• Journal of Animal Science and Technology
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• 제46권5호
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• pp.729-734
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• 2004

본 연구는 한우의 mtDNA D-loop 영역 염기 변이가 산유량에 미치는 효과를 검증하기 위하여 수행하였다. 젖소의 mtDNA D-loop 영역에서 단일염기의 치환이 20 개의 지역에서 확인되었다. 그중 8, 169, 16042, 16051, 16057, 16093, 16119, 16122, 16209, 16255, 16302 bp 지역에서 높은 빈도의 염기치환이 검출되었다. 16119bp 지역에서는 T가 C로 치환된 빈도가 0 .138로 검출되었으며, 이 위치에서의 염기치환에 의한 산유량 효과는 -1.61 (p<0.1)로 나타났다. 16185bp 지역에서의 염기치환에 의한 산유량 효과는 3.557(p<0.05)로 나타났으며 G가 A로 치환된 빈도는 0.063로 나타났다. 본 연구에서 검출한 한우 다유계통 집단의 mtDNA내 D- loop 영역의 염기서열 변이 빈도와 유량과의 연관성분석 결과 등은 한우 집단의 유전적 변이성 추정과 좀 더 다양한 경제형질과의 관련성 분석으로 다양한 유전적 지표인자 발굴에 도움 될 것이며 이를 통해 분자유전학적 기법을 이용한 한우의 육종 전략을 확립하논데 기초 자료가 되는 것은 물론 우려나라 고유 품종인 한우의 유전자원 보존과 분자육종학적인 연구에 기초 자료로서 유용하게 활용 할 수 있을 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제32권7호
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• pp.911-917
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• 2022

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

• 생명과학회지
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• 제30권3호
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• pp.313-319
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• 2020

최근, 국내에서 발생하는 대가축의 질병은 바이러스 혹은 세균 등과 같은 병원체가 사료 섭취, 가축 간의 신체접촉, 호흡 등 다양한 경로를 통해 전파되어 발병되는 전염성 질병이다. 전염성 질병은 가축의 건강을 위협하고 생산성을 감소시키기 때문에 현장에서 조기 진단하여 개체 격리와 같은 통제 관리가 필수적이다. 기존 사용되고 있는 진단 키트들은 현장에서 사용하기에 용이하지 않으며 극소량의 감도에서 진단이 제한적인 단점을 가지고 있다. 그러므로, 현장에서 극소량의 감도와 진단의 편이성을 고려하여 DNA와 RNA 수준에서 진단할 수 있는 CRISPR/Cas 시스템은 최적의 시스템이라 할 수 있다. 본 연구논문에서는 대가축의 전염성 질병들을 현장에서 조기 진단함에 있어 CRISPR/Cas 시스템의 활용전략에 대해 소개하고자 한다. 최근 발견된 CRISPR/Cas 효소들은 2개의 클래스와 6가지 하위유형으로 분류되었다. 이 중에서 클래스 2에 포함되는 Cas 효소들은 대표적으로 제 2형에 Cas9, 제 5형에 Cas12a와 Cas12b, 제 6형에 Cas13a와 Cas13b가 있다. 현재까지 개발된 CRISPR/Cas 시스템들은 간단한 시각 신호를 통해 표적에 대한 정량 및 다중 감지가 가능하고 특히, 극소량 수준의 초고감도에서도 표적만을 진단할 수 있으며 단시간 이내에 진단 결과를 얻을 수 있다. 하지만 초고감도 DNA 혹은 RNA를 진단하기 위해 최적의 신호 증폭 방법과 결합되어야 하고 표적 DNA 혹은 RNA를 진단에 적합하도록 DNA를 RNA로, RNA를 DNA로 전변해야 하는 단점이 있다. 따라서, 현장에서 대가축의 전염성 질병을 조기에 진단할 수 있는 CRISPR/Cas 바이오센서를 개발하는데 있어 가축의 전염 매개체로부터 추출되는 병원체 유형(DNA 혹은 RNA)을 고려하여 최적의 Cas 효소를 선정하여야 하고 이에 따른 적절한 신호 증폭 방법이 결합되어야 한다. 따라서, CRISPR/Cas 시스템은 유전자 편집 방법을 사용하는 빠르고 효율적인 진단 도구이며 이 시스템은 소의 전염병을 조기에 진단하고 감염 확산방지에 도움될 수 있을 것으로 판단 되어진다.

