• Title/Summary/Keyword: ITS-PCR

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Isolation and Characterization of the sod2$^{2+}$ Gene Encoding a Putative Mitochondrial Manganese Superoxide Dismutase in Schizosaccharomyces bombe

  • Jeong, Jae-Hoon;Kwon, Eun-Soo;Roe, Jung-Hye
    • Journal of Microbiology
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    • v.39 no.1
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    • pp.37-41
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    • 2001
  • The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

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Molecular Cloning and Characterization of DNA Repair Related Gene in Yeast

  • Kang, Seon-Ah;Park, In-Soon
    • Journal of Life Science
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    • v.10 no.1
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    • pp.40-44
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    • 2000
  • The SNF2/SW ATPase/helicase family comprises proteins form a variety of species with in vivo functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. Here, we reported the characterization of h게2+gene which was iolated by PCR amplification using the conserved domain of SNF2 motifs. Sequence analysis of PCR product showed striking evolutionary conservation among the SNF2 family of proteins. Two transcripts of 6.7 and 3.4 Lb were detected by Northern blot analysis. furthermore, the intensities of these two bands were increased by ultraviolet(UV) irradiation. These results indicate that the hrp2+ is a novel member of the SNF2 family of proteins and is one of the UV-inducible genes in S. pombe. To determine the level of transcripts of hrp2+ gene during cellular growth, Northern blot analysis were performed. This result indicates that the level of hrp2+transcript reached its maximum before cells entered the exponential growth phase. This suggests that hrp2+ gene is experssed mainly at the early stage of cell growth.

Polymorphism of Insulin-like Growth Factor 1 Receptor Gene in 12 Pig Breeds and Its Relationship with Pig Performance Traits

  • Wang, Wenjun;Ouyang, Kehui;Su, Xifan;Xu, Mingsheng;Shangguan, Xinchen
    • Asian-Australasian Journal of Animal Sciences
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    • v.19 no.11
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    • pp.1541-1545
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    • 2006
  • The polymorphism of insulin-like growth factor I receptor (IGFIR) gene in 12 pig breeds (total n = 593) was detected by PCR-SacII-restriction fragment length polymorphism and allele A (379 bp) or allele B (235 bp and 144 bp) observed. In the studied breeds, it was found that European pigs principally carried allele A, while Chinese native pig breeds principally carried allele B. In addition, the role of pig IGFIR was investigated in 156 Wanbai pigs and 212 Large Yorkshire pigs. Growth related variables including body weight at birth, 2-, 4- and 6-mo of age and backfat thickness and lean percentage estimated by ultrasonography at 6-mo of age were recorded in analyzing the association between IGFIR gene polymorphism and growth traits. AA-genotype pigs exhibited greater (p<0.05) body weights (BW) at birth, 2- and 6-mo of age, but not at 4-mo of age, than those of the BB-genotype in Wanbai and Yorkshire breeds. Moreover, in the Yorkshire breed, AA-genotype pigs had less backfat thickness (p<0.05) and greater lean percentage (p<0.01) than the BB genotype. Based on these results, it is necessary to do more studies on IGFIR before introducing the IGFIR locus into breeding programs.

Rapid and Sensitive Detection of Hepatitis C Virus in Clinical Blood Samples Using Reverse Transcriptase Polymerase Spiral Reaction

  • Sun, Wenying;Du, Ying;Li, Xingku;Du, Bo
    • Journal of Microbiology and Biotechnology
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    • v.30 no.3
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    • pp.459-468
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    • 2020
  • This study established a new polymerase spiral reaction (PSR) that combines with reverse transcription reactions for HCV detection targeting 5'UTR gene. To avoid cross-contamination of aerosols, an isothermal amplification tube (IAT), as a separate containment control, was used to judge the result. After optimizing the RT-PSR reaction system, its effectiveness and specificity were tested against 15 different virus strains which included 8 that were HCV positive and 7 as non-HCV controls. The results showed that the RT-PSR assay effectively detected all 8 HCV strains, and no false positives were found among the 7 non-HCV strains. The detection limit of our RT-PSR assay is comparable to the real-time RT-PCR, but is more sensitive than the RT-LAMP. The established RT-PSR assay was further evaluated for detection of HCV in clinical blood samples, and the resulting 80.25% detection rate demonstrated better or similar effectiveness compared to the RT-LAMP (79.63%) and real-time RT-PCR (80.25%). Overall, the results showed that the RT-PSR assay offers high specificity and sensitivity for HCV detection with great potential for screening HCV in clinical blood samples.

