• International Journal of Industrial Entomology and Biomaterials
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• 제25권2호
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• pp.195-203
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• 2012

In reared populations of Allomyrina dichotoma, commercial insects, the skin of last instar larvae was changed softer with opaque white, and infested grubs eventually died. To clarify the cause of the symptom, we collected the larvae of A. dichotoma from five farms and examined their intestinal bacterial florae using pyrosequencing technique. From those results, a member of Paenibacillus was found only in the larvae showing the symptom of disease. Through PCR analysis using a Paenibacillus specific primer set, we obtained the partial 16S rRNA gene sequence and confirmed the microbe as Paenibacillus sp. For clear identification, a whole guts was extracted from each larva showing the sign of the disease and incubated at $70^{\circ}C$ for 15 min to isolate spore forming bacteria. After then, each content of guts was cultured on $MYPGP_{NAL}$ agar medium($12.5{\mu}g/ml$ of nalidixic acid) at $30^{\circ}C$. The 16S rRNA gene sequence analysis for the isolated bacteria showed that they were closely related to P. rigui(97.9% similarity), to P. chinjuensis(96.1% similarity), and to P. soli(95.3% similarity). Additional tests including API test and cellular fatty acid composition analysis were performed, but the strain couldn't be identified at species level, suggesting it may represent novel species of the genus Paenibacillus.

• 한국발생생물학회지:발생과생식
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• 제19권3호
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• pp.135-144
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• 2015

Heat shock protein (HSP) 70, the highly conserved stress protein families, plays important roles in protecting cells against heat and other stresses in most animal species. In the present study, we identified and characterized four Hsp70 (RuHSP4, RuHSC70, RuHSP12A, RuGRP78) family proteins based on the expressed sequence tag (EST) analysis of the Korean rose bitterling R. uyekii cDNA library. The deduced RuHSP70 family has high amino acid identities of 72-99% with those of other species. Phylogenetic analysis revealed that RuHsp70 family clustered with fish groups (HSP4, HSC70, HSP12A, GRP78) proteins. Quantitative RT-PCR analysis showed the specific expression patterns of RuHsp70 family members in the early developmental stages and several tissues in Korean rose bitterling. The expression of 4 groups of Hsp70 family was detected in all tested tissue. Particularly, Hsp70 family of Korean rose bitterling is highly expressed in hepatopancreas and sexual gonad (testis and ovary). The expression of Hsp70 family was differentially regulated in accordance with early development stage of Rhodeus uyekii.

• Asian Pacific Journal of Cancer Prevention
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• 제16권13호
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• pp.5343-5348
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• 2015

Promoter hypermethylation of the runt-related transcription factor 3 (RUNX3) gene is associated with increased risk of hepatocellular carcinoma (HCC). Oxidative stress plays a vital role in both carcinogenesis and progression of HCC. However, whether oxidative stress and RUNX3 hypermethylation in HCC have a cause-and-effect relationship is not known. In this study, plasma protein carbonyl and total antioxidant capacity (TAC) in patients with hepatitis B virus (HBV)-associated HCC (n=60) and age-matched healthy subjects (n=80) was determined. RUNX3 methylation in peripheral blood mononuclear cells (PBMC) of subjects was measured by methylation-specific PCR. Effect of reactive oxygen species (ROS) on induction of RUNX3 hypermethylation in HCC cells was investigated. Plasma protein carbonyl content was significantly higher, whereas plasma TAC was significantly lower, in HCC patients than healthy controls. Based on logistic regression, increased plasma protein carbonyl and decreased plasma TAC were independently associated with increased risk for HCC. PBMC RUNX3 methylation in the patient group was significantly greater than in the healthy group. RUNX3 methylation in hydrogen peroxide ($H_2O_2$)-treated HepG2 cells was significantly higher than in untreated control cells. In conclusion, increase in oxidative stress in Thai patients with HBV-associated HCC was demonstrated. This oxidative increment was independently associated with an increased risk for HCC development. RUNX3 in PBMC was found to be hypermethylated in the HCC patients. In vitro, RUNX3 hypermethylation was experimentally induced by $H_2O_2$. Our findings suggest that oxidative stress is a cause of RUNX3 promoter hypermethylation in HCC cells.

