• Proceedings of the Botanical Society of Korea Conference
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• 1996.07a
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• pp.61-74
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• 1996

Human and monogastric animals can not synthesize 10 out of the 20 amino asids and therefor need to obtain these from their diet. The plant seed is a major source of dietary protein. It is particular important in their study to increase nutritional quality of the seed storage proteins. The low contents of lysine, asparagine and threonenein various cereal seeds and of cystein and methionine. In legume seeds is due to the low proportions of these amino acids in the major storage proteins, we have tried to apply the three strategies; (1) mutagenesis and selection of specific amino acid analogue resistance, (2) cloning and expression study of lysine biosynthesis related gene, (3) transfomation of lysine rich soybean glycinin gene. The 5-methyltryptophan (5MT) resistant cell lines, SAR1, SAR2 and SAR3 were selected from anther derived callus of rice (Oryza sativa L. "Sasanishiki"). Among these selected cell lines, two (SAR1 and SAR3) were able to grow stably at 200 mg/L of 5MT. Analysis of the freed amino acids in callus shows that 5MT resistant cells (SAR3) accumulated free tryptophan at least up to 50 times higher than those that of the higher than of SAS. These results indicated that the 5MT resistant cell lines are useful in studies of amino acid biosynthesis. Tr75, a rice (Oryza sativa L., var. Sasanishiki) mutant resistant to 5MT was segregated from the progenies of its initial mutant line, TR1. The 5MT resistant of TR75 was inherited in the M8 generations as a single dominant nuclear gene. The content of free amino acids in the TR75 homozygous seeds increased approximately 1.5 to 2.0 fold compared to wild-type seeds. Especially, the contents of tryptophan, phenylalanine and aspartic acid were 5.0, 5.3 and 2.7 times higher than those of wild-type seeds, respectively. The content of lysine is significantly low in rice. The lysine is synthesized by a complex pathway that is predominantly regulated by feedback inhibition of several enzymes including asparginase, aspatate kinase, dihydrodipicolinat synthase, etc. For understanding the regulation mechanism of lysine synthesis in rice, we try to clone the lysine biosynthetic metabolism related gene, DHPS and asparaginase, from rice. We have isolated a rice DHPS genomic clone which contains an ORF of 1044 nucleotides (347 amino acids, Mr. 38, 381 daltons), an intron of 587 nucleotides and 5'and 3'-flanking regions by screening of rice genomic DNA library. Deduced amino acid sequence of mature peptide domain of GDHPS clone is highly conserved in monocot and dicot plants whereas that of transit peptide domain is extremely different depending on plant specie. Southern blot analysis indicated that GDHPS is located two copy gene in rice genome. The transcripts of a rice GDHPS were expressed in leaves and roots but not detected in callus tissues. The transcription level of GDHPS is much higher in leaves indicating enormous chloroplast development than roots. Genomic DNA clones for asparaginase genes were screened from the rice genomic library by using plaque hybridization technique. Twelve different genomic clones were isolated from first and second screening, and 8 of 12 clones were analyzed by restriction patterns and identified by Southern Blotting, Restriction enzyme digestion patterns and Southern blot analysis of 8 clones show the different pattern for asparaginase gene. Genomic Southern blot analysis from rice were done. It is estimated that rice has at least 2-3 copy of asparaginase gene. One of 8 positive clones was subcloned into the pBluescript SK(+) vector, and was constructed the physical map. For transformation of lysine rich storage protein into tobacco, soybean glycinin genes are transformed into tobacco. To examine whether glycinin could be stably accumulated in endosperm tissue, the glycinin cDNA was transcriptionally fused to an endosperm-specific promotor of the rice storage protein glutelin gene and then introduced into tobacco genomic via Agrobacterium-mediated transformation. Consequently the glycinin gene was expressed in a seed-and developmentally-specific manner in transgenic tobacco seeds. Glycinin were targeted to vacuole-derived protein bodies in the endosperm tissue and highly accumulated in the matrix region of many transgenic plant (1-4% of total seed proteins). Synthesized glycinin was processed into mature form, and assembled into a hexamer in a similar manner as the glycinin in soybean seed. Modified glycinin, in which 4 contiguous methionine residues were inserted at the variable regions corresponding to the C - teminal regions of the acidic and basic polypeptides, were also found to be accumulated similarly as in the normal glycinin. There was no apparent difference in the expression level, processing and targeting to protein bodies, or accumulation level between normal and modified glycinin. glycinin.

• Animal cells and systems
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• v.3 no.2
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• pp.229-236
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• 1999

The recA gene plays a central role in genetic recombination and SOS DNA repair in Escherichia coli (E. coli). We have previously identified a 42 kDa RecA-like protein inducible by a variety of DNA damages from a fluorescent Pseudomonas strain sp. and characterized its inducible kinetics. In the present study, we cloned and characterized the gene encoding the RecA-like protein by immunological screening of Pseudomonas genomic expression library using polyclonal E. coli anti-RecA antibodies as a probe. From 10$^{5}$ plaques screened, five putative clones were finally isolated. Southern blot analysis indicated that four clones had the same DNA inserts and the recA-like gene was located within the 3.2 kb EcoRI fragment of Pseudomonas chromosomal DNA. In addition, the cloned recA-like gene was transcribed into an RNA transcript approximately 1.1 kb in size, as judged by Northern blot analysis. The cellular level of RNA transcript of the cloned recA-like gene was increased to an average of 5.15- fold upon treatment with DNA damaging agents such as ultraviolet (UV)- light, nalidixic acid (NA), methyl methanesulfonate (MMS), and mitomycin-C (MMC). These results suggest that the cloned gene is inducible by DNA damage similarly to the recA gene in E. coli. However, the cloned gene did not restore the DNA damage sensitivity of the E. coli recA-mutant.

