• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.113-113
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• 2017

Downy mildew (DM), caused by several species in the Peronosclerospora and Scleropthora genera, is a major maize (Zea mays L.) disease in tropical or subtropical regions. DM is an obligate parasite species in the higher plants and spreads by oospores, wind, and mycelium in seed surface, soil, and living hosts. Owing to its geographical distribution and destructive yield reduction, DM is one of the most severe maize diseases among the maize pathogens. Positional cloning in combination with phenotyping is a general approach to identify disease resistant gene candidates in plants; however, it requires several time-consuming steps including population or fine mapping. Therefore, in the present study, we suggest a new combination strategy to improve the identification of disease resistant gene candidates. Downy mildew (DM) resistant maize was selected from five cultivars using the spreader row technique. Positional cloning and bioinformatics tools identified the DM resistant QTL marker (bnlg1702) and 47 protein coding genes annotations. Eventually, 5 DM resistant gene candidates, including bZIP34, Bak1, and Ppr, were identified by quantitative RT-PCR without fine mapping of the bnlg1702 locus. Specifically, we provided DM resistant gene candidates with our new strategy, including field selection by the spreader row technique without population preparation, the DM resistance region identification by positional cloning using bioinformatics tools, and expression level profiling by quantitative RT-PCR without fine mapping. As whole genome information is available for other crops, we propose applying our novel protocol to other crops or for other diseases with suitable adjustment.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.133-137
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• 1986

17S-ribosomal RNA gene from the hygromycin resistant protozoan Tetrahymena thermophila hmr 3 was cloned on E. coli vector pBR 322 as part of study to work the 17S-rRNA structure and the mechanism of hygromycin resistance. The 17S-rDNA was inserted into the Hind 111 site of pBR 322. The clones having recombinant plasmid were selected by the method of colony hybridization with a 17S-rDNA probe of wild type B1868. The orientation of 17S-rDNA insert was located near the tetracycline resistant gene of pBR 322 in a clone 5-19 with the recombinant plasmid.

• Archives of Pharmacal Research
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• v.21 no.4
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• pp.470-474
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• 1998

A kanamycin producer, Streptomyces kanamyceticus IFO 13414 is highly resistant to kanamycin. Cloning of the kanamycin resistance genes in S. lividans 1326 with pIJ702 gave several kanamycin resistant transformants. Two transformants, S. lividans SNUS 90041 and S. lividan. SNUS 91051 showed similar resistance patterns to various aminoglycoside antibiotics. Gene mapping experiments revealed that plasmids pSJ5030 and pSJ2131 isolated from the transformants have common resistant gene fragments. Subcloning of pSJ5030 gave a 1.8 Kb gene fragment which showed resistance to kanamycin. Cell free extracts of S. lividans SNUS 90041, S. lividans SNUS 91051 and subclone a S. lividans SNUS 91064 showed kanamycin acetyltransferase activity. The detailed gene map is included.

• Proceedings of the Ginseng society Conference
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• 1990.06a
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• pp.65-67
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• 1990

The sequence of ginseng peptide gene was designed and synthesized by the solid phase plasmid pUC19. Escherichia coli JM101 cells were transformed with above hybrid plasmids. Ampicillin resistant transformants were screened and identified by in situ colony hybridization and Southern blot techinques. Finally the gene sequencing was done by the Sanger dideoxy method using primer extension.

• Journal of Ginseng Research
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• v.14 no.2
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• pp.207-209
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• 1990

The sequence of ginseng peptide gene was designed and synthesized by the solid phase phosphoramidiate method. Synthetic segments were isolated, pllrified and joined to the plasmid pUC19. E.icherichiu coli JM101 cells were transformed with above hybrid plasmids. AmpiciIBin resistant transformants were screened and identified by in situ colony hybridization and Southern blot techniques. Finally the gene sequencing was done by the Sanger dideoxy method using primer extension.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.42-46
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• 1992

From the soil environment which is treated with herbicides. we isolated strain having high resistance against glyphosate. After being identified. the isolated strain was turned out to be Pseudomc~nu.c~c ~paciua nd to have intense tolerance lo the 10 mM glyphosate. The isolated strain shows slow, growth rate about twenty hours in glyphosate comparing with that in inorganic phosphate. As a result of confirming the p~~sitioonf glyphosate resistant gene. it was proved to exist in chromosome. After cloning it into E coli C600, transformants E. coli THC-101 and plasmid pGR19 were obtained.

