• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.113.2-114
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• 2003

Five pepper-infecting potyviruses, Pepper mottle virus (PepMoV), Chilli veinal mottle virus (CVMV), Pepper veinal mottle virus (PVMV), Pepper severe mosaic virus (PSMV) and Tobacco each virus (TEV), are known filamentous virus and can be infected pepper crops systemically. To understand pathology and genome information of the five viruses on pepper plants, host reactions and sequences were compared to the 5 viruses. Five potyviruses were inoculated onto some typical cultivars of hot peppers and compared their symptoms, and virus accumulations. A set of degenerate primers for potyviruses were applied to 5 viruses and RT-PCR was performed. RT-PCR products containing partial nuclear inclusion b and coat protein (CP) genes were cloned. Then, oligo dT primer and species-specific primer were redesigned to amplify the C-terminal part of CP and 3' noncoding regions of each viruses. Sequences of the viruses were analyzed and compared to serological relationships among the viruses. The data can be useful for screening of potyviruses in pepper plants and pathogen-derived transgenic pepper plant development.

• Mycobiology
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• v.42 no.1
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• pp.46-51
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• 2014

A degenerated strain of Pleurotus eryngii KNR2312 was isolated from a commercial farm. Random amplified polymorphic DNA analysis performed on the genomic DNA of the normal and degenerated strains of this species revealed differences in the DNA banding pattern. A unique DNA fragment (1.7 kbp), which appeared only in the degenerated strain, was isolated and sequenced. Comparing this sequence with the KNR2312 genomic sequence showed that the sequence of the degenerated strain comprised three DNA regions that originated from nine distinct scaffolds of the genomic sequence, suggesting that chromosome-level changes had occurred in the degenerated strain. Using the unique sequence, three sets of PCR primers were designed that targeted the full length, the 5' half, and the 3' half of the DNA. The primer sets P2-1 and P2-2 yielded 1.76 and 0.97 kbp PCR products, respectively, only in the case of the degenerated strain, whereas P2-3 generated a 0.8 kbp product in both the normal and the degenerated strains because its target region was intact in the normal strain as well. In the case of the P2-1 and P2-2 sets, the priming regions of the forward and reverse primers were located at distinct genomic scaffolds in the normal strain. These two primer sets specifically detected the degenerate strain of KNR2312 isolated from various mushrooms including 10 different strains of P. eryngii, four strains of P. ostreatus, and 11 other wild mushrooms.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1290-1297
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• 2008

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting $G_{1PAO},\;G_{2PAO},\;and\;G_{3PAO}$ groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non-Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (GINPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the $G_{4PAO}$ group of Accumulibacter phosphatis, which suggests that GINPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.64-64
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• 2003

The sex differentiation of fishes occurs under the control of genetic and various environmental factors. DM-domain containing genes are novel zinc finger transcription factors and play key roles in sex determination. In order to isolate the wrasse DMRT (wDMRT) cDNA from the protogynous wrasse (Halichoeres tenuispinnis), the wrasse testis cDNA library was screened using the $^{32}$ P-labeled PCR products, which were amplified with the degenerate primers from conserved DM-domain regions of several DMRT genes. Among a few positives obtained through screening, the full length wDMRT cDNA of 2.9kb size encoding a predicted 300 amino acid residues was isolated. The sequence analysis exhibited 60%, 43% sequence identity with rainbow trout and tilapia DMRT1, respectively. RT-PCR assay showed that wDMRT was expressed specifically in male testis. Also, wDMRT gene was strongly expressed in May during reproductive season, when the reproductivity of wrasse is most active. This results suggested that wDMRT gene function in testis differentiation The conserved DM-domain regions were amplified using PCR from DMRT genes of several species among Labridae, and their sequences were determined. The sequence of DM-domain region of Halichoeres. tenuispinis was identical to those of Pseudolabrus japonicus, Pteragogus flagellifera, and showed 94% identity with that of Halichoeres poecioptrerus.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.420-422
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• 2005

TMC (Tautomycetin) is a liner polyketide immunosuppressive antifungal compound produced by Streptomyces spp. Inhibition of T cell proliferation with TMC was observed highly efficient at 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A. To elucidate the biosynthetic pathway of TMC, a genomic DNA library was constructed using a E. coil-Streptomyces shuttle cosmid vector, pOJ446. The DNA libraries were screened by colony blot hybridization using several polyketide ${\beta}-ketosynthase$ (KS) probes amplified from TMC-producing Streptomyces genomic DNA using polymerase chain reaction (PCR), of which the degenerate primers were designed based on the highly conserved sequences present in KS domains of various type I polyketide synthase genes in Streptomyces species. This library construction and screening approach led to the isolation of several positive cosmid clones representing type I polyketide biosynthetic gene clusters. In addition, a Streptomyces regulatory gene called afsR2 (a global regulatory gene stimulating antibiotic production in both S. coelicolor and S. lividans) was successfully integrated into the TMC-producing Streptomyces chromosome via E. coil-Streptomyces heterologous conjugation mehtod. The more detailed results of production improvement and genetic characterization of TMC-producing Streptomyces spp. will be discussed.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.147.1-147
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• 2003