• 미생물학회지
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• 제45권2호
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• pp.170-176
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• 2009

제주도에 서식하는 2종의 해양 해면, Dictyonella sp.와 Spirastrella abata의 공생세균 군집구조를 16S rDNA-DGGE(denaturing gradient gel electrophoresis) 방법에 의해 분석하였다. 해면으로부터 total genomic DNA를 추출하여 GC clamp가 추가된 세균에 특이적인 341f primer와 518r primer를 이용하여 16S rRNA gene의 V3 부위를 증폭한 후 DGGE 전기 영동하고 재증폭하여 염기서열을 분석하였다. 그 결과 Dictyonella sp.에서 8개, Spirastrella abata에서 7개의 band를 확인할 수 있었다. 공통된 주요 band가 없는 패턴을 나타내었으며, DGGE band로부터 DNA를 추출하여 부분 염기서열을 분석한 결과, NCBI에 등록된 서열들과 93%~98%의 유사도를 나타내었다. Dictyonella sp.의 주요 해면 공생세균은 uncultured Gammaproteobacteria, Spirastrella abata의 경우 uncultured Alphaproteobacteria, Firmicutes에 각각 포함되어 해면 종에 따른 숙주 특이적 분포를 보이는 것으로 나타났다.

• Molecules and Cells
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• 제26권3호
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• pp.250-257
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• 2008

Microsatellites or simple sequence repeats (SSR) are widely distributed in eukaryotic genomes and are informative genetic markers. Despite many advantages of SSR markers such as a high degree of allelic polymorphisms, co-dominant inheritance, multi-allelism, and genome-wide coverage in various plant species, they also have shortcomings such as low polymorphic rates between genetically close lines, especially in Capsicum annuum. We developed an alternative technique to SSR by normalizing and alternating anchored primers in random amplified microsatellite polymorphisms (RAMP). This technique, designated reverse random amplified microsatellite polymorphism (rRAMP), allows the detection of nucleotide variation in the 3' region flanking an SSR using normalized anchored and random primer combinations. The reproducibility and frequency of polymorphic loci in rRAMP was vigorously enhanced by translocation of the 5' anchor of repeat sequences to the 3' end position and selective use of moderate arbitrary primers. In our study, the PCR banding pattern of rRAMP was highly dependent on the frequency of repeat motifs and primer combinations with random primers. Linkage analysis showed that rRAMP markers were well scattered on an intra-specific pepper map. Based on these results, we suggest that this technique is useful for studying genetic diversity, molecular fingerprinting, and rapidly constructing molecular maps for diverse plant species.

• Parasites, Hosts and Diseases
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• 제58권1호
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• pp.61-65
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• 2020

Majority of the imported malaria cases in Korea is attributed to Plasmodium falciparum and P. vivax infections, whereas P. malariae and P. ovale infections are very rare. Falciparum and ovale malaria are mostly imported from Africa, while most of the vivax malaria cases are imported from Southeast Asia. Here, we report 6 Korean imported ovale malaria cases (4 males and 2 females) who had visited in Africa during 2013-2016. These subjects were diagnosed with P. ovale based on microscopic findings, Plasmodium species-specific nested-PCR, and phylogenetic clade using 18S rRNA gene sequences. We identified 2 P. ovale subtypes, 1 P. ovale curtisi (classic type) and 5 P. ovale wallikeri (variant type). All patients were treated with chloroquine and primaquine, and no relapse or recrudescence was reported for 1 year after treatment. With increase of travelers to the countries where existing Plasmodium species, the risk of Plasmodium infection is also increasing. Molecular monitoring for imported malaria parasites should be rigorously and continuously performed to enable diagnosis and certification of Plasmodium spp.

• Mycobiology
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• 제48권4호
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• pp.313-320
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• 2020

In Pleurotus sp., green mold, which is considered a major epidemic, is caused by several Trichoderma species. To develop a rapid molecular marker specific for Trichoderma spp. that potentially cause green mold, eleven Trichoderma species were collected from mushroom farms and the Korean Agricultural Culture Collection (KACC). A dominant fungal isolate from a green mold-infected substrate was identified as Trichoderma pleuroticola based on the sequences of its internal transcribed spacer (ITS) and translation elongation factor 1-α (tef1) genes. In artificial inoculation tests, all Trichoderma spp., including T. atroviride, T. cf. virens, T. citrinoviride, T. harzianum, T. koningii, T. longibrachiatum, T. pleurotum, and T. pleuroticola, showed pathogenicity to some extent, and the observed symptoms were soaked mycelia with a red-brown pigment and retarded mycelium regeneration. A molecular marker was developed for the rapid detection of wide range of Trichoderma spp. based on the DNA sequence alignment of the ITS1 and ITS2 regions of Trichoderma spp. The developed primer set detected only Trichoderma spp., and no cross reactivity with edible mushrooms was observed. The detection limits for the PCR assay of T. harzianum (KACC40558), T. pleurotum (KACC44537), and T. pleuroticola (CAF-TP3) were found to be 500, 50, and 5 fg, respectively, and the detection limit for the pathogen-to-host ratio was approximately 1:10,000 (wt/wt).