• Journal of Animal Science and Technology
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• v.45 no.2
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• pp.169-182
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• 2003

To understand the structure and regulation of bovine ADRP (Adipocyte Differentiation Related Protein) gene, we have isolated the genomic clone of bovine ADRP and determined its sequence. A genomic Southern blot analysis confirmed that ADRP gene is present as a single copy in bovine genome and the ADRP gene spans 12 kb. Bovine ADRP genomic clone, HwADRPg-1, had 8 exons and 7 introns, and all splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly. Analysis of the upstream 649 bp of the sequence of HwADRPg-1 showed that it does not contain any canonical TATAA boxes; however Sp1 binding sites and CAAT boxes are found. The promoter contained potential binding sites for AP-1, AP-2 and several putative transcription factor binding sites. The 5'-flanking region of HwADRPg-1 contained muscle specific transcription activator Myo G and C/EBP (CCAAT/ enhancer binding protein) recognizing site. These results suppose that the Myo G transcription activator regulate the transcription of bovine ADRP gene in muscular tissue and its transcriptional activity was triggered by degree of muscular development. Our results provide the necessary analysis for other flanking sequences are needed in addition to the proximal cis elements of this promoter to confer adipocyte differentiation-dependent or growth-dependent transcriptional control.

• The Korean Journal of Mycology
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• v.25 no.1 s.80
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• pp.1-5
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• 1997

The cDNA encoding ${\beta}-tubulin$ of Pleurotus sajor-caju was isolated using an internal gene segment probe amplified by polymerase chain reaction (PCR) of genomic DNA and by cDNA library screening. The cDNA was consisted of 1560 nucleotides(nt), including a 5'-untranslation region (UTR) of 27nt, an open reading frame (ORF) of 1341nt, and a 3'-UTR of 191nt. The ORF encoded a protein of 446 amino acids(aa), which shows over 80% homology with ${\beta}-tubulins$ of other filamentous fungi. Southern hybridization analysis showed that there were two isotypes of ${\beta}-tubulin$ genes in P. sajor-caju. Through sequence analysis we found that ${\beta}-tubulin$ had a unusual $Cys^{165}$ residue, which might be a significant factor for the insensitivity of fungi to fungicide benomyl.

• The Plant Pathology Journal
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• v.30 no.4
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• pp.375-383
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• 2014

Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC) transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.). Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1) gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5'-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.77-80
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• 2001

A variety of different methods to generate diverse proteins, including random mutagenesis and recombination, are currently available, and most of them accumulate the mutations on the target gene of a protein, whose sequence space remains unchanged. On the other hand, a pool of diverse genes, which is generated by random insertions, deletions, and exchange of the homologous domains with different lengths in the target gene, would present the protein lineages resulting in new fitness landscapes. Here we report a method to generate a pool of protein variants with different sequence spaces by employing green fluorescent protein (GFP) as a model protein. This process, designated functional salvage screen (FSS), comprises the following procedures： a defective GFP template expressing no fluorescence is firstly constructed by genetically disrupting a predetermined region(s) of the protein, and a library of GFP variants is generated from the defective template by incorporating the randomly fragmented genomic DNA from E. coli into the defined region(s) of the target gene, followed by screening of the functionally salvaged, fluorescence-emitting GFPs. Two approaches, sequence-directed and PCR-coupled methods, were attempted to generate the library of GFP variants with new sequences derived from the genomic segments of E. coli. The functionally salvaged GFPs were selected and analyzed in terms of the sequence space and functional property. The results demonstrate that the functional salvage process not only can be a simple and effective method to create protein lineages with new sequence spaces, but also can be useful in elucidating the involvement of a specific region(s) or domain(s) in the structure and function of protein.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.462-469
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• 2012

A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from $50^{\circ}C$ to $55^{\circ}C$. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.

• 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.

• The Journal of Natural Sciences
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• v.15 no.1
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• pp.79-88
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• 2005

There are five isoforms of thiol peroxidase in yeast. Each isoform was named after its subcellular localization such as cytoplasmic TPx I, cTPx II, cTPx III, mitochondrial TPx (mTPx), and nuclear TPx (nTPx). Recently, we reported that unlike other TPx null mutants, cTPx IInull mutant showed a slow-growth phenotype. This observation suggests that cTPx II might be involved in yeast cell growth. In this study, for a first step toward to investigate the physiological function of cTPx II in yeast, we have identified a novel interaction between cTPx II and various proteins by using the yeast two-hybrid system.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.346-353
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• 2005

A 50-kb chromosome DNA region was isolated from Streptomyces kanamyceticus by screening the fosmid genomic library, using the 16S rRNA methylase gene (kmr) as a probe. Sequence analysis of this region revealed 42 putative open reading frames (ORFs), which included biosynthetic genes such as genes responsible for 2-deoxystreptamine (2­DOS) biosynthesis as well as genes for resistance and regulatory function. Also, the kanamycin acetyltransferase gene (kac) was characterized by in vitro enzyme assay, which conferred E. coli BL21 (DE3) with 10, 50, and 80-times higher resistance to kanamycin A, tobramycin, and amikacin, respectively, than the control strain had, thus strongly indicating that the isolated gene cluster is very likely involved in kanamycin biosynthesis. This work provides a solid basis for further elucidation of the kanamycin biosynthesis pathway as well as the productivity improvement and construction of new hybrid antibiotics.