• Title/Summary/Keyword: Gene isolation

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Isolation of the Regulator Gene Responsible for Overproduction of Catalase A in $H_2O$$_2$-resistant Mutant of Streptomyces coelicolor

  • Hahn, Ji-Sook;Oh, So-Young;Keith F. Chater;Roe, Jung-Hye
    • Journal of Microbiology
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    • v.38 no.1
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    • pp.18-23
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    • 2000
  • Streptomyces coelicolor produces three kinds of catalases to cope with oxidative stress and to allow normal differentiation. Catalase A is the major vegetative catalase which functions in removing hydrogen peroxide generated during the process of aerobic metabolism. To understand the regulatory mechanism of response against oxidative stress, hydrogen peroxide-resistant mutant (HR4O) was isolated from S. coelicolor J1501 following UV mutagenesis. The mutant overproduced catalase A more than 50-fo1d compared with the wild type. The mutation locus catRI was mapped closed to the mthB2 locus by genetic crossings. An ordered cosmid library of S. coelicolor encompassing the mthB2 locus was used to isolate the regulator gene (catR) which represses catalase overproduction when introduced into HR4O. A candidate catR gene was found to encode a Fur-like protein of 138 amino acids (15319 Da).

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An Oligonucleotide Microarray Bait for Isolation of Target Gene Fragments

  • Shi, Rong;Ma, Wen-li;Liu, Cui-Hua;Song, Yan-Bin;Mao, Xiang-Ming;Zheng, Wen-Ling
    • BMB Reports
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    • v.37 no.2
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    • pp.148-152
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    • 2004
  • A new molecular-baiting method was studied by retrieving targeted gene fragments from an oligonucleotide microarray bait after hybridization. To make the microarray bait, 70-mer oligonucleotides that were designed to specifically represent the SSA1 gene of Saccharomyces cerevisiae were printed on the slide. Samples of the Saccharomyces cerevisiae mRNA were extracted and labeled by the RD-PCR (Restriction Display PCR) method using the Cy5-labelled universal primer, then applied for hybridization. The sample fragments that hybridized to the microarray were stripped, and the eluted cDNAs were retrieved and cloned into the pMD 18-T vector for transformation, plasmid preparation, and sequencing. BLAST searching of the GenBank database identified the retrieved fragments as being identical to the SSA1 gene (from 2057-2541bp). A new method is being established that can retrieve the sample fragments using an oligo-microarray-bait.

Isolation and Characterization of Rice OsHRL Gene Related to Bacterial Blight Resistance

  • Park, Sang-Ryeol;Moon, Seok-Jun;Shin, Dong-Jin;Kim, Min-Gab;Hwang, Duk-Ju;Bae, Shin-Chul;Kim, Jeong-Gu;Yi, Bu-Young;Byun, Myung-Ok
    • The Plant Pathology Journal
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    • v.26 no.4
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    • pp.417-420
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    • 2010
  • The expression of HR-like lesion inducing gene of Oryza sativa (OsHRL) was slightly increased by Xanthomonas oryzae pv. oryzae (Xoo) infection. Transgenic rice plants over-expressing OsHRL gene were challenged with Xoo and the development of disease symptoms were examined to investigate the effect of OsHRL gene expression on plant defense responses. The over-expression of OsHRL increased disease resistance against Xoo compared with wild type plants.

The first virus isolation and partial characterization of equine herpesvirus-4 in a horse, South Korea

  • Choi, Eun-Jin;Lee, Hyun-Kyoung;Lee, Kyoung-Hyun;So, Byoung-Jae;Song, Jae-Young;Do, Jae-Chul;Yang, Seon-Joo;Lee, Hyun-Chul;Yang, Young-Jin
    • Korean Journal of Veterinary Service
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    • v.38 no.2
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    • pp.141-144
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    • 2015
  • An equine herpesvirus-4 (EHV-4) was isolated in nasal swabs collected in a horse showing respiratory clinical signs. Equine dermis cells inoculated with the sample were observed with characteristic viral cytopathic effects after 3 days of postinoculation and the infected cells exhibited bright intracelluar fluorescence by indirect immunofluorescence assay. At the nucleotide level, the partial glycoprotein B gene of the Korean EHV-4 isolate (K001) had 99.9% identity to 1942 strain (GenBank No. M26171). To author's knowledge, the report describes the first isolation and partial characterization of EHV-4 in Korea. The virus can be used for further study of EHV-4.

Isolation of Multidrug-Resistant Salmonella typhimurium DT104 from Swine in Korea

  • Lee, Ki-Eun;Lee, Yeon-Hee
    • Journal of Microbiology
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    • v.45 no.6
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    • pp.590-592
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    • 2007
  • We report the isolation of Salmonella enterica serotype Typhimurium phage type DT104 (CCARM 8104) from swine in Korea. The CCARM 8104 isolate was resistant to nalidixic acid and showed reduced susceptibility to quinolones. The CCARM 8104 isolate had a missense mutation, Asp87Asn, in the quinolone resistance-determining region in gyrA and produced PSE-1. The CCARM 8104 isolate carried two different class 1 integrons, and the PSE-1 ${\beta}$-lactamase gene was inserted into a 1,200 bp class 1 integron. The presence of DT104 with pse-1 in an integron located in a plasmid and reduced susceptibility to quinolone in swine pose a significant threat of possible horizontal spread between swine and humans.