Identification of Differentially Expressed Genes in Murine Hippocampus by Modulation of Nitric Oxide in Kainic Acid-induced Neurotoxic Animal Model

  • Suh, Yo-Ahn;Kwon, O-Min;Yim, So-Young;Lee, Hee-Jae;Kim, Sung-Soo
    • The Korean Journal of Physiology and Pharmacology
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    • v.11 no.4
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    • pp.149-154
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    • 2007
  • Kainic acid (KA) causes neurodegeneration, but no consensus has been reached concerning its mechanism. Nitric oxide may be a regulator of the mechanism. We identified differentially expressed genes in the hippocampus of mice treated with kainic acid, together with or without L-NAME, a nonselective nitric oxide synthase inhibitor, using a new differential display PCR method based on annealing control primers. Eight genes were identified, including clathrin light polypeptide, TATA element modulatory factor 1, neurexin III, ND4, ATPase, $H^+$ transporting, V1 subunit E isoform 1, and N-myc downstream regulated gene 2. Although the functions of these genes and their products remain to be determined, their identification provides insight into the molecular mechanism(s) involved in KA-induced neuronal cell death in the hippocampal CA3 area.

Morphology, Pathogenicity and Molecular analysis of Alternaria Isolates from Solanaceous Crops (oral)

  • Cho, H.S.;Park, M.S.;Kim, B.R.;Yu, S.H.
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.112.3-113
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    • 2003
  • More than 30 isolates of Alternaria were obtained from various solanaceous crops in Korea. For all isolates, morphological characteristics of the conidia were determined and compared with those of representative isolates of A. solani and A. tomatophila. Pathogenicity test was performed to Potato, tomato, egg plant and red Pepper and molecular characteristics of them including the representative isolates were determined using sequence analyses of ITS rDNA and histone H3 gene, and URP-PCR analysis. Based on morphological characteristics, the isolates from the solanaceous crops were grouped as identical or very similar to either A. tomatophila(ATO), A. solani(ASO), and unidentified Altemaria sp.(ASP). Among the molecular markers used in this study, the URP-PCR analysis was found to be appropriate for taxonomic resolution of these species. Based on the conidial morphology, pathogenicity test and molecular characteristics, A. tomatophila(early blight of tomato) could be distinguished from A. solani(early blight of potato), and the Alternaria sp.(ASP) from potato, which was closely related to A. solani in conidial morphology, was considered as a new species.

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The occurrence trend of the RSV and its coining of coat protein of korean strain.

  • Park, Jo-Im;Lee, Bong-Choon;Hong, Yeon-Kyu;Kwak, Do-Yeon;Oh, Byeong-Geon;Park, Sung tae
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.114.2-115
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    • 2003
  • Rice stripe virus causes severe damage to rice in Korea, Japan and China. RSV is a type member of the tenuivirus group and transmitted by the small brown planthopper, Laodeiphax striatellus, in a persistant manner. Until now, occurrence of RSV is limited in of southern part of Korea. But recently occurrence of RSV is increasing and spreading in central part of Korea including Chungcheong and Kyonggi province. So we analyzed recent occurrence trend of RSV which is increased and cloned and sequenced coat protein gene for isolating of RSV strain. Infected rice of each species(Ilpumbyeo, Sindongjinbyeo, Keumobyeo-2, Dongjinbyeo, Jongnambyeo, Samcheonbyeo, etc.)is collected, we extracted total RNA from infected leaves and detected RSV viral RNA by reverse transcription(RT)-PCR using specific primer of coat protein gene. The result of RT-PCR, we observed specific band. We already cloned cDNA from the band, is analyzing sequence variety and homology of each species.

