• Applied Biological Chemistry
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• v.38 no.6
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• pp.522-527
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• 1995

To develop an antiviral agent for the rice black-streaked dwarf virus (RBSDV), a hammerhead type ribozyme, which has a potential target site on the genome segment 3, was designed. Oligonucleotides for the ribozyme and its substrate were synthesized, annealed, and cloned into a plasmid pBluescript II KS(+). Ribozyme and substrate RNAs were then synthesized by in vitro transcription with $T_3$ RNA polymerase, obtaining RNAs in expected size, 193 and 182 nucleotides, respectively. The substrate RNA was efficiently cleaved into two fragments when incubated with the ribozyme at $55^{\circ}C$, while the cleavage was not detected at $37^{\circ}C$. In addition, the segment 3 RNA of RBSDV was also cleaved into two fragments by the same ribozyme at $55^{\circ}C$. Taken together, our results demonstrated that the hammerhead ribozyme has an in vitro endonucleolytic activity and may be used as an antiviral agent in transgenic plants.

• Korean Journal of Plant Tissue Culture
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• v.21 no.5
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• pp.315-318
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• 1994

To obtain transformed plants, we cocultured cotyledonary explants of Codonopsis lanceolata with Agrobacterium tumefaciens LBA4404, a disamed strain harboring a binary vector pBI121 carrying the CaMV35S promoter-$\beta$-glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker in MS liquid medium with 1mg/L BA. After 48 h of culture, explants were transferred onto MS solid medium with Img/L BA, 250mg/L carbenicillin, and 100mg/L kanamycin sulfate and cultured in the dark. Numerous adventitious buds formed on the cut edges of the explants after 2 weeks of culture. When subjected to GUS histochemical assay buds showed a positive response at a frequency of 15%. Explants formed adventitious shoot at a frequency of 56.7%, after 6 weeks of culture. Upon transfer onto the basal medium, most of the shoots were rooted and subsequently the regenerants were transplanted to potting soil. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants.

• Journal of Mushroom
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• v.2 no.3
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• pp.115-126
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• 2004

Somatic hybrids of inter-compatible and inter-incompatible strains were obtained by protoplast fusion. The fusion products between compatible strains, Pleurotus ostreatus and P. florida, formed heterokaryons, while fusants between incompatible strains such as P. cornucopiae + P. florida, P. ostreatus + Ganoderma applanatum, P. florida + Ganoderma lucidum, and P. ostreatus + Flammulina velutipes formed synkaryons that retained genes from both parents. The heterokaryons showed the same level of basidioma development. In contrast, the synkaryons showed unique characteristics including clamp connection formation at mitosis, either partner basidioma development, and abnormal segregation and recombination compared with inter-compatible strains. Synkaryons can be classified into homokaryoyic and heterokaryotic type. A comparison of somatic hybrids with compatible and incompatible strains was made using random amplified polymorphic DNA (RAPD) analysis. The heterokaryons between compatible species showed the same level of variability and contained both parental RAPD bands. In contrast, most of the synkaryons between incompatible species showed similarity to those of either parental bands and non-parental RAPD bands. Synkaryons can be classified into microgenome insertion type and macrogenome insertion type. A tetrapolar mating system was found among monospore isolates in somatic hybrids and wild type P. ostreatus. Homokaryons from each somatic hybrid combination were paired with tester homokaryons of the initial wild type of P. ostreatus. The changed mating types were identified in progenies. The pattern of mating type switching in somatic hybrids depends on compatibility of fusion partner. There are several factors related to the mechanism of clamp connection formation and fruiting body development of synkaryons. Of these,the major factor may be associated with self-fertility and mating type switching such as homokaryotic fruiting of wild type P. ostreatus. This review will discuss these aspects.