• 한국균학회지
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• 제27권2호통권89호
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• pp.124-131
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• 1999

본 실험은 진흙버섯류 7종 15균주에 대한 5.85 rDNA와 ITS 부위의 염기서열을 비교 분석함으로서 종간 및 종내의 유연관계를 조사하였다. 5.8S rDNA와 ITS 부위를 증폭하고자 18S rDNA의 3'말단 부위와 28S rDNA의 5'말단 부위에 두 개의 primer를 이용하여 PCR증폭을 행하였다. 5.8S rDNA와 ITS 부위를 증폭하여 염기서열을 비교 분석한 결과 본 실험에 공시된 Phellinus속의 제균종은 크게 4개의 cluster를 형성하였다. 첫 번째 cluster는 Phellinus hartigii IMSNU 32041, Phellinus robustus IMSNU 32068로 이루어졌고, 두 번째 cluster는 Phellinus linteus KCTC 6190, IMSNU 31014, DGUM 25003, DGUM 25004, Phellinus sp. DGUM 25007, Namsan No. 1과 Phellinus weirianus IMSNU 32021, 세 번째 cluster는 Phellinus laevigatus KCTC 6229, KCTC 6230과 Phellinus igniarius KCTC 6227, KCTC 6228로 이루어졌으며, Phellinus chrysoloma KCTC 6225와 KCTC 6226이 마지막 cluster를 형성하였다. 결과적으로 ITS 염기서열의 결과만으로 볼 때 Phellinus linteus와 Phellinus weirianus는 명확하게 종 단위의 개념을 정립할 수 없었다. 따라서 정확한 분류를 위해 생리학적, 분자생물학적 인 분류방법이 첨가되어야 하며, type strain에 대한 ITS 염기서얼도 결정되어야 한다. Phellinus속의 균들에서는 ITS2부위에 비해 ITS1부위의 변이율이 높았다. ITS 염기서열은 종 구분에 유용한 도구이며, 다른 균종들과 비교해 보았을 때 Phellinus linteus와 Phellinus weirianus에서만 ITS1 부위에서 특이적인 염기서열을 가지고 있었다.

• Journal of Microbiology and Biotechnology
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• 제30권4호
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• pp.533-539
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• 2020

A quantitative real-time polymerase chain reaction (QPCR) was applied to estimate biokinetic coefficients of Clostridium cadaveris and Clostridium sporogenes, which utilize protein as carbon source. Experimental data on changes in peptone concentration and 16S rRNA gene copy numbers of C. cadaveris and C. sporogenes were fitted to model. The fourth-order Runge-Kutta approximation with non-linear least squares analysis was employed to solve the ordinary differential equations to estimate biokinetic coefficients. The maximum specific growth rate (μ_max), half-saturation concentration (K_s), growth yield (Y), and decay coefficient (K_d) of C. cadaveris and C.sporogenes were 0.73 ± 0.05 and 1.35 ± 0.32 h^-1, 6.07 ± 1.52 and 5.67 ± 1.53 g/l, 2.25 ± 0.75 × 10¹⁰ and 7.92 ± 3.71 × 10⁹ copies/g, 0.002 ± 0.003 and 0.002 ± 0.001 h^-1, respectively. The theoretical specific growth rate of C. sporogenes always exceeded that of C. cadaveris at peptone concentration higher than 3.62 g/l. When the influent peptone concentration was 5.0 g/l, the concentration of C.cadaveris gradually decreased to the steady value of 2.9 × 10¹⁰ copies/ml at 4 h Hydraulic retention time (HRT), which indicates a 67.1% reduction of the initial population, but the wash out occurred at HRTs of 1.9 and 3.2 h. The 16S rRNA gene copy numbers of C. sporogenes gradually decreased to steady values ranging from 1.1 × 10¹⁰ to 2.9 × 10¹⁰ copies/ml. C. sporogenes species was predicted to wash out at an HRT of 1.6 h.

• 식물병연구
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• 제15권2호
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• pp.88-93
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• 2009

고추 유묘의 신초가 말라죽고 잎에 수침상점무의 증상으로부터 병원균을 분리하여 균총의 색깔과 형태, 포자의 모양과 크기를 관찰한 결과 균총의 색깔은 처음에는 핑크색 띄나 차츰 회색으로 변하였고, 분생포자는 방추형으로 크기가 $8.1-17.0{\times}2.0-3.8{\mu}m$이며 생장최적온도 $25-27^{\circ}C$이다. 종 특이적 프라이머를 이용하여 고추 유묘에서 분리한 균을 동정한 결과 C. acutatum에 특이적인 프라이머인 CaINT에서만 496 bp의 증폭산물을 얻었다. 이들 결과를 바탕으로 유묘에서 분리한 균은 Collectotrichum acutatum으로 동정하였다. 고추 유묘에서 분리한 균주를 고추묘 생육단계별로 병원성 검정한 결과 유묘 뿐 아니라 식물체 전 생육기에 잎 탄저병 증상을 나타내었으며 또한 고추 열매에서도 강한 병원성을 나타내었다.