• Asian-Australasian Journal of Animal Sciences
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• v.15 no.3
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• pp.421-426
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• 2002

The pyruvate dehydrogenase complex (PDC), a member of $\alpha$-keto acid dehydrogenase complex, catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and $H^+$. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, we have partially cloned the gene for E3BP in human. Nine putative clones were isolated by human genomic library screening with 1.35 kb fragment of E3BP cDNA as a probe. For investigation of cloned genes, Southern blot analysis and the construction of the restriction map were performed. One of the isolated clones, E3BP741, has a 3 kb-SacI fragment, which contains 200 bp region matched with E3BP cDNA sequences. The matched DNA sequence encodes the carboxyl-terminal portion of lipoyl-bearing domain and hinge region of human E3BP. Differences between yeast E3BP and mammalian E3BP coupled with the remarkable similarity between mammalian E2 and mammalian E3BP were confirmed from the comparison of the nucleotide sequence and the deduced amino acid sequence in the cloned E3BP. Cloning of human E3BP gene and analysis of the gene structure will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

• Proceedings of the Korean Society of Crop Science Conference
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• 1999.05a
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• pp.162-163
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• 1999

The goal of this research was (1) to describe the conditions and parameters required for the cell cycle synchronization and the accumulation of large number of metaphase cells in maize and other cereal root tips, (2) to isolate intact metaphase chromosomes from root tips suitable for characterization by flow cytometry, and (3) to construct chromosome-specific libraries from maize. Plant metaphase chromosomes have been successfully synchronized and isolated from many cereal root-tips. DNA synthesis inhibitor (hydroxyurea) was used to synchronize cell cycle, follwed by treatement with trifluralin to accumulate metaphase chromosomes. Maize flow karyotypes show substantial variation among inbred lines. thish variation should be sueful in isolating individual chromosome types. In addition, flow cytometry is a useful method to measure DNA content of individual chromosomes in a genotyps, and to detect chromosomal variations. Individual chromosome peaks have been sorted from the maize hybrid B73/Mol7. Libraries were generated form the DOP-PCR amplification product from each peak. To date, we have analyzed clones from a library constructed from the maize chromosome 1 peak. Hybridization of labeled genomic DNA to clone inserts indicated that $24\%,\;18\%,\;and\;58\%$ of the clones were highly repetitive, medium repetitive, and low copy, respectively. Fifty percent of putative low cpoy clones showed single bands on inbred screening, blots, and the remaining $50\%$ were low copy repeats. Single copy clones showing polymorphism will be mapped using recombinant inbred mapping populations. Repetitive clones are being characterized by Southern blot analysis, and will be screened by in situ hybridization for their potential utility as chromosome specific markers.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.65.2-65
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• 2003

To protect the plant against several soil-borne pathogens, we are currently constructing disease-resistant transgenic root stock for the growth of cucurbitaceae vegetable plants, watermelon and gourd. We made a watermelon cDNA library from Cladosporium cucumerinum-Infected leaves for substractive hybriazation and differential screening. We isolated the several pathogen inducible cDNA clones, such as caffeoyl-CoA-methyltransferase, LAA induced protein, receptor-like kinase homolog, hydroxyproline-rich glycoprotein, catalase, calmodulin binding protein, mitochondrial ATPase beta subunit, methyl tRNA synthetase and WRKY transcription factors. We previously obtained CaMADS in pepper and galactinol synthase ( CsGolS) in cucumber that were confirmed to be related with disease-resistance. CaMADS and CsGolS2 were transformed into the inbred line 'GO701-2' gourd, the inbred line '6-2-2' watermelon and the Kong-dye watermelon by Agrobacterium tumerfaciens LBA4404. Plant growth regulators (zeatin, BAP and IAA) were used for shoot regeneration and root induction for optimal condition. Putative transgenic plants were selected in medium containing 100mg/L kanamycin and integration of the CaMADS and CsGO/S2 into the genomic DNA were demonstrated by the PCR analysis. We isolated major soil-borne pathogens, such as Monosporascus cannonballus, Didymella bryoniae, Cladosporium cuvumerinum from the cultivation area of watermelon or root stock, and successfully established artificial inoculation method for each pathogen. This work was supported by a grant from BioGreen 21 program, Rural Development Administration, Republic of Korea.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1197-1203
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• 2007

