• Journal of the korean academy of Pediatric Dentistry
• /
• v.33 no.2
• /
• pp.304-310
• /
• 2006

Prevotella intermedia is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. This study has identified a hemin-binding P. intermedia protein by expression of a P. intermedia genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. The genomic library of P. intermedia was constructed into plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$, and screened for recombinant clones using heminbinding activity by plating onto hemin-containing agar. Approximately 5,000 recombinant E. coli colonies were screened onto LB-amp-hemin agar, single clone(pHem1) was exhibited a clearly pigmented phonotype. The 2.5kb insert DNA of pHem1 was determined by restriction enzyme mapping. Southern blot analysis of BamHI, BglII, EcoRI, HindIII and PstI-digested P. intermedia DNA indicated that single copy of the gene was present in the genome. Northern blot analysis revealed that the size of transcript was approximately 1.8 kb. The cloned gene contained a single ORF, consisting of approximately 850-residue amino acids. A BLAST search of the Institute for Genomic Research genes with similar nucleotide sequence revealed no significant similarity It needs further investigation to clarify the mechanisms of heme uptake in P. intermedia.

• Journal of Life Science
• /
• v.16 no.2 s.75
• /
• pp.360-364
• /
• 2006

Fibrin clots of blood vessels are one of the serious factor caused cardiovascular disease. The development of a antithrombotic and thrombolysis solvent is necessary to prevent and treat these diseases. It has been reported that a strong fibrin-specific fibrinolytic enzyme was produced from a Korean fermented soybean paste similar to Japanese miso. We have been screened the known or novel fibrinolytic enzymes by activity-based and sequence-based screening from soil DNA metagenome library containing all kinds of environmental genomic DNA. The activity-based screening was determined the protease activity on 0.5% skim milk. For sequence-based screening, we designed a set of primer expanding gene sequence of fibrinolytic enzyme, performed PCR and selected clones showing the expected size of amplicons from metagenome library. Transformation of the gene encoding fibrinolytic enzyme was carried out with commercial vectors and their transformants were selected. Finally, we found 15 positive clones from metagenome library. Then each of sequences were analyzed and identified as similar or known the clones of nattokinase. We are going to perform full sequence of each clones, ligate with expression vector, transform into competent cells and then determine activity of expressed enzymes.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.9
• /
• pp.1196-1206
• /
• 2014

A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at $50^{\circ}C$ and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of $20{\sim}50^{\circ}C$ and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thin-layer chromatography suggested that CS10 is an endo-${\beta}$-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.4
• /
• pp.844-854
• /
• 2005

We constructed a defined physical and genetic map of H. pylori strain 51, previously isolated from a Korean patient with a duodenal ulcer, by combining a restriction analysis by pulse-field gel electrophoresis with the construction of a BAC library. A Notl-digest of H. pylori strain 51 genome yielded seven fragments, from which the genomic size was estimated to be 1,698$\pm$24 kb. The BAC library was constructed from 50 to 200 kb fragments of HindIII-digested genomic DNA. From 700 BAC clones, an ordered overlapping maxi-set of 82 BAC clones was assembled that covered the entire genome. The positions of 15 genes were localized in the strain 51 genome with 4-22 kb of resolution and were compared with their orthologues in strain 26695 and strain J99. The arrangement of the 15 genes was identical in strain 51 and strain J99, except for flaA and hpaA. The plasticity zone of strain 51, like that of strain J99, was located in the single region, and was shorter than those of strain 26695 and strain J99. The strain 51 plasticity zone consisted of ORFs common only to strain 51 and J99 or to strain 51 and 26695, as well as strain 51-specific ORFs. Three genetic translocations and/or inversions were found between orthologue ORFs in strain 51 and strain J99. These results show that the chromosomal organization of strain 51 differs from Western strains such as strain 26695 and strain J99.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1993.04a
• /
• pp.134-134
• /
• 1993