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Analysis genetic diversity of Plasmodiophora brassicae using RFLP and RAPD(oral)

  • Heo, Seung-Hwan;Jang, Chang-Soon;Lee, Hyoun-Kyoung;Lee, Woo-Chung;Jang, Se-Jeong;Kim, Hong-Gi
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.112.1-112
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    • 2003
  • Genetic diversity of Plasmodiophora brassicae from major chinese cabbage cultivating areas in Korea was analyzed by using PCR-RFLP and RAPD. Single spores of P brassicae isolated from galls of club root made induce lesion on chinese cabbage successfully. The PCR-RFLP and RAPD by primers PbITS, URP 3, 6 and OPA 7 revealed that single spore isolates showed various DNA polymorphisms among them unrelated geographic origins. These results indicate that P. brassicae population in Korea showed genetic difference among them. This study could be facilitate to identify genetic characteristics ofP. brassicae based on DNA polymorphisms between single spore isolates and to get basic information which can be used to advanced resistance breeding against club root of chinese cabbage.

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Characterization of Grapevine leafroll-assoiated virus 1 and Grapevine leafroll-associated virus 3 isolated from Vitaceae in Korea.

  • Kim, Hyun-Ran;Lee, Sin-Ho;Kim, Jae-Hyun;Yoon, Gum-Ook;Kim, Jeong-Soo
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.138.2-139
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    • 2003
  • Grapevine leafroll-associated 1 virus (GLRaV-1) and Grapevine leafroll-associated 3 virus (GLRaV-3), member of the genus Ampelovirus, are important viral disease of grapevine in the world. these viruses transmitted only dicotyledonous host by vectors such as mealybugs and there is no suitable herbaceous host for virus. The diseased leaves turn yellowish or reddish depending on cultivars and viruses. Viruses are existed at low concentration and ununiformly distribution in grapevine. Using small-scale double-stranded RNA (dsRNA) extraction method, reverse transcription and polymerase chain reaction (RT-PCR) product of 1Kb long which encoded of coat protein (CP) gene for both viruses was successfully amplified with a specific primers. The RT-PCR product was cloned into the plasmid vector and its nucleotide sequences were determined from selected recombinant cDNA clones. Sequence analysis revealed that the CP of GLRaV-1 consisted of 969 nucleotide, which encoded 323 amino acid residues and CP of GLRaV-3 consisted of 942 nucleotide, which encoded 314 amino acid residues. The CP of GLRaV-1 and GLRaV-3 has 93.8% and 98.7% amino acid sequence identities, respectively.

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Bacterial Communities Developing during Composting Processes in Animal Manure Treatment Facilities

  • Yamamoto, Nozomi;Otawa, Kenichi;Nakai, Yutaka
    • Asian-Australasian Journal of Animal Sciences
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    • v.22 no.6
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    • pp.900-905
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    • 2009
  • We analyzed succession of the bacterial communities during composting of animal manure in three individual facilities. Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) targeting for the bacterial 16S rRNA gene were used to clarify the changes of bacterial community throughout each composting process. Our study revealed that the bacterial community structures differed during the composting process. The bacterial community in composting of facility A showed little change throughout the process. In the compost sample from facility B, its community had a small shift as the temperature increased. In compost from facility C, the temperature dynamically changed; it was shown that various bacterial communities appeared and disappeared as follows: in the initial phase, the members of phylum Bacteroidetes dominated; in the thermophilic phase, some bacteria belonging to phylum Firmicutes increased; towards the end, the community structure consisted of three phyla, Bacteroidetes, Firmicutes, and Proteobacteria. This study provides some information about the bacterial community actually present in field-scale composting with animal manure.