The Effective Isolation of a Mosquitocidal Bacteria, Bacillus thuringiensis Subsp. israelensis (모기 살충성 세균 B. thuringiensis subsp. israelensis의 효과적인 분리 방법)

  • 김광현;이광배;신두만
    • Journal of environmental and Sanitary engineering
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    • v.13 no.2
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    • pp.34-39
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    • 1998
  • For more convenient and rapidly isolation of Bacillus thuringiensis subsp. israelensis(Bti), 1) heat treatment spore forming bacteria, 2) growth in enrichment culture media for Bacillus sp. and 3) selection of bacteria producing a lecithinase for Bacillus thuringiensis subsp. israelensis, were performed. Spore forming bacteria were counted 4.8 $\times $ 10$^{8}$cells/g soil on NAPGCY media, 9.2 $\times $ 10$^{7}$cells/g soil on NA media, and 3.6 $\times $ 10$^{8}$cells/g soil on NAAC media, respectively. Bacteria producing only a lecithinase were reached at 25.2% on medium contained egg york, bacteria only producing a delta-endotoxin were reached at 23.2% by phase contrast microscope, and bacteria producing a lecithinase & a delta-endotoxin simultaneously were reached at 13.7%. Bacillus thuringiensis which producing a lecithinase and a delta-endotoxin simultaneously among bacteria producing a lecithinase, were reached at 56.5%; A half of Bacillus thuringiensis was produced a delta-endotoxin, but not produced a lecithinase. Among 8 isolates of Bacillus thuringiensis, two strain of Bti which has a mosquito-cidal toxin, were detected by PCR using a specific primer of $\delta $-endotoxin gene from Bti.

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Isolation of a Multidrug Resistance Inhibitor from Aconitum pseudo-laeve var. erectum

  • Kim, Dae-Keun;Kwon, Hyog-Young;Lee, Kang-Ro;Rhee, Dong-Kwon;Zee, Ok-Pyo
    • Archives of Pharmacal Research
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    • v.21 no.3
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    • pp.344-347
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    • 1998
  • To overcome multidrug resistance (MDR) in cancer chemotherapy, we prepared various plant extracts and searched for a component which is effective for inhibition of MDR. MDR inhibition activity was determined by measuring cytotoxicity to MDR cells using multidrug resistant human fibrocarcinoma KB V20C, which is resistant to 20 nM vincristine and expresses high level of mdr1 gene. Of various plant extracts, the MeOH extract of the root of Aconitum pseudo-laeve var. erectum was found to have potent inhibitory activity on MDR. The bioassayguided fractionation of the MeOH extract of the plant led to the isolation of an alkaloid, lycaconitine, as an active principle. And the $IC_{50}$ of lycaconitne for KB V20C cells was $74\mu{g}$/ml.

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Yeasts in the Flowers of Wild Fleabane [Erigeron annus (L.) Pers.]

  • Kim, Jong-Shik;Kim, Dae-Shin
    • Korean Journal of Environmental Agriculture
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    • v.34 no.3
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    • pp.238-243
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    • 2015
  • BACKGROUND: Yeasts associated with fleabane flowers were identified using isolation methods previously applied in yeast biotechnology. A culture-based approach was required for isolation of many yeast strains associated with fleabane. METHODS AND RESULTS: We spread homogenized fleabane flowers onto GPY medium containing chloramphenicol, streptomycin, Triton X-100, and L-sorbose. We isolated 79 yeast strains from the flowers of wild fleabane, and identified the yeasts via phylogenetic analysis of isolates from agar plates. The yeast species included 39 isolates of Aureobasidium pullulans, 17 of the genus Candida, 14 of the genus Rhodosporidium, 6 of the genus Cryptococcus, and 3 of the genus Rhodotorula. CONCLUSION: Yeast isolates associated with fleabane flowers included A. pullulans (39 isolates) and other yeast species (40 isolates). Such yeast isolates may have biotechnological potential.

Isolation of atypical Aeromonas salmonicida in cultured starry flounder (Platichthys stellatus) (양식산 강도다리(Platichthys stellatus)에서의 비정형 Aeromonas salmonicida 분리)

  • Wi-Sik, Kim;Min-Su, Kwon;Hwi-Jin, Kim;Myung-Joo, Oh;Kyoung-Hui, Kong
    • Journal of fish pathology
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    • v.35 no.2
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    • pp.247-250
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    • 2022
  • About 6.7% mortality was reported in a starry flounder (Platichthys stellatus) aquaculture farm in 2022. Most of the diseased fish showed a loss of pectoral fin, hemorrhages on muscle and gills, pale gills, enlarged spleen, and nodules on kidney. Parasites, fungi or viruses (viral hemorrhagic septicemia virus and hirame novirhabdovirus) were not detected from diseased fish. However, numerous bacteria were isolated from liver, spleen and kidney. Nucleotide sequences of the A-protein-encoding virulence array protein gene (vapA) of the bacteria showed 99.93% identity with Aeromonas salmonicida subsp. masoucida. This study is the first report of isolation of atypical A. salmonicida in cultured starry flounder in Korea.

Isolation of Mouse Ig Heavy and Light Chain Genomic DNA Clones, and Construction of Gene Knockout Vector for the Generation of Humanized Xenomouse (인간 단클론 항체 생산용 Humanized Xenomouse 제작의 기초 소재인 생쥐 Ig 중사슬 및 경사슬 Genomic DNA 클론의 확보 및 유전자 적중 벡터의 제작)

  • Lee, Hee-kyung;Cha, Sang-hoon
    • IMMUNE NETWORK
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    • v.2 no.4
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    • pp.233-241
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    • 2002
  • Background: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. Methods: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain ${\lambda}$ genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. Results: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. Conclusion: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.