• Korean Journal of Microbiology
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• v.22 no.4
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• pp.229-234
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• 1984

A modified E. coli trp operon, $trpL({\Delta}att)\;trpE^{FBR}$, was conjugally transfered into Klebsiella pneumoniae $KC_{100}\;(Phe^-,\;Tyr^-,\;Trp^-,\;Rif^r,\;Kam^r)$ by in vivo cloning using the hybrid plasmid $R_{6}K::$ Mucts 61 with a transfer frequency of $5.2{\times}10^{-7}$. Two K. pneumoniae transconjugants, $KUA_{701}\;and\;KUA_{702}$, were isolated. The characters of attenuation control-free and resistance to feedback-inhibition which are characteristics of donor C. coli trp operon were normally expressed in the $KUA_{701}.\;However,\;KUA_{702}$ retained only the feedback-inhibition resistant character. $Trp^+$ phenotype and ampicillin resistant character were completely stable in the transconjugants, but streptomycin resistant character was lost in the transconjugants.

• Proceedings of the Ginseng society Conference
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• 2004.12a
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• pp.50-52
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• 2004

Introduce of gene connected with disease and transformation system of ginseng, Squalene systhase(PSS) gene cloned from and disease resistant gene were carried out for expression and transformation of plant using Agrobacterium. PSS of 35S-35S-AMV-PSS-Tnos, has been constructed which were mobilized into Agrobacterium tumefaciens strain MP 90 disarmed Ti-plasmid. PSS gene were introduced into the binary vector pRD 400. The transgenic ginseg plants were propagated using repetitive secondary embryogenesis and introduced NPTII and PSS genes of the transgenic ginseng were successfully indentified by the PCR and survival test on the medium.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.430-435
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• 2013

Multidrug resistance, especially multidrug efflux mechanisms that extrude structurally unrelated cytotoxic compounds from the cell by multidrug transporters, is a serious problem and one of the main reasons for the failure of therapeutic treatment of infections by pathogenic microorganisms as well as of cancer cells. Streptococcus mutans is considered one of the primary causative agents of dental caries and periodontal disease, which comprise the most common oral diseases. A fragment of chromosomal DNA from S. mutans KCTC3065 was cloned using Escherichia coli KAM32 as host cells lacking major multidrug efflux pumps. Although E. coli KAM32 cells were very sensitive to many antimicrobial agents, the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetracycline, kanamycin, rhodamin 6G, ampicillin, acriflavine, ethidium bromide, and tetraphenylphosphonium chloride. This suggested that the cloned DNA fragment carries a gene encoding a multidrug efflux pump. Among 49 of the multidrug-resistant transformants, we report the functional gene cloning and characterization of the function of one multidrug efflux pump, namely MdeA from S. mutans, which was expressed in E. coli KAM32. Judging from the structural and biochemical properties, we concluded that MdeA is the first cloned and characterized multidrug efflux pump using the proton motive force as the energy for efflux drugs.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.40 no.1
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• pp.52-58
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• 1995

5-methyltryptophan（5MT） resistant mutant plants （MRl） were analyzed for characterization of anthranilate synthetase （AS） and tryptophan synthetase （TS） enzymes. The enzyme was measured in crude extracts from MR1 and control seedlings of Danggin inbred line. There was no significant difference in the level of AS between MR1 and control seedlings when grown on MS medium without 5MT. However, MR1 seedlings grown on MS medium with 25mg/L 5MT showed the level of AS twice higher than that of control seedlings. The activity of AS was inhibited to 50％ in untreated plants when 4mg /L L-tryptophan was added to their extracts. Extracts from MR1 plants required about four times higher concentration of amino acid to cause equal inhibition. In the TS assay, the activity observed in MR1 seedlings was four times higher than that of control seedlings. We have also isolated and sequenced the gene which encoding the tryptophan synthetase B subunit （TSB） from maize. The gene encodes polypeptides with high homology to TSB isolated from other plants, and is expressed in all the developmental stages examined. Northern hybridization analysis indicated that the gene expression in MR1 seedlings grown on MS medium showed a higher level than in control seedlings.