• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1275-1280
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• 2015

Previously, we performed de novo RNA sequencing of Scolopendra subspinipes mutilans using high-throughput sequencing technology and identified several antimicrobial peptide candidates. Among them, a cationic antimicrobial peptide, scolopendrasin VII, was selected based on its physicochemical properties, such as length, charge, and isoelectric point. Here, we assessed the anticancer activities of scolopendrasin VII against U937 and Jurkat leukemia cell lines. The results showed that scolopendrasin VII decreased the viability of the leukemia cells in MTS assays. Furthermore, flow cytometric analysis and acridine orange/ethidium bromide staining revealed that scolopendrasin VII induced necrosis in the leukemia cells. Scolopendrasin VII-induced necrosis was mediated by specific interaction with phosphatidylserine, which is enriched in the membrane of cancer cells. Taken together, these data indicated that scolopendrasin VII induced necrotic cell death in leukemia cells, probably through interaction with phosphatidylserine. The results provide a useful anticancer peptide candidate and an efficient strategy for new anticancer peptide development.

• Journal of Microbiology and Biotechnology
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• 제31권9호
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• pp.1241-1255
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• 2021

This study was carried out to explore a non-chemical strategy for enhancing productivity by employing some antagonistic rhizobacteria. One hundred eighteen bacterial isolates were obtained from the rhizospheric zone of various crop fields of Gangwon-do, Korea, and screened for antifungal activity against Fusarium wilt (Fusarium oxysporum f. sp. lactucae) in lettuce crop under in vitro and in vivo conditions. In broth-based dual culture assay, fourteen bacterial isolates showed significant inhibition of mycelial growth of F. oxysporium f. sp. lactucae. All of the antagonistic isolates were further characterized for the antagonistic traits under in vitro conditions. The isolates were identified on the basis of biochemical characteristics and confirmed at their species level by 16S rRNA gene sequencing analysis. Arthrobacter sulfonivorans, Bacillus siamensis, Bacillus amyloliquefaciens, Pseudomonas proteolytica, four Paenibacillus peoriae strains, and Bacillus subtilis were identified from the biochemical characterization and 16S rRNA gene sequencing analysis. The isolates EN21 and EN23 showed significant decrease in disease severity on lettuce compared to infected control and other bacterial treatments under greenhouse conditions. Two bacterial isolates, EN4 and EN21, were evaluated to assess their disease reduction and growth promotion in lettuce in field conditions. The consortium of EN4 and EN21 showed significant enhancement of growth on lettuce by suppressing disease caused by F. oxysporum f. sp. lactucae respectively. This study clearly indicates that the promising isolates, EN4 (P. proteolytica) and EN21 (Bacillus siamensis), can be commercialized and used as biofertilizer and/or biopesticide for sustainable crop production.

• Plant Breeding and Biotechnology
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• 제6권4호
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• pp.313-320
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• 2018

Rice is the staple food of more than 50% of the world population. Cultivated rice has the AA genome (diploid, 2n = 24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized transposable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos have been used to generate gene-tagged populations. To date T-DNA and maize transposable element systems have been utilized as main insertional mutagens in rice. The Ac/Ds system offers the advantage of generating new mutants by secondary transposition from a single tagged gene. To enhance the efficiency of gene detection, advanced gene-tagging systems (i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of insertional mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been conducted by collaborative works in Korea.

• The Plant Pathology Journal
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• 제37권2호
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• pp.162-172
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• 2021

Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the cross-family infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.

• 생명과학회지
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• 제28권1호
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• pp.130-139
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• 2018

말을 활용한 경주는 고대 유럽의 여러 국가들에서 마차 경주 혹은 산악 경주 등의 형태로 이루어졌으며, 고대 그리스 올림픽에서 마차 경주가 정식 종목으로 채택되었다. 서러브레드종은 17세기부터 속도, 체력, 그리고 경주 능력을 위해 선택적으로 교배되었다. 그 결과, 18세기부터 귀족들이 향유하는 스포츠로서 서러브레드종을 활용한 경주가 시행되었다. 이후 여러 국가에서 각기 다양한 형태로 발달하여 현재 크게 평지 경주, 장애물 경주, 마차 경주 등으로 발달하였다. 서러브레드 경주마는 300여 년 동안 강력한 선발 육종 전략에 의하여 선택되어 왔기에, 현재 우수한 경주 능력을 갖추고 있다. 말산업은 번식, 조련, 경마 등을 통하여 막대한 경제적 효과를 유발하기에, 말의 경주 능력을 유지하고 극대화하는 것이 필요하다. 최근에 많은 양의 게놈 데이터를 처리하기 위해 차세대 시퀀싱(Next Generation Sequencing; NGS)이 개발되었으며, 이 분석 기술의 현저한 발전을 토대로 우수한 형질을 가진 동물 육종 전략을 쉽게 수행 할 수 있게 되었다. 따라서 뛰어난 경주 능력을 가진 경주마를 선발 육종하기 위해서는 최신 유전체 분석 기술을 활용하는 전략이 필요하다. 본 논문에서는 경주마의 경주 능력을 향상시키기 의한 유전체 분석의 현재의 노력을 알아보고, 마지막으로 경주마와 제주마에서 유전체 분석을 활용하는 전략을 제안할 것이며, 대한민국의 생명자원인 제주마의 선발 육종 전략을 제안할 것이다. 말 산업은 기술, 사회 및 경제 분야에서 인간에게 강력한 파급 효과를 주는 동물 중 하나이다. 우리는 국내 고부가가치 말의 원천적인 유전 정보를 확보하고 선발 육종 할 수 있는 체계적인 기술을 확립하여 생산, 연구 업무 등에 대한 일자리 확보에 기여할 수 있기를 기대한다.

