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Molecular Phylogeny of Chattonella (Raphidophyceae) Species from Deungnyang Bay, Korea Using Single-Cell PCR (Single-cell PCR을 이용하여 분석한 득량만 Chattonella 종 (Raphidophyceae)의 분자계통학적 특성)

  • Kim, Jin Joo;Song, Seon Yeung;Park, Tae Gyu
    • Journal of the Korean Society of Marine Environment & Safety
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    • v.24 no.7
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    • pp.967-972
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    • 2018
  • The genus Chattonella belonging to the class raphidophyceae, is a harmful algal bloom species. Recently, its occurrence has been increasing and expanding along the Korean coast. Species identification of the genus Chattonella only by morphological observation is difficult due to the lack of rigid cell walls. In this study, the morphological characteristics and genetic affinity of Chattonella sp. isolated from Deungnyang Bay in 2017 were examined. We carried out single-cell isolation from field samples then sequenced three different areas using the single-cell PCR method: 1) parts of ribosomal operon, the large subunit (LSU) of the rDNA, 2) the chloroplast-encoded subunit psaA of Photosystem I, and 3) rbcL encoding the large subunit of the Rubisco gene. The cells were morphologically very similar to the general genus Chattonella ($74.0{\pm}10.1{\mu}m$ in length, $33.1{\pm}3.6{\mu}m$ in width). The three partial gene sequences were insufficient to justify distinction at the species rank. However, they clustered at 99-100 % sequence similarity with C. marina, C. marina var. antiqua and C. marina var. ovata.

Genome sequence of Actinomyces georgiae KHUD_A1 isolated from dental plaque of Korean elderly woman (한국 노인 여성의 치태에서 분리된 Actinomyces georgiae KHUD_A1의 유전체 염기서열 해독)

  • Moon, Ji-Hoi;Shin, Seung-Yun;Hong, Won Young;Jang, Eun-Young;Yang, Seok Bin;Ryu, Jae-In;Lee, Jin-Yong;Lee, Jae-Hyung
    • Korean Journal of Microbiology
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    • v.55 no.1
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    • pp.74-76
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    • 2019
  • Gram-positive anaerobic bacilli Actinomyces spp. commonly reside on mucosal surfaces of the oropharynx, gastrointestinal tract, and urogenital tract. Here, we first report the draft genome sequence of Actinomyces georgiae KHUD_A1, isolated from dental plaque of a Korean elderly woman. The genome is 2,652,059 bp in length and has a GC content of 68.06%. The genome includes 2,242 protein-coding genes, 9 rRNAs, and 64 tRNA. We identified 157 KHUD_A1 strain-specific genes, including genes encoding CPBP family intramembrane metalloprotease, bile acid: sodium symporter family protein, Txe/YoeB family addiction module toxin and Phd/YefM family antitoxin. The sequence information of A. georgiae KHUD_A1 will help understand the general characteristics of the bacterial species and the genome diversity of the genus Actinomyces.

Molecular Characterization of an Isolate of Bean Common Mosaic Virus First Identified in Gardenia Using Metatranscriptome and Small RNA Sequencing

  • Zhong-Tian Xu;Hai-Tao Weng;Jian-Ping Chen;Chuan-Xi Zhang;Jun-Min Li;Yi-Yuan Li
    • The Plant Pathology Journal
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    • v.40 no.1
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    • pp.73-82
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    • 2024
  • Gardenia (Gardenia jasminoides) is a popular and economically vital plant known for its ornamental and medicinal properties. Despite its widespread cultivation, there has been no documentation of plant viruses on gardenia yet. In the present study, gardenia leaves exhibiting symptoms of plant viral diseases were sampled and sequenced by both metatranscriptome and small RNA sequencing. As a consequence, bean common mosaic virus (BCMV) was identified in gardenia for the first time and named BCMV-gardenia. The full genome sequence of BCMV-gardenia is 10,054 nucleotides (nt) in length (excluding the poly (A) at the 3' termini), encoding a large polyprotein of 3,222 amino acids. Sequence analysis showed that the N-termini of the polyprotein encoded by BCMV-gardenia is less conserved when compared to other BCMV isolates, whereas the C-termini is the most conserved. Maximum likelihood phylogenetic analysis showed that BCMVgardenia was clustered closely with other BCMV isolates identified outside the leguminous plants. Our results indicated that the majority of BCMV-gardenia virus-derived small interfering RNAs (vsiRNAs) were 21 nt and 22 nt, with 21 nt being more abundant. The first nucleotide at the 5' termini of vsiRNAs derived from BCMV-gardenia preferred U and A. The ratio of vsiRNAs derived from sense (51.1%) and antisense (48.9%) strands is approaching, and the distribution of vsiRNAs along the viral genome is generally even, with some hot spots forming in local regions. Our findings could provide new insights into the diversity, evolution, and host expansion of BCMV and contribute to the prevention and treatment of this virus.

