• Korean Journal of Microbiology
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• v.55 no.2
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• pp.103-111
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• 2019

DNA methylation is involved in diverse processes in bacteria, including maintenance of genome integrity and regulation of gene expression. CcrM, the DNA methyltransferase conserved in Alphaproteobacterial species, carries out $N^6$-adenine or $N^4$-cytosine methyltransferase activities using S-adenosyl methionine as a co-substrate. Celeribacter marinus IMCC12053 from the Alphaproteobacterial group was isolated from a marine environment. Single molecule real-time sequencing method (SMRT) was used to detect the methylation patterns of C. marinus IMCC12053. Gibbs motif sampler program was used to observe the conversion of adenosine of 5'-GANTC-3' to $N^6$-methyladenosine and conversion of $N^4$-cytosine of 5'-GpC-3' to $N^4$-methylcytosine. Exocyclic DNA methyltransferase from the genome of strain IMCC12053 was chosen using phylogenetic analysis and $N^4$-cytosine methyltransferase was cloned. IPTG inducer was used to confirm the methylation activity of DNA methylase, and cloned into a pQE30 vector using dam-/dcm- E. coli as the expression host. The genomic DNA and the plasmid carrying methylase-encoding sequences were extracted and cleaved with restriction enzymes that were sensitive to methylation, to confirm the methylation activity. These methylases protected the restriction enzyme site once IPTG-induced methylases methylated the chromosome and plasmid, harboring the DNA methylase. In this study, cloned exocyclic DNA methylases were investigated for potential use as a novel type of GpC methylase for molecular biology and epigenetics.

• Research in Plant Disease
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• v.17 no.2
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• pp.211-215
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• 2011

Cucumber mosaic virus (CMV)-like isolate was collected from Raphanus sativus (cv. Choon-hyang), which showed mosaic symptoms. The isolate was confirmed to a strain of CMV by host responses in Vigna unguiculata, Chenopodium amaranticolor and Gomphrena globosa, by viral genome composition with RT-PCR and PCR-RFLP, and by serological analysis. Symptom developed by the strain of CMV was severe in Nicotiana benthamiana, N. glutinosa, N. tabacum (cv. Samsun, cv. Xanthi), Cucumis melo (cv. Early hanover), Cucumis sativus (cv. White wonder), Capsicum annuum (cv. Chung-yang and cv. Geum-top), but mild symptom was developed in Raphanus sativus (cv. Choon-hyang), Brassica rapa ssp. pekinensis (cv. Bul-Am No. 3), and B. juncea (cv. Daenong Jukgot). Newly isolated strain of CMV could infect diverse crops including Solanaceae, Cucurbitaceae and Brassicaceae. We designated the new strain of CMV as Gn-CMV based on the novel infectivity of Brassicaceae. In double-stranded (ds) RNA analysis, Gn-CMV consisted of 3.3, 3.0, and 2.2 kb genomes likewise other strains of CMV. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed 28 kDa of the CMV coat protein. By restriction enzyme mapping using Cac8I, ClaI and MspI of RT-PCR products indicated that Gn-CMV belongs to CMV subgroup I.

• Research in Plant Disease
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• v.7 no.1
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• pp.1-7
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• 2001

An isolate of Cucumber mosaic cucumovirus(CMV) was isolated from Hydrangea macrophylla for. otaksa(Sieb. et Zucc. ) Wils. showing mosaic symptoms, and designated as Hm-CMV. Hm-CMV was characterized by the tests of host range, physical properties, serological properties, RNA and coat protein compositions, and reverse transcription and polymerase chain reaction (RT-PCR) analysis. Twelve species in 4 families were used in the host range test of Hm-CMV and could be differentiated from Y-CMV used as a control CMV by the ringspot and line pattern on inoculated leaves of several tobacco plants. Thevirus produced local lesions on inoculated leaves of Chenopodium amarticolor, C. quinoa and Vigna unguiculata. The physical properties of the virus were as follows; thermal inactivation point(TIP) was 60$\^{C}$, dilution end point (DEP) was 10$\^$-3/, and longevity in vitro (LIP) was 3∼4 days. Hm-CMV was serologically identical to Y-CMV. SDS-polyaciylamide gel electrophoresis(SDS-PAGE) showed one major protein band of about 28 kDa. In RNA or dsRNA analysis, Hm-CMV consisted of four RNA or dsRNA species, but satellite RNA was not detected. In RT-PCR using CMV-common primer and CMV subgroup I-specific primer, bothe amplified expected size of about 490 bp and 200 bp DNA fragments from Hm-CMV, respectively. Restriction enzyme analysis of the 490 bp RT-PCR products using EcoR I and Msp I showed that Hm-CMV belonged to CMV subgroup I. However, Hm-CMV could be differentiated from other CMV subgroup I isolates by RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR).