Freesia, a member of the Iridaceae family, has fragant, tubular shaped flowers and is very popular ornamental plants in the world. Diseased freesia plants showing systemic leaf streak mosaic symptoms were collected from a cultivated farm in Kyonggi province, Korea in 2003, and its causal agents were investigated. Two viruses, Cucumber mosaic virus (Fr-CMV) and a potyvirus, were identified from the leaf tissues of the diseased freesia based on sequence analysis and host range tests. CMV-Fr could infect systemically on Chenopodium quinoa, C. amaranticolor, N. glutinosa, and N. benthamiana, and this biological property is distinguishable from ordinary strains of CMV. A filamentous potyvirus-shaped virus could not infect general indicator plants by mechanical inoculation. Single RT-PCR products was successfully amplified with a set of degenerate primers specific to the Potyvirus genus and total nucleic acids from the infected tissues, and was cloned into the pGEMT-Easy vector. Nucleotide sequences confirmed it belongs to the Potyvirus genus with either a new species or an isolate of Freesia mosaic virus (no information is available for the FrMV). This is the first report of FrMV in Korea and more characterizations of the two viruses are in progress.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.529-533
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• 2001

The ${\beta}$-Amylase cDNA fragment from the oomcete Saprolegnia parasitica was cloned by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligonucleotide primers derived from conserved ${\beta}$-amylase sequences. The 5'and 3'regions of the $\beta$-amylase gene were amplified using the rapid amplification of cDNA ends (rACE) system. It consisted of an open reading frame of 1,350 bp for a protein of 450 amino acids. Comparison between the genomic and cDNA sequences revealed that the intron was not present in the coding region. The deduced amino acid sequence of the ${\beta}$-amylase gene had a 97% similarity to the ${\beta}$-amylase of Saprolegnia ferax, followed by 41% similarity to those of Arabidopsis thaliana, Hordeum vulgare, and Zea mays. The ${\beta}$-amylase gene was also expressed in Saccharomyces cerevisiae by placing it under the control of the alcohol dehydrogenase gene (ADC1) promoter.

• The Plant Pathology Journal
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• v.27 no.1
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• pp.33-36
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• 2011

The genome of a potyvirus isolate associated with chlorotic spots and vein banding symptoms on Spathiphyllum spp., in Andhra Pradesh state, India was amplified by RT-PCR using degenerate potyvirus primers, amplicons cloned, and sequence (1.6 kb) analyzed. This virus isolate shared maximum identity of 74.8% and 80.2% at coat protein (CP) gene nucleotide (906 nucleotides) and amino acid (302 amino acids) levels, respectively with Dasheen mosaic virus (DsMV)-M13 isolate reported from China. But its 3'-UTR (258 nucleotides) had maximum identity of 62.5% with DsMV-Vietnam isolate. The deduced molecular weight of CP is 33.57 kDa and it contained DAG triplet in its N-terminal region. In CP amino acid based phylogenetic analysis, this virus isolate represented a separate branch but closer to DsMV isolates cluster. Based on the molecular criteria set for the discrimination of species and genus in the Potyviridae family, the present virus isolate was identified as a distinct virus species in the genus Potyvirus and proposed the name Spathiphyllum chlorotic vein banding virus (SCVbV).

• Journal of Life Science
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• v.14 no.1
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• pp.38-44
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• 2004

Two genes, SinIFS1 and SinIFS2 from Korean soybean cultivar, Sinpaldalkong known as one of isoflavonerich cultivars, were cloned with PCR and degenerate primers. The sequences of two genes were analyzed with previously reported IFS genes of leguminous plants and their expression pattern in various environmental conditions was surveyed. The genomic clone of SinIFS1 contained 1,828bp nucleotides and encoded a polypeptide of 521 amino acids, and 1912bp nucleotides and a polypeptide of 521 amino acids for SinIFS2. Both genes included several conserved motifs, oxygen binding and activation (A/G-G-X-E/D-T-T/S), ERR triad (E...R....R), and heme binding (F-X-X-G-X-R-X-C-X-G) domain, which are typical in any member of cytochrome P45O superfamily. Very high sequence homology (>98%) was observed in the comparison with other IFSs of legumes. In the northern blot analysis to check the expression and increase of SinIFS1 to various environmental renditions (low temperature, light, dark, UV, and fungal elicitor), the most significant induction, more than 6 times of transcript level compared to the dark treatment as a control, was observed from the fungal elicitor treatment. The next up-regulated expression was from UV treatment (4${\times}$), low temperature and light conditions.

• KSBB Journal
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• v.16 no.6
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• pp.626-631
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• 2001

Lactobacilli have been considered to play important roles in the health of human vagina. They secrete inhibitory substances to prevent vaginal infection by pathogenic organisms. In a previous study, we have isolated several lactobacilli from Korean woman and one of them (KLB46) was selected and indentified as Lactobacillu crispatus which showed high antimicrobial activity. In this study. cold adaptation prior to subsequent stresses exposure was examined whether L. crispatus KLB46 maintain the viability better than the non-adapted calls under stresses. For pharmaceutical formulation, the lyophilization process is required where stresses such as freezing/thawing and dehydration are routinely applied. Formulated L. crispatus KLB46 can be used for ecological treatment of bacterial vaginosis. The response of cold-adapted cells to other environmental stresses such as acid, heat, ethanol, NaCl, and H$_2$O$_2$ was also examined. The results showed that cold-adapted cells maintained higher survival rate compared with the non-adapted cells (freezing-thawing. 3-folds; dehydration: 3-folds; acid, 3-folds; heat, 10-folds). However, we did net observe any positive effect of cold adaptation on other stresses such as ethanol, NaCl and H$_2$O$_2$. When chloramphenicol was added during cold adaptation, adaptation effect was abolished. This confirms that de novo protein synthesis is necessary during the adaptation process. Moreover, we have identified cold shock protein homolog that codes for a major cold shock protein by PCR amplification using degenerate primers.