• Microbiology and Biotechnology Letters
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• v.31 no.1
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• pp.25-31
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• 2003

Delftia acidovorans 51-A isolated from river water degrades aniline. In order to clone genes involved in aniline degradation, transposon Tn5-B20 was inserted into the strain 51-A to generate a mutant strain 10-4-2 that cannot utilize aniline as a carbon source. The mutant strain was not an auxotroph but could not degrade aniline. Southern hybridization analysis indicated that the transposon was inserted into the mutant bacterial DNA as a single copy. Flanking DNA fragment of Tn5-B2O insertion was cloned and sequenced. DNA sequence analysis revealed three ORFs encoding TdnQ, TdnT, and TdnA 1 that arc responsible for catechol formation from aniline through oxidative deamination. The analysis also confirmed that Tn5-B2O was inserted at the immediate downstream of tdnA1. The result suggests that the transposon insertion behind tdirA1 disrupted the pathway of the catechol formation from aniline, resulting in the mutant phenotype, which cannot degrade aniline. A large plasmid over 100-kb in size was detected from D. acidovorans 51-A and Southern hybridization analysis with Tn5-B2O probe showed that the transposon was inserted on the plasmid named pTDN51. Our results indicated that the tdn genes on pTDN51 of D. acidovorans 51-A are involved in aniline degradation.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.515-530
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• 1995

Coronaviridae에 속하는 transmissible gastroenteritis virus(TGEV)와 porcine epidemic diarrhea virus(PEDV)를 specific하게 detection할 수 있는 방법을 개발하고자 본 연구를 수행하였다. 두 바이러스 모두 RNA 바이러스이기 때문에 reverse transcription-polymerase chain reaction(RT-PCR)으로 nucleocapsid(N) protein gene의 cDNA를 증폭시켰다. SmaI으로 처리한 pTZ19R에 ligation시킨 후 염기서열을 밝히고자 sequencing하였다. 각각의 prototype virus와 비교하여 상동성을 밝혔다. 두 바이러스에 대한 cDNA probe를 제작하여 Southern blot hybridization을 실시하였다. TGEV의 경우 백신주인 P45와 병독주인 Miller strain을 사용하였다. cDNA를 증폭시키기 위해 N1/N1R과 N2/N2R 두 가지 primer를 이용한 결과, N1/N1R primer의 경우 586bp 크기의 PCR product를 얻을 수 있었고, N2/N2R primers로 582bp의 cDNA를 증폭시킬 수 있었다. PEDV 실험을 위하여 PED 임상 증상을 나타내는 분변을 이용하여 RT-PCR을 실시하였다. P2/P2R primer로 753bp의 PCR product를 얻을 수 있었다. TGEV의 두 가지 strain의 N protein gene을 sequencing하여 prototype인 Purdue strain과 염기서열 상동성을 조사한 결과, 97%이상의 높은 homology를 나타내었다. PED-V 역시 N protein gene을 sequencing하여 CV777과 염기서열 상동성을 조사한 결과 97%이상의 homology로 PEDV임을 알 수 있었다. TGEV와 PEDV의 염기서열을 비교한 결과 29%의 낮은 homology를 관찰할 수 있었다. 두 가지 바이러스의 N protein gene에 대한 cDNA probe를 제작하여 Southern blot hybridization을 한 결과, 각 바이러스에 매우 특이적 반응을 나타내었다.

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.116-121
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• 1987

In order to cloning of the nif genes of Enterobacter agglomerans NFB-264, the digested total DNA of the strain was ligated to pBR 322 and transformed into E. coli. Through the negative selection and colony hybridization, the transformants were obtained. The recombinant plasmids, pNEL 10 and pNES 20 were extracted from these transformants. It was known from Southern hybridization that pNEL 10 contained the 12 Mdal foreign DNA fragment hybridized with nif Q-X probe and pNES 20 included the 5 Mdal foreign DNA fragment hybridized with nif NE and nif YK probe.