• 대한의생명과학회지
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• 제6권1호
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• pp.73-82
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• 2000

양끝이 같은 형태로 이루어진 DNA조각들은 벡터에 두 가지 방향으로 삽입될 수 있다. 기존의 방법으로는 이들의 방향성을 알아내기 위하여 제한효소의 처리 혹은 DNA염기서열 분석법이 수행되어졌는데, 이들은 적절한 제한효소 인식 부위의 부재 혹은 높은 가격과 많은 샘플수 등으로 그 이용범위가 어느 정도 제한되어 있었다. 본 연구에서는, 벡터에 클로닝 된 DNA조각의 방향성을 결정하기 위한 새로운 실험기법과 이에 따르는 구체적인 방법을 기술하고 이의 직접적인 이용을 보고하고 있다. 통상적인 염기서열 분석용 oligonucleotide primer와 중합효소 연쇄반응용 (PCR) primer를 이용한 PCR에 기초한 이 방법은, 여러 후보 클론의 플라스미드 DNA를 주형으로 하여 한 차례의 반응으로, 원하는 방향으로의 DNA조각이 삽입된 클론을 찾아낼 수 있게 한다. 이 실험기법의 용이함과 정확성은 최근에 보고된 바 있는 랫트의 신경 펩타이드인 urocortin의 cDNA를 재조합 발현 벡터상에 클로닝하고 분석하는 것으로 증명할 수 있었다. 이 같은 방법으로 찾아진 유전자 재조합 클론들은 추가적인 실험을 통하여 CHO 세포주에 transfection 되었는데, 이들이 실제로 urocortin을 발현함은 면역효소 측정법으로 검증될 수 있었고, 이를 통하여 최초로 이 40개의 아미노산으로 이루어진 짧은 펩타이드를 진핵 세포상에서 재조합 단백질의 형태로 발현시키는 데 성공하였다.

• Bulletin of the Korean Chemical Society
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• 제27권5호
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• pp.657-662
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• 2006

Identification of accessible sites in targeted RNAs is a major limitation to the effectiveness of antisense oligonucleotides. A class of antisense oligodeoxynucleotides, known as the “10-23” DNA enzyme or DNAzyme, which is a small catalytic DNA, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have designed a strategy to identify accessible cleavage sites in the target RNA, which is hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. A pool of DNAzymes of 58 nucleotides-length that possess randomized annealing arms, catalytic core sequence, and fixed 5'/3'-end flanking sequences was designed and screened for their ability to cleave the target RNA. The screening procedure, which includes binding of DNAzyme pool to the target RNA under inactive condition, selection and amplification of active DNAzymes, incubation of the selected DNAzymes with the target RNA, and target site identification on sequencing gels, identified 16 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in terms of kinetics and accessibility. These selected DNAzymes were effective in cleaving the target RNA in the presence of $Mg^{2+}$. This strategy can be applicable to identify accessible sites in any target RNA for antisense oligonucleotides-based gene inactivation methods.

• Journal of Microbiology and Biotechnology
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• 제33권1호
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• pp.61-74
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• 2023