Tn3 Transposon을 이용한 Shuttle Mutagenesis방법으로 효모의 Genome 상에 무작위적으로 $\beta$-gal 표지 유전자를 삽입하고 효모생활사의 각 세포시기 마다 특이하게 발현되는 유전자를 X-Gal plate상에서 찾아내고 이들 효모 유전자의 단백질이 세포내에 어떤 위치에 분포하는가를 간접 면역현미경법으로 추적해보았다. 먼저 효모의 genomic library를 38bp의 Tn3 Termial repeat를 가지고 있지 않은 pHSS6 Vector에 patial fill-in 방법으로 조성하였으며 최종적으로 20 Genome equivalent에 해당하는 18개 Pool의 genomic library를 만들었다. 이들 library를 조사하여 본 결과 모든 클론이 평균 3kb 크기의 insert를 가지고 있었으며 이는 99.99%의 효묘 genome을 대표하였다. 특정한 유전자의 발현을 알아보기 위해 먼저 mini-Tn3로 shuttle mutagenesis를 실시하고 vegetative growth동안 발현되는 유전자를 X-gal을 사용하여 골라내었다. 지금까지 16823개의 클론을 조사하였는데 이중 13%에 해당하는 2187개가 X-gal plate상에서 양성반응을 보여주었다. 양성반응을 보여주는 융합단백질의 세포내 분포틀 anti-$\beta$-galactosidase 항체를 사용하여 추적해보았다. 항체론 이용한 형광염색결과 약 70%의 세포가 background이상의 염색을 보여주었으며 이중 novel한 염색 pattern을 나타내는 클론도 다수 탐지되었다. 이상의 결과를 종합하면 Tn3틀 이용한 Shuttle Mutagenesis 방법으로 지금까지 전통적인 유전학적인 접근 방식으로 탐지되지 않았던 다수의 새로운 효모 유전자를 찾아낼 수 있을 것으로 사료된다.tamine제중 triprolidine이 $K_{M}$ /K$_{H}$ 비가 가장 높았고 diphenidol이 가장 낮았다. 이상의 결과로 보아 항 histamine제의 muscarinic receptor 차단작용은 이들 약물의 항 alleragy 효과에 필요한 작용이 아니며 본 실험에서 추정된 항 histamine제의 H$_1$-receptor와 muscarinic receptor에 대한 상대적 역가는 이들 약물의 선택과 평가에 중요한 지표가 될수 있을 것으로 생각된다.ing ischemic insults. The nature of the receptor is being explored by molecular genetic techniques, and we have recently cloned two of the major subunits; some of the data will be presented.LIFO, 우선 순위 방식등을 선택할 수 있도록 확장하였다. SIMPLE는 자료구조 및 프로그램이 공개되어 있으므로 프로그래머가 원하는 기능을 쉽게 추가할 수 있는 장점도 있다. 아울러 SMPLE에서 새로이 추가된 자료구조와 함수 및 설비제어 방식등을 활용하여 실제 중형급 시스템에 대한 시뮬레이션 구현과 시스템 분석의 예를 보인다._3$", chain segment, with the activation energy of carriers from the shallow trap with 0.4[eV], in he amorphous regions.의 증발산율은 우기의 기상자료를 이용하여 구한 결과 0.05 - 0.10 mm/hr 의 범위로서 이로 인한 강우손실량은 큰 의미가 없음을 알았다.재발이 나타난 3례의 환자를 제외한 9례 (75%)에서는 현재까지 재발소견을 보이지 않고 있다.

• Environmental Engineering Research
• /
• v.12 no.5
• /
• pp.231-237
• /
• 2007

The microbial eco-physiology has been the vital key of microbial ecological research. Unfortunately, available methods for direct identity of microorganisms and for the investigation of their activity in complicated community dynamics are limited. In this study, metagenomics was considered as a promising functional genomics tool for improving our understanding of microbial eco-physiology. Its potential applications and challenges were also reviewed. Because of tremendous diversity in microbial populations in environment, sequence analysis for whole metagenomic libraries from environmental samples seems to be unrealistic to most of environmental engineering researchers. When a target function is of interest, however, sequence analysis for whole metagenomic libraries would not be necessary. For this case, nucleic acids of active populations of interest can be selectively gained using another cutting-edge functional genomic tool, SIP (stable isotope probing) technique. If functional genomes isolated by SIP can be transferred into metagenomic library, sequence analysis for such selected functional genomes would be feasible because the reduced size of clone library may become adequate for sequencing analysis. Herein, integration of metagenomics with SIP was suggested as a novel functional genomics approach to study microbial eco-physiology in environment.

