PCR Method Based on the ogdH Gene for the Detection of Salmonella spp. from Chicken Meat Samples

진언호,조성학,Min-Gon Kim,하상도,김근성,이규호,김광엽,정덕화,이영춘,Cheorl-Ho Kim 한국미생물학회 2004 The journal of microbiology Vol.42 No.3

In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony- forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.

참깨 myo-inositol 1-phosphate synthase 유전자의 특성과 기능분석에 관한 연구

진언호,천재안,정정한 한국생명과학회 2003 생명과학회지 Vol.13 No.4

1845 bp의 SeMIPS cDNA를 발육종자에서 분리하고 이 cDNA의 구조와 특성을 분석하였다. 이 cDNA는 엽록체로 향하는 신호펩타이드의 아미노산 서열이 존재하지 않아서 세포질형 MIPS로 예상되었다. 또한 이 cDNA의 아미노산 서열의 유사성은 다른 MIPS와 비교한 결과 60-94%의 높은 아미노산 서열 유사성을 보여주었으며, 특히 식물끼리의 유사성이 훨씬 높았다. Northern blot분석에서 볼 때 참깨의 조직별 SeMIPS mRNA는 완숙종자, 줄기, 뿌리에서는 약하게 발현되었고, 잎에서는 비교적 강하게 발현되는 현상을 보여주었다. Yeast 돌연변이체를 통한 활성 시험에서는 SeMIPS가 myo-inositol 1-phosphate synthase의 효소활성을 가지고 있다는 실험적 증거를 얻었으며, C-말단 아미노산 20개가 효소활성에 필수적이라는 사실이 본 실험에서 검증되었다. A cDNA (SeMIPS) encoding myo-inositol 1-phosphate synthase has been isolated from developing sesame (Sesamum indicum L. cv. Dan-Baek) seeds and its structure and function analyzed. The SeMIPS protein was highly homologous with those from plant species (88-94%), while a much lower degree of sequence homology (60%) was found with that of human. The functional domains commonly found in MIPS protein were identified and their amino acid residues were compared with each other. Northern blot indicated that the expression of the SeMIPS gene might be organ-specifically regulated. A complementation assay based on a yeast mutant system confirmed that the SeMIPS gene encodes a myo-inositol 1-phosphate synthase (MIPS) of sesame by showing functional expression of the SeMIPS cDNA in the yeast mutants containing the disrupted INO1 gene.

Linoleic acid 합성유전자의 특성분석과 발현에 관한 연구

진언호,김영길,정정한 한국생명과학회 2000 한국생명과학회 학술발표회 Vol.29 No.-

형질전환 참깨 Hairy Root 배양에 의한 재조합 곰팡이 Phytase의 발현과 생산물의 분리 정제

천재안,진언호,이진우,이영병,이신우,정정한 한국생물공학회 2004 한국생물공학회 학술대회 Vol.2004 No.10

Molecular Characterization of NbCHMP1 Encoding a Homolog of Human CHMP1 in Nicotiana benthamiana

양경실,배현숙,진언호,Junwon Kim,Kiwon Song,Soo Jin Kim,황인환,임용표 한국분자세포생물학회 2004 Molecules and cells Vol.17 No.2

In humans, CHMP1 encodes a protein of dual function that plays a role in both modification of chromatin structure and endosomal vesicle trafficking. Recently, it was found that sal1, a CHMP1 homolog in maize, is important for the development of the aleurone cell layers in maize endosperm. In this study, we investi-gated the structure and function of a Nicotiana ben-thamiana CHMP1 homolog designated NbCHMP1. NbCHMP1 encodes a small protein with a bipartite nuclear localization signal at its N-terminus, and good homology with the corresponding genes from diverse plants and animals. NbCHMP1 mRNA was present at comparable levels in stems, roots, flowers, and leaves. A GFP fusion of the full length NbCHMP1 protein was localized to the cytosol in distinct structures, while a GFP fusion of its N-terminal 80 aa was targeted to the nucleus, suggesting dual-targeting of NbCHMP1 in plants. Overexpression of NbCHMP1 in yeast did not affect its growth and the expressed protein was pre-sent in the cytosol in particulate form. Virus-induced gene silencing of NbCHMP1 resulted in subtle altera-tions of leaf morphology and color, without signifi-cantly affecting plant viability or development. Thus the CHMP1 homolog apparently does not play an es-sential role in the development of the vegetative tissues of N. benthamiana, in contrast to the essential role of sal1 in formation of the aleurone cell layers during maize endosperm development.

Molecular Cloning and Characterization of a Large Subunit of Salmonella typhimurium Glutamate Synthase (GOGAT) Gene in Escherichia coli

정태욱,이동익,김동수,진언호,박춘,김종국,김민곤,하상도,김근성,이규호,김광엽,정덕화,김철호 한국미생물학회 2006 The journal of microbiology Vol.44 No.3

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%. Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.