• KSBB Journal
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• v.21 no.2
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• pp.144-150
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• 2006

The exploitation of metagenome, the access to the natural extant of enormous potential resources, is the way for elucidating the functions of organism in environmental communities, for genomic analyses of uncultured microorganism, and also for the recovery of entirely novel natural products from microbial communities. The major breakthrough in metagenomics is opened by the construction of libraries with total DNAs directly isolated from environmental samples and screening of these libraries by activity and sequence-based approaches. Screening with activity-based approach is presumed as a plausible route for finding new catabolic genes under designed conditions without any prior sequence information. The main limitation of these approaches, however, is the very low positive hits in a single round of screening because transcription, translation and appropriate folding are not always possible in E. coli, a typical surrogate host. Thus, to obtain information about these obstacles, we studied the genetic organization of individual URF's(unidentified open reading frame from metagenome sequenced and deposited in GenBank), especially on the expression factors such as codon usage, promoter region and ribosome binding site(rbs), based on DNA sequence analyses using bioinformatics tools. And then we also investigated the above-mentioned properties for 4100 ORFs(Open Reading Frames) of E. coli K-12 generally used as a host cell for the screening of noble genes from metagenome. Finally, we analyzed the differences between the properties of URFs of metagenome and ORFs of E. coli. Information derived from these comparative metagenomic analyses can provide some specific features or environmental blueprint available to screen a novel biocatalyst efficiently.

• Korean Journal of Environment and Ecology
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• v.35 no.5
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• pp.503-524
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• 2021

A largemouth bass (Micropterus salmoides) is an ecosystem disturbance fish species at the highest rank in the aquatic ecosystem, causing a serious imbalance in freshwater ecosystems. Although various attempts have been made to eradicate and control largemouth bass, no effective measures were found. Therefore, it is necessary to find an approach to maximize the effective population reduction based on the unique characteristics of largemouth bass. This study used the transcriptome analysis to derive 182,887 unigene contigs and select 12 types of final target sequences for applying the CRISPR/Cas9 system in the genes of IZUMO1 and Zona pellucida sperm-binding protein, which are proteins involved in sperm-egg recognition. After synthesizing 12 types of sgRNA capable of recognizing each target sequence, 12 types of Cas9-sgRNA ribonucleoprotein (RNP) complexes to be used in subsequent studies were prepared. This study searched the protein-coding gene of sperm-egg through the Next Generation Sequencing (NGS) and edited genes through the CRISPR/Cas9 system to induce infertile individuals that produced reproductive cells but could not form fertilized eggs. Through such a series of processes, it successfully established a composition development process for largemouth bass. It is judged that this study contributed to securing the valuable basic data for follow-up studies to verify its effect for the management of ecological disturbances without affecting the habitat of other endemic species in the same water system with the largemouth bass.

• Korean Journal of Microbiology
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• v.55 no.3
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• pp.313-315
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• 2019

An anaerobic bacterial strain WCF-2 was isolated from cow dung in finding cellulose-degrading bacteria for use as silage additives. Strain WCF-2 showed a higher cellulolytic activity than Cellulosilyticum lentocellum DSM $5427^T$, the closest relative of strain WCF-2 (98.2% of 16S rRNA gene sequence similarity). We sequenced the complete genome of strain WCF-2 and compared it with that of C. lentocellum DSM $5427^T$. The OrthoANI value between the two strains was 97.9% thus strain WCF-2 was identified as C. lentocellum. The genome size of strain WCF-2 was 4,779,774 bp with a G + C content of 34.4%, 4,154 coding genes (CDS), 54 pseudo genes, and 142 RNA genes. Strain WCF-2 harbored seven cellulase genes, five of which showed low similarities with those of C. lentocellum DSM $5427^T$.

• 대한생식의학회:학술대회논문집
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• 2001.03a
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• pp.195-205
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• 2001