• The Plant Pathology Journal
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• v.26 no.4
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• pp.313-320
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• 2010

Genetic instability of the rice blast fungus Magnaporthe oryzae has been suggested as a major factor underlying the rapid breakdown of host resistance in the field. However, little information is available on the mechanism of genetic instability. In this study, we assessed the stability of repetitive DNA elements and several key phenotypic traits important for pathogenesis after serially transferring two isolates though rice plants and an artificial medium. Using isolate 70-15, we obtained a total of 176 single-spore isolates from 10 successive rounds of culturing on artificial medium. Another 20 isolates were obtained from germ tubes formed at the basal and apical cells of 10 three-celled conidia. Additionally, 60 isolates were obtained from isolate KJ201 after serial transfers through rice plants and an artificial medium. No apparent differences in phenotypes, including mycelial growth, conidial morphologies, conidiation, conidial germination, appressorium formation, and virulence, or in DNA fingerprints using MGR586, MAGGY, Pot2, LINE, MG-SINE and PWL2 as probes were observed among isolates from the same parent isolate. Southern hybridization and sequence analysis of two avirulence genes, AVR-Pita1 and AVR-Pikm, showed that both genes were also maintained stably during 10 successive generations on medium and plants. However, one reversible loss of restriction fragments was found in the telomere-linked helicase gene (TLH1) family, suggesting some telomere regions may be more unstable than the rest of the genome. Taken together, our results suggest that phenotype and genotype of M. oryzae isolates do not noticeably change, at least up to 10 successive generations on a cultural medium and in host plants.

• The Plant Pathology Journal
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• v.22 no.3
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• pp.215-221
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• 2006

Gibberella zeae (anamorph: Fusarium graminearum) is an important pathogen of cereal crops. This fungus produces a broad range of secondary metabolites, including polyketides such as aurofusarin (a red pigment) and zearalenone (an estrogenic mycotoxin), which are important mycological characteristics of this species. A screen of G. zeae insertional mutants, generated using a restriction enzyme-mediated integration (REMI) procedure, led to the isolation of a mutant (Z43R606) that produced neither aurofusarin nor zearalenone yet showed normal female fertility and virulence on host plants. Outcrossing analysis confirmed that both the albino and zearalenone-deficient mutations are linked to the insertional vector in Z43R606. Molecular characterization of Z43R606 revealed a deletion of at least 220 kb of the genome at the vector insertion site, including the gene clusters required for the biosynthesis of aurofusarin and zearalenone, respectively. A re-creation of the insertional event of Z43R606 in the wild-type strain demonstrated that the 220-kb deletion is responsible for the phenotypic changes in Z43R606 and that a large region of genomic DNA can be efficiently deleted in G. zeae by double homologous recombination. The results showed that 52 putative genes located in the deleted genomic region are not essential for phenotypes other than the production of both aurofusarin and zearalenone. This is the first report of the molecular characterization of a large genomic deletion in G. zeae mediated by the REMI procedure.

• IMMUNE NETWORK
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• v.11 no.5
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• pp.253-257
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• 2011

Background: The active metabolite (1, 25- dihydroxycholecalciferol) of vitamin D (25-hydroxycholecalciferol) leads to activation of macrophages and deficiency of vitamin D seems to be involved in the risk of tuberculosis. The effects of vitamin D are exerted by interaction with the vitamin D receptor (VDR) and may be influenced by polymorphism in the VDR gene. In this study, variation in the VDR gene was investigated in Korean population with tuberculosis. Methods: We typed three VDR polymorphisms of restriction endonuclease sites for TaqI, BsmI and FokI in 155 patients with tuberculosis and 105 healthy volunteers. Results: The frequencies of FokI genotypes determined from TB patients were 29.13% for FF, 56.31% for Ff, and 14.56% for ff. We observed 1.4-fold increased prevalence of the Ff genotype in TB patients compared with normal healthy groups (p=0.0857). However, there was no significant association between the genotype groups, TB patient and normal control, for FokI polymorphism. There was also no significant association between VDR gene and tuberculosis in another polymorphism (BsmI and TaqI). Conclusion: Three polymorphisms (TaqI, BsmI and FokI) in the VDR gene do not appear to be responsible for host susceptibility to human tuberculosis in Korean population.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.130-130
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• 2003

