• Journal of Korean Society of Environmental Engineers
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• v.28 no.10
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• pp.1055-1064
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• 2006

Extremely slow growing anammox(anaerobic ammonium oxidation) bacteria were cultivated using a combination of UASB(Upflow Anaerobic Sludge Blanket) reactor seeded with anaerobic granular sludge and carbon-fiber cultivating reactor. After 180 days of continuous cultivation, average nitrogen removal rate showed 0.54 kg $N/m^3-day$ when 0.6 kg $N/m^3-day$ of nitrogen loading was applied. The black granule was changed to brown and red granule as continuous operation, and the red granule was highly dependant on the high anammox activity. Microbial community structure of red granule in the UASB reactor was analyzed by molecular methods such as gene cloning, phylogenetic tree analysis, and FISH(Fluorescence In Situ Hybridization) method. As a result of gene cloning and phylogenetic tree analysis, 5 kinds of phylum were found to be Planctomycetes, Proteobacteria, Acidobacteria, Chlorobi and Chloroflexi. 13 clones were matched to anammox bacteria among 51 clones in the red anammox granule. In-silico test which used cloning information and FISH probe of the AMX368 was conducted to detect the presence of anammox bacteria in the red anammox granule. As a result of in-silico test only one clone was exactly matched to AMX368 but 11 clones was mutated one base among 18 bases representing all 12 clones are anammox bacteria. A filamentous Chloroflexi might be related to the granulation of anammox bacteria. As a result of FISH analysis, anammox bacteria was abundant in the red anammox granule.

• International Journal of Industrial Entomology and Biomaterials
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• v.15 no.2
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• pp.115-122
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• 2007

As the genome of B.mori is available in GenBank and the EST database of B.mori is expanding, identification of novel genes of B.mori is conceivable by data-mining techniques. We used the in silico cloning method to get the vacuolar-type $H^+-ATP$ synthetase (V-ATPase) c subunit (16 kDa proteolipid subunit) gene of B.mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and sequencing. The V-ATPase c subunit cDNA contains a 468 bp ORF. The ORF encoded a 155-residue protein that showed extensive homology with V-ATPase c subunits from other 15 species and contained four membrane-spanning helices. Tissue expression pattern analysis revealed that V-ATPase c expressed strongly in Malpighian tubules, not in fat body. This gene has been registered in GenBank under the accession number EU082222.

• International Journal of Industrial Entomology and Biomaterials
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• v.14 no.2
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• pp.69-74
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• 2007

As the genome of B. mori is available in GenBank and the EST database of B. mori is expanding, identification of novel genes of B. mori was conceivable by datamining techniques and bioinformatics tools. In this study, we used the in silico cloning method to get the 6-Phosphogluconolactonase (6PGL) gene of B. mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and prokaryotic expression. The 6PGL cDNA comtains a 702 bp ORF. The deduced protein has 233 amino acid residues, with the predicted molecular weight of 25946. 72 Da, isoelectric point of 5.41, and contains conserved NagB domains. This gene has been registered in GenBank under the accession number EF198104.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.10
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• pp.1473-1477
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• 2007

Four-and-a-half LIM-only protein 3 (FHL3) is a member of the LIM protein superfamily and can participate in mediating protein-protein interaction by binding one another through their LIM domains. In this study, the 5'- and 3'- cDNA ends were characterized by RACE (Rapid Amplification of the cDNA Ends) methodology in combination with in silico cloning based on the partial cDNA sequence obtained. Bioinformatics analysis showed FHL3 protein contained four LIM domains and four LIM zinc-binding domains. In silico mapping assigned this gene to the gene cluster MTF1-INPP5B-SF3A3-FHL3-CGI-94 on pig chromosome 6 where several QTL affecting intramuscular fat and eye muscle area had previously been identified. Transcription of the FHL3 gene was detected in spleen, liver, kidney, small intestine, skeletal muscle, fat and stomach, with the greatest expression in skeletal muscle. The A/G polymorphism in exon II was significantly associated with birth weight, average daily gain before weaning, drip loss rate, water holding capacity and intramuscular fat in a Landrace-derived pig population. Together, the present study provided the useful information for further studies to determine the roles of FHL3 gene in the regulation of skeletal muscle cell growth and differentiation in pigs.

• International Journal of Oral Biology
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• v.42 no.2
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• pp.63-70
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• 2017

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.

