• BMB Reports
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• 제31권3호
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• pp.303-306
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• 1998

Serine proteinase cDNA fragment from protozoan parasite Acanthamoeba culbertsoni was amplified by the reverse transcription-polymerase chain reaction (RTPCR) using degenerate oligonucleotide primers derived from conserved serine proteinase sequences. The amplified DNA fragment was subcloned and sequenced. The sequence analysis and alignment showed significant sequence similarity to other eukaryotic serine proteinases and conservation of the His, Asp, and Ser residues that form the catalytic triad. The cDNA fragment was cloned into the pGEMEX-1 expression vector and expressed in Escherichia coli. A resulting fusion protein of 56 kDa had proteolytic activity. The fusion protein reacted with sera of mice immunized with purified serine proteinase of A. culbertsoni in Western blot. Immune recognition of the fusion protein by mouse antisera suggested that the fusion protein may be valuable as a diagnostic reagent.

• BMB Reports
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• 제29권3호
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• pp.272-275
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• 1996

The expression of genes induced by Dichloroacetate (DCA) treatment was analyzed by mRNA differential display. Purified total RNAs from rat liver treated with saline or DCA (100 mg/100 g b.w.) were reverse transcribed by using a set of oligonucleotide primers. The PCR products were resolved on a denaturing sequencing gel. PCR band representing mRNA expressed specifically in DCA-treated liver was excised and reamplified by PCR. A 120-bp c-DNA clone named IC1 was isolated and the DNA sequence of IC1 was analyzed. IC1 revealed 50% homology with 3' end of a mouse fibroblast growth factor mRNA This result indicates that DCA induces the expression of a gene which has a 50% homology with a Mouse fibroblast growth factor, and expression of this gene might be involved in non genotoxic process caused by DCA.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.881-884
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• 2000

A polymerase chain reaction (PCR) was performed to rapidly identify Lactobacillus plantarum from type strains and kimchi samples. The PCR experiments were carried out using specific oligonucleotide primer sets based on the 16S rRNA gene sequences of L. plantarum. The expected DNA amplificate of 419 bp was obtained when either purified DNA or whole cells of L. plantarum strains reacted with LP primers, yet not with any of the other strains. The PCR product was confirmed by DNA sequencing. Accordingly, since the PCR method used is simple, specific, and rapid, it will be useful for monitoring and evaluation L. plantarum in the mixed microbial population found in kimchi.

• BMB Reports
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• 제32권5호
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• pp.502-505
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• 1999

cDNA fragments of ovine liver pyridoxal kinase were amplified by PCR using degenerate oligonucleotide primers derived from partial amino acids sequences of the enzyme. Using PCR products as probes, several overlapping cDNA clones were isolated independently from an ovine liver and a human brain cDNA library. The largest cDNA clone for each was selected for sequence analysis. The ovine liver cDNA encodes a polypeptide of 297 amino acid residues with Mr of 32,925, whereas the human clone is comprised of an open reading frame encoding 312 amino acid residues with Mr of 35,102. The deduced sequence of the human brain enzyme is completely identical to that of human testes cDNA recently reported (Hanna et al., 1997). The ovine enzymes have approximately 77% sequence identity with the human enzyme although the two sequences are completely different in the N-terminus comprising 32 residues. This result suggests that pyridoxal kinase is highly homologous in mammalian species.

• 한국작물학회지
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• 제44권3호
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• pp.253-255
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• 1999

Reverse transcription and polymerase chain reaction (RT-PCR) assay was used to detect SMV strains. A pair of oligonucleotide primers were designed to include the cylindrical inclusion (CI) coding region between 4,176 to 5,560 nt. Amplification from the total RNA extracted from infected plants with SMV yielded a 1,385 bp DNA fragment. RT-PCR was shown to be $10^3$ times more sensitive than the ELISA assay and it could detect a virus in $10^{-6}$ dilution. Restriction enzyme analysis of RT- PCR products using EcoR I showed that SMV isolates were classified into six groups according to the patterns of restriction fragments.

• 한국식물병리학회지
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• 제14권6호
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• pp.589-593
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• 1998

DNA amplification by polymerase chain reaction (PCR) was used to specifically detect Plasmodiophora brassicae, causing clubroot of crucifers. On the basis of DNA sequence informations, an oligonucleotide primer set specific for the pathogen was designed form small subunit gene (18S-like) and internal transcribed spacer (ITS) region of ribosomal DNA. Primer ITS 5/PB-C produced an amplification product of approximately 520 bp in length with DNA from P. brassicae. However, no amplification product was produced with DNAs from several soil-borne fungi, Didymella bryoniae and Rhizopus stolonifer. Using these primers, the clubroot pathogen was readily detected from infected roots of crucifers, but not from healthy roots. Southern hybridization analysis further confirmed that the amplification product was originated from P. brassicae.