• Genomics & Informatics
• /
• v.8 no.4
• /
• pp.177-184
• /
• 2010

The target of rapamycin (TOR) signaling pathway is a conserved pathway that regulates eukaryotic cell growth in response to environmental cues. Chemical genomic approaches that profile rapamycin sensitivity of yeast deletion strains have given insights into the function of TOR signaling pathway. In the present study, we analyzed the rapamycin sensitivity of yeast deletion library strains on synthetic medium. As a result, we identified 130 strains that are hypersensitive or resistant to rapamycin compared with wild-type cells. Among them, 36 genes are newly identified to be related to rapamycin sensitivity. Moreover, we found 16 strains that show alteration in rapamycin sensitivity between complex and synthetic media. We suggest that these genes may be involved in part of TOR signaling activities that is differentially regulated by media composition.

• Journal of Applied Biological Chemistry
• /
• v.44 no.1
• /
• pp.6-11
• /
• 2001

An ${\omega}-3$ fatty acid desaturase gene which is involved in de novo synthesis of -Iinolenate was isolated from cDNA library of Perilla frutescens. A cDNA library was constructed with mRNA extracted from perilla seeds of 12 DAF. The cDNA clone consisting of 1317-bp open reading frame encoding 438 amino acids with a relative MW of 50kDa, was isolated and showed 65-83% similarities to other known genes. This cDNA is deduced to encode a plastidal ${\omega}-3$ fatty acid desaturase based on the fact that it has higher homology to plastidal ones than to microsomal ones and its N-terminal sequence shares several characteristics of transit peptides of chloroplast proteins. Southern blot analysis of genomic DNA indicated that more than one gene or alleles for ${\omega}-3$ fatty acid desaturase are present in the genome of perilla. Northern blot analysis showed that the ${\omega}-3$ fatty acid desaturase gene is mainly revealed in early developing seeds and has different expression patterns depending on tissue types compared to the microsomal ones.

• 한국생물공학회:학술대회논문집
• /
• 2005.10a
• /
• pp.243-246
• /
• 2005

A novel esterase showing high enantioselectivity to (S)-ketoprofen ethyl ester was selected from fosmid environmental DNA library which is provided by Microbial Genomic & Applications Center. As a result of Blast search, the gene wasn't registerated in Gene Bank yet. And as we know, conserved domain region of esterase , G-X-S-X-G, wasn't discovered.$^{4)}$ And it is similar to Beta-lactamase. The DNA sequence of cloned esterase include an open reading frame consisting of 1170 bp, designated as EST-Y29, encoding a protein of 389 amino acids with a molecular mass of about 42.8 kDa. And amino acid sequence analysis revealed only a few identity (28%) to tile known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases. when being comparison to other esterase revealed , this enzyme seems to be classified as a new member of esterase family. EST-Y29 was functionally overexpressed in a soluble form in E. coli with maximum conversion yield of (S)-ketoprofen at $65^{\circ}C$. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzyme from a metagenome.

• Korean Journal Plant Pathology
• /
• v.14 no.3
• /
• pp.240-246
• /
• 1998

A cDNA encoding desiccation-related protein was isolated from a flower bud cDNA library of Chinese cabbage (C-DH) and its nucleotide sequence was characterized. It contains 679 bp nucleotides with 501 bp open reading frame. The amino acid sequence of the putative protein showed the highest amino acid sequence homology (79 % identity) to dehydrin protein in Gossypium hirsutum. Also, the C-DH shares 48-52% amino acid sequence identity with the other typical dehydrin proteins in plant cells. When the amino acid sequence of their proteins were aligned, several peptide motifs were well conserved, of which function has to be solved. Particularly the C-DH contains 15 additional amino acids at its N-terminus. Genomic Southern blot analysis using the coding region of C-DH showed that the C-DH consists of a single copy gene in Chinese cabbage genome. The C-DH mRNA, whose transcript size is 0.7 kb, was expressed with a tissue-specific manner. It was highly expressed in seed, flower buds and low expression as detected in root, stem or leaf tissues of Chinese cabbage. And the transcript level of C-DH was significantly induced by the treatment of plant hormone, abscisic acid and water-deficit conditions.