• Journal of Microbiology
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• 제44권6호
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• pp.591-599
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• 2006

To address the diversity of cyclodextrin-producing P. graminis strains isolated from wheat roots and rhizospheres of maize and sorghum sown in Australia, Brazil, and France, restriction fragment length polymorphism analysis of part of genes encoding RNA polymerase (rpoB-RFLP) and DNA gyrase subunit B (gyrB-RFLP) was used to produce genetic fingerprints. A phylogenetic tree based on rpoB gene sequences was also constructed. The isolates originated from Brazil could be separated from those from Australia and France, when data from the rpoB-based phylogenetic tree or gyrB-RFLP were considered. These analyses also allowed the separation of all P. graminis strains studied here into four clusters; one group formed by the strains GJK201 and $RSA19^T$, second group formed by the strains MC22.02 and MC04.21, third group formed by the strains TOD61, TOD 221, TOD302, and TOD111, and forth group formed by all strains isolated from plants sown in Cerrado soil, Brazil. As this last group was formed by strains isolated from sorghum and maize sown in the same soil (Cerrado) in Brazil, our results suggest that the diversity of these P. graminis strains is more affected by the soil type than the plant from where they have been isolated.

• 식물병연구
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• 제24권2호
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• pp.138-144
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• 2018

In December 2016, stem rot symptoms were observed on Persian buttercup (Ranunculus asiaticus) plants in Chilgok, Gyeongbuk, Korea. In the early stage of the disease, several black spots appeared on the stem of infected plants. As the disease progressed, the infected stem cleaved and wilted. The causal agent was isolated from a lesion and incubated on Reasoner's 2A (R2A) agar at $25^{\circ}C$. Total genomic DNA was extracted for phylogenetic analysis. Based on the 16S rRNA gene analysis, the isolated strain was found to belong to the genus Pseudomonas. To identify the isolated bacterial strain at the species level, the nucleotide sequences of the gyrase B (gyrB) and RNA polymerase D (rpoD) genes were obtained and compared with the sequences in the GenBank database. As the result, the causal agent of the stem rot disease was identified as Pseudomonas marginalis. To determine the pathogenicity of the isolated bacterial strain, it was inoculated into the stem of healthy R. asiaticus plant, the inoculated plant showed a lesion with the same characteristics as the naturally infected plant. Based on these results, this is the first report of bacterial stem rot on R. asiaticus caused by P. marginalis in Korea.

• Mycobiology
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• 제32권3호
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• pp.123-127
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• 2004

Sweet persimmons have been increasingly cultivated in the southern part of Korea. However, anthracnose disease caused by Colletotrichum species is one of the major hindrances in cultivation and productions. In this study, we used polymerase chain reaction(PCR) to detect Colletotrichum species with the AFLP(amplified fragment length polymorphism) method. In AFLP, we used E3(5'-GACTGCGTACCAATTCTA-3') and M1(5'-GATGAGTCCTGAGTAACAG-3') primer combination and, as a result, 262 bp segment was observed in Colletotrichum species only. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless sweet persimmon plants. Based on sequence data for specific segments, Co.B1(5'-GAGAGAGTAGAATTGCGCTG-3') and Co.B2(5'-CTACCATTCTTCTA GGTGGG-3') were designed to detect Colletotrichum species. The 220 bp segment was observed in Colletotrichum species only, but not in other fungal and bacterial isolates.

• Asian-Australasian Journal of Animal Sciences
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• 제32권5호
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• pp.629-636
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• 2019

Objective: The study was designed to determine the cytotoxic effect of gallic acid (GA), obtained by the hydrolysis of tannins, on mice TM4 Sertoli cells apoptosis. Methods: In the present study, non-tumorigenic mice TM4 Sertoli cells were treated with different concentrations of GA for 24 h. After treatment, cell viability was evaluated using WST-1, mitochondrial dysfunction, cells apoptosis and necrosis was detected using JC-1, Hoechst 33342 and propidium iodide staining. The expression levels of Cyclin B1, proliferating cell nuclear antigen (PCNA), Bcl-2-associated X protein (BAX), and Caspase-3 were also detected by quantitative real-time polymerase chain reaction and Western-blotting. Results: The results showed that 20 to $400{\mu}M$ GA inhibited viability of TM4 Sertoli cells in a dose-dependent manner. Treatment with $400{\mu}M$ GA significantly inhibited PCNA and Cyclin B1 expression, however up-regulated BAX and Caspase-3 expression, caused mitochondrial membrane depolarization, activated Caspase-3, and induced DNA damage, thus, markedly increased the numbers of dead cells. Conclusion: Our findings showed that GA could disrupt mitochondrial function and caused TM4 cells to undergo apoptosis and necrosis.