• Molecules and Cells
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• v.23 no.1
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• pp.115-121
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• 2007

Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.48-48
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• 2003

본 실험은 spermatogonia 단계에 발현하는 유전자를 찾기 위하여 suppression subtractive hybridization를 수행하였다. 기존에 mouse에서는 spermatogonia 특이적인 유전자들이 밝혀져 있기 때문에 pig에 특이적인 유전자를 찾기 위하여 pig 250days testis와 pig 60days testis를 재료로 하여 실험하였다. SSH를 통하여 254days testis에 특이적으로 발현되는 후보유전자를 7개 찾았고 25days testis와 60days testis 의 Northern blot을 통하여 25days에 과발현하고 60days에 발현의 양이 대폭 줄어드는 spermatogonia 유전자로 생각되는 후보유전자 2개를 선택하여 pig tissue northern blot, genomic DNA southern blot, RT-PCR 그리고 In-situ hybridization을 수행하였다. Tissue northern blot과 RT-PCR을 통하여 후보자 1번은 간과 폐, 난소, 정소에서 발현하고, 후보유전자 15번은 난소와 정소에서만 특이적으로 발현함을 알았다. DNA sequence analysis와 NCBI Blast search를 통하여 후보자 1번은 다른 종에서 밝혀진 유전자였고 후보유전자 15번은 어느 종에서도 밝혀지지 않은 새로운 유전자였다. Degenerated primer를 통하여 후보자 1번의 pig full sequence를 밝히고 NCBI에 등록하였다. 그리고 In-situ hybridization을 통하여 후보유전자득이 20일째 testis의 Leydic cell에서 많이 발현되고 adult testis에서는 발현이 감소하는 결과를 얻었다. 이것으로 보아 위의 두 후보유전자는 spermatogonia에 직접 관련된 유전자이기 보다는 spermatogonia의 발달에 영향을 주는 leydic cell 특이발현을 가진 유전자로 사료되어진다.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.04a
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• pp.14-14
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• 1999

• Journal of Periodontal and Implant Science
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• v.32 no.2
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• pp.269-280
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• 2002

The purpose of this study is to develop species-specific DNA probes and polymerase chain reaction (PCR) primers for detection and identification of Prevotella nigrescens (P. nigrescens) 9336. This study procedure includes (1) whole-genomic DNA extraction of P. nigrescens 9336 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse Dot Hybridization method, (4) confirmation of strain-specific DNA probe by Southern blot analysis, (5) determination of nucleotide sequences of strain-specific DNA probe. Thirty-five restriction fragments of P. nigrescens 9336 genomic DNA digested with the Hind III were obtained. Reverse dot hybridization and Southern blot analysis data showed that three of them, Pn10, Pn23, and Pn35, could be P. nigrescens 9336-specific DNA probes. These data indicated that these DNA probes could be useful in detection and identification of the P. nigrescens 9336.

• Journal of Life Science
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• v.14 no.6 s.67
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• pp.1018-1022
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• 2004

Optimized conditions for Agrobacterium-mediated lisianthus pollen transformation were adjusted using various factors such as temperature, pH and sucrose concentration. Pollen tube growth was successfully achieved in a medium (pollen germination medium; PGM) containing $7-15\%$ sucrose with pH in the range of 5.5-7.0 at temperature of $20-27^{\circ}C$. Lisianthus pollen was vacuum-infiltrated with Agrobacterium cell suspension for 20 min, and transformed pollen was confirmed by GUS histochemistry and Southern hybridization following RT-PCR. Transgenic pollen system may be utilized for establishing an area of plant transient expression systems based on the convenient pollen transformation procedure presented in here.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.319-323
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• 2004

Soluble latex protein fraction excreted from Euporbia lathyris laticifer was resolved by 10% SDS-polyacrylamide gel electrophoresis to identify distinctively displayed latex major protein bands including ELp65, ELp55, ELp43, ELp32 and ELp23. Among them, ELp65 was purified by ammonium sulfate precipitation, gel permeation chromatography and ion exchange chromatography. Its N-terminal amino acid sequencing revealed its homology to the leading region of mature peptide of tomato p69a subtilisin-like protease, suggesting a certain role involved in plant defense system. In the analysis of Southern blot hybridization using PCR-amplified tomato p69a probe DNA, E. lathyris genome was suggested to have a gene family consisting of 3-5 gene members putatively encoding subtilisin-like proteases.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.393-399
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• 1998

Enterobacter sp.B54 which shows antagonistic activity to Phytophthora capsici on potato dextrose agar was selected among 112 strains isolated from Korean soil. After Tn5 lac-induced mutants were obtained through Pl :: Tn5 lac mutagenesis, 2 mutants for loss of antibiosis and 1 mutant for increased antibiosis were screened by using in vitro fungal inhibition assay. When the 3 mutants affected in antibiosis were analyzed by southern hybridization with pRZ102 (ColEl :: Tn5) as a probe, its results suggest that Tn5 lac was randomly inserted into different chromosomal sites in these mutants.