• Biomedical Science Letters
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• v.14 no.1
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• pp.47-53
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• 2008

Hox genes are known to be transcription factors controlling vertebrate pattern formation along the anteroposterior body axis by regulating many target gene expressions during vertebrate embryogenesis. In order to isolate in vivo Hox responsive target genes, ChIP-cloning technique has been applied using Hoxc8 antibody. Here murine embryo of day 11.5 post coitum (E11.5) highly expressing Hoxc8 gene was used after removing head and tail portions where Hoxc8 is rarely expressing. After fixation with formaldehyde, the chromatin DNAs harboring bound proteins were isolated. After sonication, about 0.5- to 1 Kb chromatin DNAs were immunoprecipitated with anti Hoxc8 antibody. After removing the bound proteins with proteinase K, DNAs were isolated, cloned into the pBluescsript II SK vector, and then sequenced. Total 33 random clones sequenced were anlalyzed to be located at 12 different genomic regions. Among these, 8 turned out to be introns and 4 were intergenic regions localized in random chromosomes. The base composition of total cloned genomic sequences (6608 bp) were AT-rich, i.e., 40% GC. When the Hoxc8 core binding sites, such as TAAT, ATTA, TTAT, and ATAA were analyzed total number of 55, 45, 54, and 55 were found, respectively, which are than twice as many as expected number of 26. Although this in silico analysis does not mean that the ChIP-cloned sequence is real Hoxc8 regulatory element in vivo, these results strongly imply that the DNA fragments cloned through chromatin immunoprecipitation could be very much likely the putative Hoxc8 downstream target genes.

• International Journal of Industrial Entomology and Biomaterials
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• v.15 no.2
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• pp.137-143
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• 2007

Lactate dehydrogenase (LDH) is a ubiquitous enzyme that plays a significant role in the clinical diagnosis of pathologic processes. Discovery of the LDH (BmLDH) gene in B. mori may shed light on its role in the biology of Lepidoptera species, and afford further understanding of the function of the enzyme. In this study, we used the bioinformatics tools to identify LDH gene in B. mori. Sequence analysis showed that BmLDH cDNA contains a 996 bp open reading frame, encoding 331 AA proteins, with seven introns. Compared with hHLDH (human heart LDH), BmLDH contained the same key active sites. Domain search and protein fold recognition analyses provide compelling evidences that the deduced protein is a LDH. Using the computer program MEGA3, we conducted a search for homologs of BmLDH among many eukaryotic species and confirmed that the BmLDH was conserved in all organisms investigated. This gene has been registered in GenBank under the accession number EU000385.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.192.2-193
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• 2003

Capsaicin, a pungent ingredient in chili pepper, is known to excite sensory neurons that mediate pain sensation. This effect of capsaicin is determined by unique receptors and the capsaicin receptor (transient receptor potential subfamily V, member 1 (TRPV1)) was cloned recently. TRPV1 contains six transmembrane domains and three ankyrin repeats at N-terminal. This characteristic architecture is common in other ion channel in TRPV families. (omitted)

• Proceedings of the Korea Information Processing Society Conference
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• 2001.10a
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• pp.105-108
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• 2001

cDNA(complementary DNA)를 복제(cloneing)하여 염기 서열화 한 EST(Expressed Sequence Tag) 데이터는 여러 생물체들의 염기서열 정보들과 비교를 통해 유사점을 찾거나 기능적 부위 검색을 통해 유전자 기능을 추정한 수 있어 기능 유전체 연구에 많이 사용되고 있다. EST 데이터를 식물은 특정종(Species)별로, 동물의 경우 종의 조직별로 클러스터링 함으로써 아직 알려지지 않은 종의 유전자를 밝혀낼 수 있음은 물론 유전자의 발현에 따른 단백질의 기능도 알아낼 수 있다. 따라서 이 논문에서는 NCBI에서 flatfile 형태로 제공하는 EST 데이터를 분석하여 관계형 데이터베이스로 모델링하고 구축하였다. 또한 EST 데이터의 효율적인 사용을 위하여 데이터를 특정 종의 조직별로 클러스터링하여 제공하는 시스템을 설계하고 구현하였다.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.9
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• pp.1237-1241
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• 2005

Mannose-P-dolichol utilization defect 1 (MPDU1) gene is required for utilization of the mannose donor MPD in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols (GPI) which are important for functions such as protein folding and membrane anchoring. The full length cDNA of the porcine MPDU1 was determined by in silico cloning and rapid amplification of cDNA ends (RACE). The deduced amino acid showed 91% identity to the corresponding human sequence with five predicted transmembrane regions. RT-PCR was performed to detect its expression pattern in five tissues and results showed that it is expressed ubiquitously among the tissues checked. A single nucleotide substitution resulting in the amino acid change (137 Tyr-137 His) was detected within exon 5. Allele frequencies in six pig breeds showed distinctive differences between those Chinese indigenous pigs breeds and European pigs. Using the pig/rodent somatic cell hybrid panel (SCHP), we mapped the porcine MPDU1 gene to SSC12, which is consistent with the comparative mapping result as conservative syntenic groups presented between human chromosome 17 and pig chromosome 12.