Identification and molecular characterization of the chitinase gene, EaChi, from the midgut of the earthworm, Eisenia andrei (붉은줄지렁이 (Eisenia andrei) 중장에서 발현되는 chitinase 유전자, EaChi의 동정 및 분자생물학적 특성에 관한 연구)

  • Tak, Eun Sik;Kim, Dae hwan;Lee, Myung Sik;Ahn, Chi Hyun;Park, Soon Cheol
    • Journal of the Korea Organic Resources Recycling Association
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    • v.18 no.3
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    • pp.31-37
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    • 2010
  • Chitinases (EC 3.2.1.14) hydrolyze the ${\beta}$-1,4-linkages in chitin, the second most abundant polymer of N-acetyl-${\beta}$-D-glucosamine which is a structural component of protective biological matrices such as fungal cell walls and insect exoskeletons. The glycosyl hydrolases 18 family including chitinases is an ancient gene family widely expressed in archea, prokaryotes and eukaryotes. Since earthworms live in the soil with a lot of microbial activities and fungi are supposed to be a major component of the diet of earthworm, it has been reported that there would be appropriate immune system to protect themselves from microorganisms attacks. In this study, the novel chitinase, EaChi, from the midgut of earthworm, Eisenia andrei, were identified and characterized. To obtain full-length cDNA sequence of chitinase, RT-PCR and RACE-PCR analyses were carried out by using the previously identified EST sequence amongst cDNA library established from the midgut of E. andrei. EaChi, a partial chitinase gene, was composed of 927 nucleotides encoding 309 amino acids. By the multiple sequence alignments of amino acids with other different species, it was revealed that EaCHI is a member of glycosyl hydrolases 18 family, which has two highly conserved domains, substrate binding and catalytic domain.

Association study of polymorphism in leukotriene C4 synthase and cysteinyl leukotriene receptor 1 genes with phenotype of asthma and clinical parameters in Korean children (Leukotrienes C4 synthase와 cysteinyl leukotriene receptor 1 유전자 다형성과 한국 소아 천식 표현형 및 임상 지표와의 연관성 연구)

  • Shim, Jung Yeon;Kim, Byung-Joo;Song, Young Hwa;Kang, Mi-Jin;Lee, So-Yeon;Kim, Hyo-Bin;Yu, Jinho;Hong, Soo-Jong
    • Clinical and Experimental Pediatrics
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    • v.52 no.6
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    • pp.680-688
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    • 2009
  • Purpose : Cysteinyl leukotrienes are important proinflammatory mediators in asthma. Recently, it was suggested that a promoter polymorphism in the genes encoding for leukotriene C4 synthase (LTC4S), a key enzyme in the leukotriene synthetic pathway, and cysteinyl leukotriene receptor 1 (CysLTR1) might be associated with aspirin-intolerant asthma. We investigated whether polymorphisms in LTC4S and CysLTR1 genes or their interactions were associated with the asthma phenotype, lung function, or bronchial hyperreactivity (BHR) in Korean children. Methods : A total of 856 asthmatic children and 254 non-asthmatic controls were enrolled; a skin prick test, lung function test and bronchial provocation test were performed. Of those enrolled, 395 children underwent exercise challenge tests. The LTC4S A(-444)C and CysLTR1 T(+927)C were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis. Results : Of those enrolled, 699 children were classified as having atopic asthma and 277 children, as having exercise-induced asthma (EIA). LTC4S and CysLTR1 polymorphisms were not associated with atopic asthma, EIA, or asthma per se. Lung function and BHR were not significantly different between the wild type (AA or TT) and the variant (AC+CC or TC+CC) genotypes in asthmatics, atopic asthmatics, and EIA (+) asthmatics, while total eosinophil counts were higher in the variant type of LTC4S than in the wild type in atopic asthmatics. There were no associations between the gene-gene interactions of LTC4S and CysLTR1 genotypes and the asthma phenotypes. Conclusion : LTC4S A(-444)C and CysLTR1 T(+927)C polymorphisms and their gene-gene interactions are not associated with asthma phenotype, lung function, or BHR in Korean children.