A genomic DNA library of the bacterium Paenibacillus sp. DG-22 was constructed and the ${\beta}-xylosi-dase-positive$ clones were identified using the fluorogenic substrate $4-methylumbelliferyl-{\beta}-D-xylopyr-anoside$ $({\beta}MUX)$. A recombinant plasmid was isolated from the clone and 4.3-kb inserted DNA was sequenced. The ${\beta}-xylosidase$ gene (xylA) was comprised of a 2,106 bp open reading frame (ORF) en-coding 701 amino acids with a molecular weight of 78,710 dalton and a pI of 5.0. The deduced amino acid sequence of the xylA gene product had significant similarity with ${\beta}-xylosidases$ classified into family 52 of glycosyl hydrolases. The xylA gene was subcloned into the pQE60 expression vector to fuse with six histidine-tag. The recombinant ${\beta}-xylosidase$ $(XylA-H_6)$ was purified to homogeneity by heat-treatment and immobilized metal affinity chromatography. The pH and temperature optima of the $XylA-H_6$ enzyme were pH 5.5-6.0 and $60^{\circ}C$, respectively.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.844-854
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• 2005

We constructed a defined physical and genetic map of H. pylori strain 51, previously isolated from a Korean patient with a duodenal ulcer, by combining a restriction analysis by pulse-field gel electrophoresis with the construction of a BAC library. A Notl-digest of H. pylori strain 51 genome yielded seven fragments, from which the genomic size was estimated to be 1,698$\pm$24 kb. The BAC library was constructed from 50 to 200 kb fragments of HindIII-digested genomic DNA. From 700 BAC clones, an ordered overlapping maxi-set of 82 BAC clones was assembled that covered the entire genome. The positions of 15 genes were localized in the strain 51 genome with 4-22 kb of resolution and were compared with their orthologues in strain 26695 and strain J99. The arrangement of the 15 genes was identical in strain 51 and strain J99, except for flaA and hpaA. The plasticity zone of strain 51, like that of strain J99, was located in the single region, and was shorter than those of strain 26695 and strain J99. The strain 51 plasticity zone consisted of ORFs common only to strain 51 and J99 or to strain 51 and 26695, as well as strain 51-specific ORFs. Three genetic translocations and/or inversions were found between orthologue ORFs in strain 51 and strain J99. These results show that the chromosomal organization of strain 51 differs from Western strains such as strain 26695 and strain J99.

• Applied Biological Chemistry
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• v.38 no.4
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• pp.313-319
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• 1995

The involvement of the cell-wall degrading enzymes in Rhizobium has long been an unsolved question about the infection process in the formation of root nodule. To assess the contribution of the cellulase to the nodulation of rhzobia, here we report the production of cellulase from R. meliloti TAL1372 which degrade carboxymethylcellulose (CMC) model substrate with CMC-plate method. We constructed a genomic library by cloning Sau3A-digested genomic DNA from R. meliloti TAL1372 into the BamHI site of the cosmid vector pLAFR3. Out of more than one thousand transductants of E. coli, one clone (pRC8-71) had CM-cellulase activity and contained pLAFR3 cosmid with 30 kb insert of R. meliloti DNA The product of CM-cellulase gene was analyzed by native PAGE. About 45 kD protein was considered to be a product of the gene. Tn5 mutagenesis reveals that the structural gene located in a ca. 3 kb KpnI fragment. The cellulase-minus mutants of R. meliloti TAL1372 were obtained by Tn5 mutagenesis of pRC8-71 and marker exchange techniques. Analyses of the nodulation ability of these Tn5 mutants showed that the CM-cellulase gene of R. meliloti TAL1372 may be involved in early nodulation development on alfalfa (Medicago satiua).