Gummy stem blight pathogen is very difficult not only to monitor the inoculum levels prior to host infection, and also it is destructive and hard to control in field condition. We have applied RAPD technique to elucidate the genetic diversity of the genomic DNA of Didymella bryoniae and also to generate specific diagnostic DNA probe useful for identification and detection. The 40 primers produced clear bands consistently from the genomic DNA of twenty isolates of Didymella bryoniae, and two hundred seventy-three amplified fragments were produced with 40 primers. The combined data from 273 bands was analyzed by a cluster analysis using UPGMA method with an arithmetic average program of NTSYS-PC (Version 1.80) to generate a dendrogram. At the distance level of 0.7, two major RAPD groups were differentiated among 20 strains. RAPD group (RG) I included 8 isolates from watermelon except one isolate from melon. RAPD group (RG) IV included 12 isolates from squash, cucumber, watermelon and melon.. In amplification experiment with SCAR specific primer RG1F-RG1R resulted in a single band of 650bp fragment only for 8 isolates out of 20 isolates that should be designated as RAPD Group 1. However, same set of experiment done with RGIIF-RGIIR did not result in any amplified product.. Our attempts to detect intraspecific diversity of ITS region of rDNA by amplifying ITS region and 17s rDNA region for 20 isolates and restriction digestion of amplified fragment with 12 enzymes did not reveal polymorphic band. In order to develop RAPD markers for RGIV specific primer, a candidate PCR fragment( ≒1.4kb) was purified and Southern hybridized to the amplified fragment RGIV isolates. This promising candidate probe recognized only RGIV isolates

• Korean Journal of Microbiology
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• v.23 no.2
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• pp.138-146
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• 1985

A Bacillus licheniformis ATCC31667 gene coding for a glucose isomerase has been cloned and expressed in glucose isomerase negative mutant of Escherichia coli. A recombinant plasmid, constructed by ligation of a EcoRI fragment of B.licheniformis chromosomal DNA to vector plasmid pBR322, was expressed glucose isomerase positive in E.coli LE392-6 with growth on minimal medium containing xylose as a sole carbon source. This recombinant plasmid, designated pBGI6, had the insery of 4.1Kb of Bacillus gene in EcoRI site, and restriction map of the plasmid was established. The plasmid pBG16 was very stable after 10days of serial transfer to a fresh medium. The activity of glucose isomerase from the transformed cell containing pBGI6 was increased about 20 fold than its wild type of host.

• The Korean Journal of Mycology
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• v.29 no.1
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• pp.9-14
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• 2001

Colletotrichum species are important fungal pathogens that cause great damages on various host plant species worldwide. In Korea, Colletotrichum species cause massive economic losses on apple, peach, grape, and especially, sweet persimon productions. In the past, identification of the pathogen and the studies on the genetic relationships among the pathogenic isolates were mainly based on morphology, cultural characteristics, and the difference in pathogenicity. However, in recent years, these traditional methods have been replaced with molecular methods including AFLP. AFLP method with the merits of both RAPD and RFLP has been widely used for the genetic relationship studies of various organisms. Therefore, in this study, AFLP method was employed for the studies of genetic relationships among the different isolates of Colletotrichum species collected from various parts of sothern Korea. As a result, two specific band pattern groups were observed among different isolates of Colletotrichum species.

• Korean Journal of Microbiology
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• v.23 no.1
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• pp.56-63
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• 1985

We have characterized the plasmids of zymomonas, and constructed E. coli-Zymomonas shuttle vector. Plasmids have been detected in four strains of Zymomonas mobilis. All strains tested had at least one plasmid ranging in size from about 1.7 to 46kb. Antibiotics resistances of Z. mobilis were tested to select the host strain. All strains were very sensitive to tetracycline and chloramphenicol. Homology tests between the plasmids in four strains showed that the plasmids of ATCC10988 is highly homologous to those of ZM1, and that there is no homology between plasmids of ZM4 and Agll. The 1.7kb plasmid of ATCC10988, named as pZM886, also has no homology with plasmids of ZM4. A hybrid plasmid, designated to pBZ41, was constructed from pZM886 and pBR322. A restriction map of pBZ41 was established. Replicon of pZM886 didn't operate in E.coli and pBR322 seemed not to replicate in Zymomonas. pBZ41 was transfered from E. coli to Zymomonas by conjugal mobilization. The transconjugants were resistant to tetracycline and maintained pBZ41 stably.