificity of the transposon during a transposition event, the result is either the insertion of a gene of interest at a specific chromosomal site, or the creation of knockout mutants. The former situation includes the integration of conjugative transposons via site-specific recombination, several transposons preferring a target site of a conserved AT-rich sequence, and Tn7 being site-specifically inserted at attTn7, the downstream of the essential glmS gene. The latter situation is exploited for random mutagenesis in many prokaryotes, including IS (insertion sequence) elements, mariner, Mu, Tn3 derivatives (Tn4430 and Tn917), Tn5, modified Tn7, Tn10, Tn552, and Ty1, enabling a variety of genetic manipulations. Randomly inserted transposons have been previously employed for a variety of applications such as genetic footprinting, gene transcriptional and translational fusion, signature-tagged mutagenesis (STM), DNA or cDNA sequencing, transposon site hybridization (TraSH), and scanning linker mutagenesis (SLM). Therefore, transposon-mediated genetic engineering is a valuable discipline for the study of bacterial physiology and pathogenesis in living hosts.

• Mycobiology
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• v.38 no.4
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• pp.331-335
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• 2010

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/${\mu}g$ of DNA in $1{\times}10^7$ protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.505-509
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• 2005

For the purpose of developing a direct label-free electrochemical detection system, we have systematically investigated the electrochemical signatures of each step in the preparation procedure, from a bare gold electrode to the hybridization of label-free complementary DNA, for the streptavidin-modified electrode. For the purpose of this investigation, we obtained the following pertinent data; cyclic voltammogram measurements, electrochemical impedance spectra and square wave voltammogram measurements, in $Fe(CN)_6^{3-}/Fe(CN)_6^{4-}$ solution (which was utilized as the electron transfer redox mediator). The oligonucleotide molecules on the streptavidin-modified electrodes exhibited intrinsic redox activity in the ferrocyanide-mediated electrochemical measurements. Furthermore, the investigation of electrochemical electron transfer, according to the sequence of oligonucleotide molecules, was also undertaken. This work demonstrates that direct label-free oligonucleotide electrical recognition, based on biofunctional streptavidin-modified gold electrodes, could lead to the development of a new biosensor protocol for the expansion of rapid, cost-effective detection systems.

• Plant Disease and Agriculture
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• v.5 no.1
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• pp.1-7
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• 1999

The grain rot of caused by Bukholderia glumae was fist reported in japan in 1955 and then reported in other countries as well as in Korea in 1986. The pathogen causes both seedling and grain rot of rice but it cannot attack any other parts of adult rice plant. Bacterial colonies grow slowly, and are circular and greyish white. The causal bacterium is Gram-negative and rod shape with 1-3 polar flagella, and produce a diffusible yellow-greenish nonfluorescent pigment on King's medium B. Biochemical characteristics such as negative in arginine dehydrolase, oxidase reaction and nitrate reduction and positive in lecithinase, and the utilization of L-arginine and inositol are useful in differentiation of this from other nonfluorescent bacteria pathogenic to rice. This pathogenic bacterium had belonged to the genus of Pseudomonas but recently was transferred to the new genus Burkholderia on the basis of physiological characteristics and DNA-DNA hybridization data. However, other characteristics such as colony heterogenicity or colonial variation after subcultures, phytotoxin, secreting antibiotics, and relationship between yellow greenish pigment production and pathogenicity need to be clarified more. To develop an effective control strategy for this disease, understanding of detailed life cycle of the disease and critical environmental factors affecting disease development is prerequisite. Although 5,435 ha of rice paddy in Korea was infested during 1998, there is no exact estimation of yield losses and distribution of the pathogen. The review will focus on recent progress on the understanding of the bacteriological and ecological characteristics of the causal bacterium and control means of the disease.

• Microbiology and Biotechnology Letters
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• v.13 no.3
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• pp.251-255
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• 1985

Nitrogen-fixing bacteria were isolated from the rice rhizosphere of various paddy fields in our country. The screening of 235 isolates for nitrogen-fixing ability resulted in the isolation of Enterobacter agglomerans NFB264 and three Klebsiella pneumoniae NFB 3, NFB 320. Plasmids of various molecular weight from 1.7 to more than 84 Mal. were detected by agarose gel electrophoresis in three out of four isolates. But, these plasmids had not any nitrogen-fixing genes. Hybridization experiments using Klebsiella pneumoniae M5al nitrogen-fixing genes, nif Q-K and nif DH, as probes revealed the presence of homologous sequences in the chromosomal DNA of all isolates. However the restriction patterns of nif genes of the isolates by various restriction endonucleases were different to those of Klebsiella pneumoniae M5al.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.18-24
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• 2001

To identify genes implicated in the control of pluripotency as well as characteristics of stem cells, we analyzed expression profiles of genes derived from mouse morulas, blastocysts, embryonic stem cells, mesenchymal stem cells, and uterus tissue using cDNA microarray. Comparative analyses of their expression profiles identified putative clones that expressed specifically in specific samples or not in a specific sample. The expression pattern of these candidate clones was analyzed using RT-PCR and non-radioactive in situ hybridization. Functional annotation of these clones on pluripotency and stem cell plasticity is in ongoing. These studies may further our understanding on the nature of the stem cells and molecular mechanisms underlying many facets of mammalian development and differentiation.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.386-390
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• 1996

The ${\beta}$-galactosidase gene from Lactococcus lactis subsp. lactis ATCC 7962 was cloned and its enzymatic properties were characterized, with a view to assessing its potential use as a selection marker in the food-grade cloning vector. Chromosomal DNA from L. lactis subsp. lactis 7962 was cleaved with PstI and ligated into pBR322 for transformation into Escherichia coli TGl. Transformants showing ${\beta}$-galactosidase activity possessed the pBR322 plasmid containing a 10 kilobase (kb) PstI fragment and this plasmid was named pCKL11. The cloned ${\beta}$-galactosidase gene came from the chromosomal DNA of L. lactis subsp. lactis 7962 was confirmed by Southern hybridization. A restriction map of pCKL11 was constructed from the cleavage of both pCKL11 and the purified 10kb insert fraqment. The. optimum pH of the ${\beta}$-galactosidase determined with the E. coli harboring the pCKL11 was 7.0. The optimum temperature was $50^{\circ}C$, while the pI of the enzyme was 7.4. These values were the same as those of the enzyme from the parent strain.

• Biomedical Science Letters
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• v.18 no.1
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• pp.1-9
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• 2012

Differentially expressed genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were identified in order to evaluate them as dioxin-sensitive markers and crucial signaling molecules to understand dioxin-induced toxic mechanisms in human bronchial cells. Gene expression profiling was analyzed by cDNA microarray and ten genes were selected for further study. They were cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1), S100 calcium binding protein A8 (calgranulin A), S100 calcium binding protein A9 (calgranulin B), aldehyde dehydrogenase 1 family, member A3 (ALDH6) and peroxiredoxin 5 (PRDX5) in up-regulated group. Among them, CYP1B1 was used as a hallmark for dioxin and sharply increased by TCDD exposure. Down-regulated genes were IK cytokine, interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), nuclease sensitive element binding protein 1 (NSEP1), protein tyrosine phosphatase type VI A, member 1 (PTP4A1), ras oncogene family 32 (RAB32). Although up-regulated 4 genes in microarray were coincided with northern hybridization, down-regulated 5 genes showed U-shaped expression pattern which is sharply decreased at lower doses and gradually increased at higher doses. These results introduce some of TCDD-responsive genes can be sensitive markers against TCDD exposure and used as signaling cues to understand toxicity initiated by TCDD inhalation in pulmonary tissues.