Cloning and Characterization of a 5-Enolpyruvyl Shikimate 3-Phosphate Synthase (EPSPS) Gene from Korean Lawn Grass (Zoysia japonica) (들잔디 5-Enolpyruvyl Shikimate 3-Phosphate Synthase(EPSPS) 유전자 클로닝 및 특성)

  • Lee, Hye-Jung;Lee, Geung-Joo;Kim, Dong-Sub;Kim, Jin-Beak;Ku, Ja-Hyeong;Kang, Si-Yong
    • Horticultural Science & Technology
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    • v.28 no.4
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    • pp.648-655
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    • 2010
  • This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$ $japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.

Association of the CYP1B1 Gene Polymorphism with the Risk of Advanced Endometriosis in Korean Women (한국 여성에서 중증자궁내막증과 CYP1B1 유전자 다형성과의 관련성에 관한 연구)

  • Cho, Yeon Jean;Hur, Sung-Eun;Lee, Ji Young;Song, In Ok;Koong, Mi Kyoung;Moon, Hye Sung;Chung, Hye-Won
    • Clinical and Experimental Reproductive Medicine
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    • v.33 no.2
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    • pp.85-95
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    • 2006
  • Objective: To investigate whether polymorphisms of gene encoding CYP1B1 is associated with the risk of endometriosis in Korean women. Methods: We investigated 199 patients with histopathologically confirmed endometriosis rAFS stage III/IV and 183 control group women who were surgically proven to have no endometriosis. The genetic distribution of four different CYP1B1 polymorphisms at $G^{119}-T$, $G^{432}-C$, $T^{449}-C$, and $A^{453}-G$ were analyzed by polymerase chain reaction(PCR) and restriction fragment length polymorphism of PCR products. Results: We found no overall association between each individual CYP1B1 genotype and the risk of endometriosis. The odds ratio of genotype GG/GC+GG/TC+TT/AA compared to GG/CC/CC/AA(reference) was calculated as 2.06 with a 95% confidence interval of 1.003~4.216. Conclusion: This results suggest that CYP1B1 genetic polymorphism may be associated with development of endometriosis in Korean women.

Association between the Human Surfactant Protein-A(SP-A) Gene Locus and Chronic Obstructive Pulmonary Disease in Korean Population (한국인에서 만성폐쇄성폐질환과 인체 폐 표면 활성제 단백-A 유전자 대립형질의 상관관계)

  • Na, Joo Ock;Oh, Myung Ho;Choi, Jae Sung;Seo, Ki Hyun;Kim, Yong Hoon
    • Tuberculosis and Respiratory Diseases
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    • v.60 no.6
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    • pp.638-644
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    • 2006
  • Backgrounds: This study investigated whether or not a polymorphism in the gene encoding the surfactant protein A(SP-A) has any bearing on the individual susceptibility to the development of chronic obstructive pulmonary disease(COPD) in a genetically homogenous Korean population. Methods: The genotypes of 19 COPD patients and 20 healthy neonates as controls were tested using a polymerase chain reaction followed by restriction fragment length polymorphism analysis for the SP-A gene. Results: The specific frequencies of the 6A2 and 6A18 alleles of SP-A1 and the 1A2 allele of SP-A2 were much higher in the COPD group than control group (p<0.05). However, the frequencies of the 6A3 and 6A4 alleles of SP-A1 and the 1A0 allele of SP-A2 in the COPD group were significantly lower than the control group. In the COPD group, the frequencies of the +50 locus genotypes GG of SP-A1 and the +9 locus genotypes CC of SP-A2 were 85.0% and 60.6%, respectively, and 19.7% and 24.8% in the control group, respectively. The frequencies of the polymorphic genotypes or alleles showed a statistically significant difference between the COPD group and the control group (P<0.05). Conclusion: A genetic polymorphism in SP-A is associated with the development of COPD in the Korean population.

Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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