• Journal of Plant Biology
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• 제38권4호
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• pp.359-364
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• 1995

As the first step to elucidate the relationship between the structure and function of CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) in plants, the partial nucleotide sequence of soybean cytidylyltransferase cDNA was determined using a polymerase chain reaction (PCR). Degenerate oligonucleotide primers were synthesized from the conserved region revealed from the rat and yeast cytidylyltransferase DNA sequences. The catalytic domain region showed 78 and 76% homology with the rat and yeast amino acid sequences, respectivly. The hydropathy profile indicated that the C-terminal non-catalytic portion of the protein was very hydrophilic, and in the region between the catalytic domain and the C-terminal region, there was a large amphipathic $\alpha$-helical domain that was believed to bind the membrane surface in the active formation. There are 7 potential sites for phosphorylation by protein kinase C and 4 potential sites for phosphorylation by Ca2+/calmodulin kinase within the determined sequence.

• Journal of Microbiology
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• 제40권2호
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• pp.151-155
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• 2002

A Superoxide Dismutase (SOD) gene from the obligate intracellular bacterium Orientia tsutsugamushi has been cloned by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Nucleotide sequencing revealed that the predicted amino acid sequence was significantly more homologous to known iron-containing SODs (FeSOD) than to manganese-containing SODs (MnSOD). Conserved regions in bacterial FeSOD could also be seen. Isolation of the oriential SOD gene may provide an opportunity to examine its role in the intracellular survival of this bacterium.

• Parasites, Hosts and Diseases
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• 제35권4호
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• pp.295-298
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• 1997

요코가와흡충과 미야타흡충의 genomic DNA를 RAPD 분석을 이용하여 비교하였다. 상업적으로 구 입한 60-70%의 G+C 성분을 가진 무작위 10-mer oligonucleotide 표지자 (Kit A, Operon Technologies Inc., CalifDmia, USA) 20개 중에서 다음의 8개를 이용하여 두 홉충간에 구별이 가능한 밴드양상을 관찰할 수 있었다: OPA-02,5-TCCCGAGCTG-3; OPA-09,5-GGGTAACGCC-3; OPA-10, 5-GTGATCGCAG-3; OPA-11, 5-CAATCGCCGT-3; OPA-13, 5-CAGCACCCAC-3; OPA-17, 5-GACCGCTGT-3; OPA-19,5-CAAACGTCGG-3; OPA-20, 5-GTrCCGATCC-3. 이 연구의 결과로 미야타흡충은 요코가와흡충과 서로 다른 유전자 염기 서열을 가지고 있음이 암시 되었다.

• Journal of Microbiology
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• 제44권4호
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• pp.403-408
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• 2006

The purpose of this study was to determine the efficacy of a nucleic acid sequence-based amplification (NASBA) method of detecting noroviruses in artificially and naturally contaminated shellfish. We used 58 fecal samples that tested positive for noroviruses with electron microscopy (EM) to develop an NASBA assay for these viruses. Oligonucleotide primers targeting the polymerase coding region were used to amplify the viral RNA in an isothermal process that resulted in the accumulation of RNA amplicons. These amplicons were detected by hybridization with digoxigenin-labeled oligonucleotide probes that were highly specific for genogroup I (GI) and genogroup II (GII) of noroviruses. The expected band of 327bp appeared in denaturing agarose gel without any nonspecific band. The specific signal for each amplicon was obtained through Northern blotting in many repeats. All fecal samples of which 46(79.3%) belonged to GII and 12(20.6%) belonged to GI were positive for noroviruses by EM and by NASBA. Target RNA concentrations as low as 5pg/ml were detected in fecal specimens using NASBA. When the assay was applied to artificially contaminated shellfish, the sensitivity to nucleic acid was 100pg/1.5g shellfish tissue. The potential use of this assay was also confirmed in naturally contaminated shellfish collected from different ponds in Guangzhou city of China, of which 24 (18.76%) out of 128 samples were positive for noroviruses; of these, 19 (79.6%) belonged to GII and 5 (20.4%) belonged to GI. The NASBA assay provided a more rapid and efficient way of detecting noroviruses in fecal samples and demonstrated its potential for detecting noroviruses in food and environmental samples with high specificity and sensitivity.