• Journal of Life Science
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• v.22 no.4
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• pp.456-465
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• 2012

Heterodimeric recombinant human bone morphogenetic proteins (rhBMPs) are powerful tools for bone tissue engineering. However, BMPs have several important limitations in their application to bone regeneration. BMPs have a short half-life and must be used in high concentrations, which may be cost-inefficient. To overcome these problems, we established a stable cell line that expressed the fusion protein comprised of recombinant human BMP2/7 heterodimer protein and PTD (rhBMP2/7-PTD). This stable cell line enabled high process yields by continuously expressing rhBMP2/7-PTD products at high levels throughout cultivation. This synthesized BMP7 was fused to a BMP2 protein with four glycine residues (to allow free bond rotation of the domains) and PTD. To demonstrate that the rhBMP2/7-PTD protein that was secreted from an rhBMP2/7-PTD-expressing stable cell line exhibited biological activity consistent with its role as an osteogenic differentiation induction growth factor, we evaluated BMP-induced ALP activity. Our results suggest that this cell line may be a powerful and efficient tool for applications such as bone tissue regeneration.

• Journal of The Korean Society of Grassland and Forage Science
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• v.26 no.2
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• pp.69-76
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• 2006

To optimize tissue culture responses for genetic transformation of Kentucky bluegrass, the effects of culture medium supplements on tissue culture responses were investigated with mature seeds of a cultivar 'Newport' as explant tissues. The optimal concentration of 2,4-D (2.4-dichloro phenoxy acetic acid) for the induction of embryogenic callus from mature seed was 3 mg/L. Plant regeneration frequency was 54% when embryogenic callus was cultured on the regeneration medium supplemented with 1 mg/L 2,4-D and 3 mg/L of BA (6-benzyladenine). Addition of 1 g/L of casein hydrolysate and 500 mg/L of L-proline improved frequencies of embryogenic callus induction and plant regeneration up to 60.8% and 58.3%, respectively. Regenerated plants were grown normally when shoots transplanted to the soil. A rapid and efficient plant regeneration system established in this study. We suggest that the results may be useful for molecular breeding of Kentucky bluegrass through genetic transformation.

• Journal of Plant Biotechnology
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• v.39 no.4
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• pp.288-294
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• 2012

In order to establish an efficient adventitious shoot regeneration conditions from leaf explants for Asian pear 'Whangkeumbae', the effect of concentration and kinds of plant growth regulator and carbon source was investigated. Leaf explants of cultures grown on Murashige and Skoog (MS) medium containing 8 g/L plant agar were used. When the medium contained 0.25 mg/L thidiazuron (TDZ) and 0.3 mg/L indolebutyric acid (IBA), the adventitious shoot regeneration rate (ASRR) was greater as 61.1% than others treated and higher TDZ concentrations (2.5 and 5 mg/L) treatment significantly reduced the ASRR. As the effect of IBA and indoleacetic acid (IAA) concentration on the ASRR, 0.5 mg/L TDZ plus different concentration of IAA exhibited relatively high ASRR and 0.5 mg/L TDZ plus 0.3 mg/L IAA showed the highest ASRR of 76.7%. Also the effect of sucrose and sorbitol as carbon source on regeneration was examined. The highest ASRR and the most shoots per explants averaged 94.4% and 3.49 by treatment of 30 mg/L sorbitol, respectably. Sorbitol is considered better carbon source than sucrose for shoot regeneration of 'Whangkeumbae' pear.

• KSBB Journal
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• v.10 no.5
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• pp.499-509
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• 1995

PU.1, a tissue-specific transcription activator, binds to a purine-rich sequence(5'-GAGGAA-3') called PU box. The PU.1 cDNA consists of an open reading frame of 816 nucleotides coding for 272 amino acids. The amino terminal end is highly acidic, while the carboxyl terminal end is highly basic. Transcriptional activation domain is located at the amino terminal end, while DNA binding domain is located at the carboxyl terminal end. Activation of PU.1 transcription factor is supposed to be accomplished by the phosphorylation of serine residue(s). There exist 22 serines in the PU.1. Five(the 41, 45, 132$.$133, and 148th) of the serines(plausible phosphorylation site by casein kinase II), are the primary targets of interest in elucidating the molecular mechanism(s) of the action of the PU.1 gene. In this study, PU.1 cDNA coding for the five serine residues(41th AGC, 45th AGC, 132$.$133th AGC$.$TCA, and 148th TCT), was mutated to alanine codon(41th GCC, 45th GCC, 132$.$133th GCC$.$GCA, and 1481h GCT), respectively, by Splicing-Overlapping-Extension(SOE) using Polymerase Chain Reaction(PCR). And each mutated cDNA fragments was ligated into pBluescript KS＋ digested with HindIII and Xba I, to generate mutant clones named pKKS41A, pRKS45A, pMKS132$.$133A, and pMKS148A. The clones will be informative to study the "Structure and Function" of the immu-nologically important gene, PU.1.