• Korean Journal of Medicinal Crop Science
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• v.22 no.6
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• pp.429-434
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• 2014

This study describes the efficient method for the discrimination of 'Cheonryang' in Panax ginseng Meyer using a STS primer. A total of 208 STS primers were applied to polymerase chain reaction (PCR) amplification for discriminating Korean ginseng cultivars. Co-dominant polymorphic band patterns were generated with two primers, MFGp 0019, MFGp 0248, and successful identification of 'Cheonryang' was achieved from out of 11 Korean ginseng cultivars. Two different sizes of DNA band patterns were detected with MFGp 0019 primer. Ten Korean ginseng cultivars shared the same size of amplified DNAs (389 bp), but 'Cheonryang' showed a different size. Thus 'Cheonryang' can be efficiently distinguished from the other ten ginseng cultivars by using the MFGp 0019 primer. In the case of MFGp 0248, two different sizes of DNA band patterns were detected in the eleven ginseng cultivars. Same sized amplified DNA bands (307 bp) were shown in five cultivars (Chunpoong, Gopoong, Kumpoong, Cheongsun, Sunhyang) and 254 bp sized DNA bands were identified in the other 6 cultivars (Yunpoong, Sunpoong, Sunun, Sunone, Cheonryang, K-1). In conclusion, the two STS primers, MFGp 0019, and MFGp 0248, provide a rapid and reliable method for the specific identification of 'Cheonryang' cultivar from a large number of samples.

• Journal of Life Science
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• v.20 no.3
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• pp.411-415
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• 2010

Anisakis simplex is one of the parasitic nematodes, and has a complex life cycle in crustaceans, fish, squid or whale. When people eat under-processed or raw fish, it causes anisakidosis and also plays a critical role in inducing serious allergic reactions in humans. However, no web-based database on A. simplex at the level of DNA or protein has been so far reported. In this context, we constructed a web-based database for Anisakis research. To build up the web-based database for Anisakis research, we proceeded with the following measures: First, sequences of order Ascaridida were downloaded and translated into the multifasta format which was stored as database for stand-alone BLAST. Second, all of the nucleotide and EST sequences were clustered and assembled. And EST sequences were translated into amino acid sequences for Nuclear Localization Signal prediction. In addition, we added the vector, E. coli, and repeat sequences into the database to confirm a potential contamination. The web-based database gave us several advantages. Only data that agrees with the nucleotide sequences directly related with the order Ascaridida can be found and retrieved when searching BLAST. It is also very convenient to confirm contamination when making the cDNA or genomic library from Anisakis. Furthermore, BLAST results on the Anisakis sequence information can be quickly accessed. Taken together, the Web-based database on A. simplex will be valuable in developing species specific PCR markers and in studying SNP in A. simplex-related researches in the future.

• Journal of Microbiology
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• v.39 no.1
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• pp.37-41
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• 2001

The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

• Journal of Microbiology
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• v.35 no.4
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• pp.309-314
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• 1997

The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on oxidative stress=responsible enzymes in the Candida sp., the gene encoding the MnSOD was isolated and examined in this study. A specific primer was designed based on conserved regions of MnSOD sequences from other organisms, and was used to isolate the gene by PCR on reverse-transcribed Candida poly($A^{+}$) RNA. The PCR product was used to screen a Candida genomic lambda library and the nucleotide wequence of positive clone was determined. The deduced primary sequence encodes a 25kDa protein which has the conserved residues for enzyme activity and metal binding. The 28 N-terminal amino acids encoded by the Candida cDNA comprise a putatice mitochondrial transit peptide. Potential regulatory elements were identified in the 5' flanking sequences. Northern blot analysis showed that the transcription of the MnSOD gene is induced 5-to 10-fold in response to mercury, cadmium ions and hydrogen peroxide.