• Title, Summary, Keyword: $DgF3'H$

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Isolation and Characterization of a Novel Flavonoid 3'-Hydroxylase (F3'H) Gene from a Chrysanthemum (Dendranthema grandiflorum) and Its Gamma-ray Irradiated Mutants (감마선 처리에 의한 스프레이형 국화 화색변이체로부터 Flavonoid 3'-Hydroxylase(F3'H) 유전자의 분리 및 특성 구명)

  • Chung, Sung-Jin;Lee, Geung-Joo;Kim, Jin-Baek;Kim, Dong-Sub;Kim, Sang-Hoon;Kang, Si-Yong
    • Horticultural Science & Technology
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    • v.30 no.2
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    • pp.162-170
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    • 2012
  • The objectives of this study were to isolate and the sequence of novel $F3'H$ gene related to an anthocyanin pathway, and to confirm the expression patterns of the gene involved in the flower color variations of chrysanthemum mutants. In this study, we isolated the full-length cDNAs and the genomic DNAs of an $F3'H$ gene from a wild type (WT) chrysanthemum (cv. Argus) and its three color mutants. The sequence analysis revealed a putative open reading frame of 1,527 bp that encodes a polypeptide of 509 amino acids. Sequence homology ranged from 97% to 99% between 'Argus' and its three color mutants. The sequence analysis from the genomic DNA revealed that the chrysanthemum $DgF3'H$ gene consisted of three exons and two introns spanning a 3,830 bp length. The sizes of the gene for three mutants ranged from a shorter size of 3,828 bp to a longer size of 3,838 bp when compared to the size of WT. The total size of the two introns was 2,157 bp for WT, but those for three color mutants ranged from 2,154 bp to 2,159 bp. A result of an RT-PCR analysis indicated that the color variations of the mutants AM1 and AM2 can be partly explained by the structural modification derived from the sequencial changes in the gene caused by gamma ray. A Southern blot analysis revealed that the $DgF3'H$ gene existing as multiple copies in the chrysanthemum genome. A systemic study will be further needed to provide a genetic mechanism responsible for the color mutation and to uncover any involvement of genetic elements for the expression of the $DgF3'H$ gene for the color variation in chrysanthemum.

Formation and Fusion of Protoplasts from the Cellulolytic Fungi, Aspergillus niger MAN-831 and Aspergillus wentii MAW-538 (Cellulase를 생산하는 Aspergillus niger MAN-831과 Aspergillus wentii MAW-538의 원형질체 형성 및 융합)

  • 박석규;이상원;문일식;손봉수;강성구
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.24 no.6
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    • pp.964-969
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    • 1995
  • For the effective utilization of cellulosic biomass, conidial protoplast fusion between Aspergillus niger MAN-831(${\beta}-glucosidase$) and A. wentii MAW-538(CMCase and avicelase), which produced potently cellulolytic enzymes was carried out. Optimal conditions for formation and regeneration of protoplast were conidiospore age-5 dyuas. $2-DG-30\mu\textrm{g}/ml$, preincubation time-4 hours, osmotic stabilizer-0.7M KCl, novozyme(7mg/ml)+driselase(2.5mg/ml) and reaction time of enzyme-5 hours. Optimal conditions for protoplast fusion were obtained by treatment of protoplasts with 15mM CaCl2 and 25% polyethylene glycol 4000(pH 6~7) as fusogenic agent at $36^{\circ}C$ for 25~30 minutes. The frequency was then $7.94{\times}10^{-4}$. CMCase, avicelase and ${\beta}-glucosidase$ activity of fusant F-208 strain was 1.5, 1.3, 1.2 times higher than those of parental strains, respectively.

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Rapid Establishment of CHO Cell Lines Producing the Anti-Hepatocyte Growth Factor Antibody SFN68

  • Song, Seong-Won;Lee, Song-Jae;Kim, Chang-Young;Han, Byungryeul;Oh, Jong-Won
    • Journal of Microbiology and Biotechnology
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    • v.23 no.8
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    • pp.1176-1184
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    • 2013
  • Anti-hepatocyte growth factor (anti-HGF) monoclonal antibodies (mAbs) are potential therapeutics against various cancers. Screening for high-producer clones is a time-consuming and complex process and is a major hurdle in the development of therapeutic mAbs. Here, we describe an efficient approach that allows the selection of high-producer Chinese hamster ovary (CHO) cell lines producing the novel anti-HGF mAb SFN68, which was generated previously by immunizing HGF bound to its receptor c-Met. We selected an SFN68-producing parental cell line via transfection of the dihydrofolate reductase-deficient CHO cell line DG44, which was preadapted to serum-free suspension culture, with an SFN68-expression vector. Subsequent gene amplification via multiple passages of the parental cell line in a methotrexate-containing medium over 4 weeks, followed by clonal isolation, enabled us to isolate two cell lines, 2F7 and 2H4, with 3-fold higher specific productivity. We also screened 72 different media formulated with diverse feed and basal media to develop a suboptimized medium. In the established suboptimized medium, the highest anti-HGF mAb yields of the 2F7 and 2H4 clones were 842 and 861 mg/l, respectively, which were about 10.5-fold higher than that of the parental cell line in a non-optimized basal medium. The selected CHO cell lines secreting high titers of SFN68 would be useful for the production of sufficient amounts of antibodies for efficacy evaluation in preclinical and early clinical studies.