• Korean journal of applied entomology
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• v.61 no.1
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• pp.197-210
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• 2022

Entomopathogenic fungi can be used to control a variety of sucking and chewing insects, with little effect on beneficial insects and natural enemies. Approximately 170 entomopathogenic fungal insecticides have been registered and used worldwide, with the recent focus being on the mode of action and mechanism of insect-fungal interactions. During the initial period of research and development, the industrialization of entomopathogenic fungi focused on the selection of strains with high virulence. However, improvement in productivity, including securing resistance to environmental stressors, is a major issue that needs to be solved. Although conidia are the primary application propagules, efforts are being made to overcome the limitations of blastospores to improve the economic feasibility of the production procedure. Fungal transformation is also being conducted to enhance insecticidal activity, and molecular biology is being used to investigate functions of various genes. In the fungi-based pest management market, global companies are setting up cooperative platforms with specialized biological companies in the form of M&As or partnerships with the aim of implementing a tank-mix strategy by combining chemical pesticides and entomopathogenic fungi. In this regard, understanding insect ecology in the field helps in providing more effective fungal applications in pest management, which can be used complementary to chemicals. In the future, when fungal applications are combined with digital farming technology, above-ground applications to control leaf-dwelling pests will be more effective. Therefore, for practical industrialization, it is necessary to secure clear research data on intellectual property rights.

• Microbiology and Biotechnology Letters
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• v.38 no.1
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• pp.40-45
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• 2010

The gene encoding $\beta$-agarase (agaB) which hydrolyzes $\beta$-1,4 linkages of agarose from Zobellia galactanivorans was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal ($MF{\alpha}1$), in which the transcription of $MF{\alpha}1$-AgaB was under the control of AOX1 (alcohol oxidase 1, methanol inducible) promoter. The constructed plasmid pPIC-AgaB (9 kb) was integrated into HIS4 gene locus of Pichia pastoris genome. Successful integration was confirmed by performing colony PCR. The transformed cells showed red halos around its colonies in methanol agar plate by adding iodine solution, indicating the active expression of agaB in P.pastoris. By SDS-PAGE and zymographic analysis, the molecular weight of $\beta$-agarase was estimated to be a 53 kDa and about 15% N-linked glycosylation was occurred. The activity of extracellular $\beta$-agarase reached 1.34, 1.42 and 1.53 units/mL by inducing 0.1, 0.5, and 1% methanol, respectively, at baffled flask culture of P.pastoris GS115/pPIC-AgaB for 48 hr. Most of the enzyme activity was found in the extacellular fraction and the secretion efficiency showed 98%. Thermostability of recombinant $\beta$-agarase was also increased by glycosylation.

• Journal of Life Science
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• v.8 no.3
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• pp.263-271
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• 1998

One of the better-characterized transcription factor of E. coli is the cAMP receptor protein(CRP) and the CRP binds cAMP and DNA. The cAMP-CRP complex is involved in regulation of many genes at bacteria. The cAMP-CRP regulatory element represents, in some respects, a global regulatory network. The aim of this work was to study the structure and the mechanisms controlling the expression of CRP in Serratia marcescens. We have been get 5 different clones from Serratia which stimulated the cells to use maltose as a sole carbon source in E. coli TP2139. The crp gene clone, pCKB12, was confirmed by Southern hybridization with E. coli crp gene. The location of the crp gene was determined by construction subclones carrying various portions of pCKB12. To investigate the potential role of CRP in E. coli, lacZ fused plasmids were constructed and investigated the ${\beta}$-galactosidase activity of the fused plasmid. The Serratiamarcescens cAMP receptor protein can substitute the E. coli CRP in transcriptional activation at the lacZ gene. These results suggest that Serratia marcescens cAMP receptor protein complex functions to regulate several promoters in E. coli.