• 대한수의학회지
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• 제38권4호
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• pp.763-770
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• 1998

Salmonella typhimurium is a causitive agent of diarrhea, fever, gastroenteritis, septicemia and sudden death in piglet. The currently used methods such as IFA, ELISA, DNA hybridization assay is needed a long-time and difficult to detect the organism in carrier animal or contaminated sample with other agents. However, it is important to detect rapidly and sensitively S typhimurium in piglet with other infectious pathogens to minimize an economic loss. Two sets of PCR primer, rfbJ forward primer(5'-AGAATATGTAATTGTCAG-3') and reverse primer(5'-TAACCGTTTCAGTAGTTC-3') were designed to amplify a 882 by fragment of Salmonella serovar type B gene. The target genomic DNA for PCR was extracted from the cultivated materials with various enrichment periods in a nonselective enrichment agar and broth with clinical specimens. The PCR is carried out here made it possible to detect the gene from two hours. Also, the amplified fragment with PCR was cloned into pGEM-T vector and digested with restrict enzyme, and sequenced for the identification of Salmonella serotype B rfbJ gene. Duplicated cultivation agar-broth followed by PCR were performed to develop a rapid and sensitive detection of S typhlmurium based on serovar type. This duplicated cultivation-PCR method provides a sensitive and rapid diagnostic tool to detect Salmonella from infected piglet with improved sensitivity.

• 대한기계학회논문집B
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• 제35권12호
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• pp.1309-1316
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• 2011

본 논문에서는 올리고-dT 자성입자와 측면방향 자기영동 기술을 기반으로 하는 초고속 RNA 추출칩을 소개한다. 센자성 와이어에 유도된 고구배자장에 의해 RNA가 결합된 올리고-dT 자성입자를 분리함으로써 용해된 혈액으로부터 고속으로 RNA를 추출하였다. 유속이 20 ml/h까지 자성입자를 80% 이상의 효율로 분리할 수 있었으며, 분리시간은 총 1분 이내였다. 추출된 시료로부터 단백질에 대한 RNA 흡광비율(absorbance ratio of RNA to protein: A260/A280)이 1.7 이상임을 확인하였고, 따라서 추출된 RNA가 매우 순수함을 보였다. 추출된 RNA를 사용하여 cDNA 합성과 PCR을 수행하였으며, 이로부터 개발된 초고속 RNA 추출칩이 적은 양의 시료만으로 간편하며 빠르고 정교한 RT-PCR을 수행하는데 실용적임을 확인하였다.

• Animal cells and systems
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• 제1권1호
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• pp.129-134
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• 1997

HLA-A2 is one of the most diversified HLA-class I antigen with 17 subtypes so far identified at the molecular level. HLA-A*02 subtyping has significant implications on the tissue typing for organ and bone marrow transplantations. Recently, DNA-based typing methods have been successfully applied to the elucidation of HLA gene polymorphisms. In the present study, HLA-A*O2 genotyping was established by using nested polymerase chain reaction-sequence specific primers (PCR-SSP) and distribution of A*O2 alleles were determined in Korean individuals. Genomic DNA prepared from four B-lymphoblastoid cell lines and lymphocytes from serologically defined 48 HLA-A2 Korean individuals by phenol/chloroform extractions was typed. The results of the four B-lymphoblastoid cells were consistent with the previous data typed by PCR analysis. Five A*O2 alleles-A*0201, A*0203, A*0206, A*0207 and A*0210-were commonly observed in a total of 17 A*02 alleles. Of these, A*0207 (f=49.0%) was the most frequent allele in Korean population. A*0206 (f=28.3%) and A*0201 (f=17.0%) were also found frequently while A*0203 and A*0210 types were observed in less than 5%. In conclusion, the high level of discrimination for HLA-A*O2 alleles will prove useful and informative in the study of transplant survival, and may identify the importance of allelic differences not readily detectable by serology on host and donor compatibility.