The global increase in multidrug-resistant (MDR) bacteria has inspired researchers to develop new strategies to overcome this problem. In this study, 23 morphologically different, soil-isolated actinomycete cultures were screened for their antibacterial ability against MDR isolates of ESKAPE pathogens. Among them, isolate BOGE18 exhibited a broad antibacterial spectrum, so it was selected and identified based on cultural, morphological, physiological, and biochemical characteristics. Chemotaxonomic analysis was also performed together with nucleotide sequencing of the 16S rRNA gene, which showed this strain to have identity with Streptomyces lienomycini. The ethyl acetate extract of the cell-free filtrate (CFF) of strain BOGE18 was evaluated for its antibacterial spectrum, and the minimum inhibitory concentration (MIC) ranged from 62.5 to 250 ㎍/ml. The recorded results from the in vitro anti-biofilm microtiter assay and confocal laser scanning microscopy (CLSM) of sub-MIC concentrations revealed a significant reduction in biofilm formation in a concentration-dependent manner. The extract also displayed significant scavenging activity, reaching 91.61 ± 4.1% and 85.06 ± 3.14% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), respectively. A promising cytotoxic ability against breast (MCF-7) and hepatocellular (HePG2) cancer cell lines was obtained from the extract with IC₅₀ values of 47.15 ± 13.10 and 122.69 ± 9.12 ㎍/ml, respectively. Moreover, based on gas chromatography-mass spectrometry (GC-MS) analysis, nine known compounds were detected in the BOGE18 extract, suggesting their contribution to the multitude of biological activities recorded in this study. Overall, Streptomyces lienomycini BOGE18-derived extract is a good candidate for use in a natural combating strategy to prevent bacterial infection, especially by MDR pathogens.

• 한국양식학회지
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• 제6권4호
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• pp.323-338
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• 1993

이 연구의 목적은 Sequencing Batch Reactor(SBR) 처리 장치를 사용하여 pentachlorophenol (PCP)를 함유한 폐수를 처리할 때 활성 슬럿지의 대사 작용에 미치는 PCP의 독성 효과를 시험 분석하는 것이다. 이 연구에서 SBR 처리 장치는 두 가지 운전 주기 1, 2 (8시간과 12시간)와 각각의 운전 주기에 대한 두 가지 운전 조건 (I, II)으로 운전되었다. 운전 조건 I은 유입 폐수를 일시에 (5분 안에) 반응조에 부가하고 2시간 동안 폭기없이 교반만 하는 것이고, 운전 조건 II는 운전 조건 I과 똑같은 조건에서 유입 폐수를 2시간 동안 서서히 반응조에 부가하는 것이다. 각 반응조의 슬럿지 일령은 15일이었다. 합성 폐수가 유입 폐수고서 사용되었고, 그것의 COD는 대략 380mg/L이었다. 각 반응조에 대하여 기본 운전이 끝난 후 0.1, 1.0, 5.0mg/L의 PCP가 틀어 있는 유입 폐수를 사용하여 8시간과 12시간 운전 주기의 정상 상태 운전이 실시되었다. 사용된 유입 폐수의 PCP 농도에서 COD 제거는 저해를 받지 않았다 5.0mg/L의 유입 폐수 PCP 농도와 운전 주기 2의 처리 장치에서, MLVSS 농도는 감소하였고 미생물의 선택성이 증가하므로 생태 반응의 영역이 줄어들었다. 또한 SOUR이 증가하므로 활성 슬럿지가 PCP에 의하여 저해를 받았음을 보여주었고, 활성 슬럿지의 침전이 좋지 않았다. 운전 주기 2의 처리 장치에서 질산화는 사용된 유입 폐수의 PCP 농도에서 어느 정도까지 일어났으나 운전 주기 1의 처리 장치에서는 질산화가 거의 일어나지 않았다. 그래서 질산화가 일어나는 시기는 PCP의 저해 작용으로 인하여 COD의 제거가 늦어지는 만큼 지체될 것이다. 생물학적인 인 제거는 운전 주기 I, 운전 조건 I 그리고 저농도의 PCP에서 운전되는 처리 장치에서 일어났으나 그 과정은 불안정하였고 쉽게 정지되었다. 그러나 운전 주기 2, 운전 조건 I과 II에서 운전되는 처리 장치에서 생물학적인 인제거는 유입 폐수의 PCP 농도가 1.0mg/L로 증가할 때까지 안정하게 일어났다. 유입 폐수의 PCP 농도가 5.0mg/L로 증가했을 때 생물학적인 인 제거 능력은 정지되었고 쉽게 회복되지 않았다.

• The Plant Pathology Journal
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• 제20권3호
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• pp.229-233
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• 2004

The bacterial leaf blight, which is caused by Xantho-monas oryzae pv. oryzae, is the most damaging and intractable disease of rice. To identify the genes involved in the virulence mechanism of transposon TnS complex, which possesses a linearized transposon and transposase, was successfully introduced into X. oryzae pv. oryzae by electroporation. The transposon mutants were selected and confirm the presence of transposition in X. oryzae pv. oryzae by the PCR amplification of transposon fragments and the Southern hybridization using these mutants. Furthermore, transposon insertion sites in the mutant bacterial chromosome were deter-mined by direct genomic DNA sequencing using transposon-specific primers with ABI 3100 Genetic Analyzer. Efficiency of transposition was influenced mostly by the competence status of X. oryzae pv. oryzae cells and the conditions of electroporation. These results indicated that the insertion mutagenesis strategy could be applied to define function of uncharacterized genes in X. oryzae pv. oryzae.