태반 영양배엽 (trophoblast)은 포유동물의 발생과정 중 가장 먼저 분화되는 세포로서, 자궁환경내에서 배아가 착상, 발생, 및 분화하기 위해서 반드시 필요한 태반을 형성하는 색심적인 세포이다. 영양배엽 세포의 분화과정중의 결함은 배아의 사산이나 임신질환 등의 치명적 결과를 초래한다. 하지만, 영양배엽 세포의 분화를 조절하는 분자생물학적인 메카니즘은 아직 규명되지 않고 있다. 영양배엽 세포의 분화를 조절하는 경로를 규경하기 위한 선결과제는 분화된 영양배엽 세포에서만 발현하는 많은 유전자들이 밝혀져야만 한다. 본 연구팀은 최근에 분화된 영양배엽 세포에서만 발현하는 두 종류의 새로운 유전자들을 찾았다. 한 종류는 homeobox를 보유하고 있는 조절 유전자 Psx이고, 다른 한 종류는 임신호르몬인 태반 프로락틴 라이크 단백질 유전자 PLP-C${\beta}$이다. 본 연구과제의 목표는 이들 유전자의 기능과 조절 메카니즘을 규명함으로써, 영양배엽 세포의 분화를 조절하는 조절경로를 밝히는 것이다. 이를 위하여 다음과 같은 일련의 연구를 수행할 것이다. 1) Psx 유전자가 분화된 영양배엽 세포에서만 발현케 하는 조절 메카니즘을 규명하기 위해 functional assays, in vitro footprinting, gel mobility shift assays, 생쥐형질전화, UV crosslinking, Southwestern blot 등의 방법을 통해 Psx 유전자의 cis-acting 요인과 trans-acting factor를 밝혀 분석한다. 2) 영양배엽 세포의 분화조절 경로를 규명하기 위해 random oligonuclotide library screening, DD-PCR, subtractive screening 등의 방법을 이용하여 Psx 유전자에 의해 조절되는 하부유전자를 밝힌다. 3) Psx 유전자를 knock-out시켜 영양배엽 세포가 발달 및 분화하는데 미치는 역할을 밝힌다. 4) Yeast two-hybrid screening방법을 이용하여 태반 프로락틴 유전자의 수용체를 찾아 이들의 신호전달 기전을 밝힌다. 제1차년 연구결과로서, mouse와 rat으로부터 각각 Psx 유전자의 genomic DNA를 클로닝하여, 유전자 구조를 비교한 결과, mouse Psx (mPsx2)는 4개의 exons으로 이루어져 있는 반면에, rat Psx (Psx3)는 3개의 exons으로 구성되어 있었다. 즉, rPsx3는 mPsx2의 exon1이 없었다. Notrhern blot과 in situ hybridization 분석에 의해 mouse와 rat에서 Psx 유전자가 다르게 발현 조절되는 현상을 밝혔다. 실제로 mPsx2와 rPsx3의 5'-flanking지역을 클로닝하여 염기서열 분석 결과 전혀 homology를 찾을 수 없었다. 또한, 이들 각각 promoter의 activity를 luciferase reporter를 이용하여 조사한 결과 Rcho-1 trophoblast cells에서 각기 다른 activity를 보여 주는 것을 발견하였다. Psx 유전자의 transcription start sites는 Primer extension에 의해 밝혔다. 또한 Psx2 유전자를 knock-out 시키기 위해 targeting vector를 Osdupde1에 제작하였다. 본 과제를 시작할 때 새로운 프로락틴 유전자 하나를 클로닝하여 이 유전자를 PLP-I라고 이름을 붙였다. 이 후 이 유전자 (PLP-I)는 PLP-C${\beta}$라고 이름을 붙이게 되었다. Mouse PLP-C${\beta}$ 유전자의 counterpart를 rat에서 찾아 염기서열을 비교한 결과 mouse와 rat에서 PLP-C${\beta}$유전자의 homology는 약 79% (amino acid level)였다. 본 연구과정을 통해 또 하나의 새로운 PLP-C subfamily member를 mouse로부터 클로닝 하였고, 이 유전자를 PLP-C${\gamma}$라 하였다. PLP-C${\beta}$와 PLP-C${\gamma}$의 발현 유형은 Northern blot과 in 냐셔 hybridization 분석에 의해 태반의 제한된 spongitrophoblast와 trophoblast giant cells에서만 발현하는 것을 밝혔다. 놀랍게도 이들 두 새로운 유전자는 alternative splicing에 의해 두 종류의 isoform이 있음을 밝혔다. PLP family member 유전자로서 splicing에 의한 isoforms을 보여 주는 유전자로는 PLP-C${\beta}$와 PLP-C${\gamma}$가 최초이다. 이들 isoform mRNAs의 발현 유형은 RT-PCR 방법을 이용하여 규명하였다. 또 하나의 새로운 발견은 PLP-C${\beta}$와 PLP-C${\gamma}$가 독특한 유전자 구조를 갖고 있었다. 즉, PLP-C${\beta}$는 exon3의 alternative splicing에 의해 5개 혹은 6개의 exons을 갖는 two isoforms이 생긴다. 반면에 PLP-C${\gamma}$는 exon2가 alternative splcing이 되면서 7개의 exons을 갖거나 6개의 exons을 갖는 isoforms을 만든다. 그리고, PLP-C${\gamma}$의 promoter activity를 trophoblast Rcho-l${\gamma}$ 세포주를 이용하여 PLP-C${\gamma}$ 의 1.5 kb 5'-flanking 지역이 trophoblast-specific promoter activity를 갖고 있음을 밝혔다. PLP-C${\gamma}$ 유전자의 transcription start site는 Primer extension에 의해 밝혔다. 제 1차 년도의 연구결과를 토대로, 2차년에서는 다음단계의 연구를 수행하고자 한다. 즉, 1) mPsx2와 rPsx3의 promoter를 비교분석 함으로서 mouse와 rat에서 Psx 유전자가 다르게 조절되는 메카니즘 규명, 2) Psx와 PLP-C 유전자의 promoter에 있는 cis-acting elements 탐색, 3) Psx2와 Psx3의 단백질을 이용하여 이들이 binding하는 target sequence 규명, 4) 제작한 Psx2 targeting vector를 이용하여 ES cells에서 Psx2 유전자 knock-out, 5) Psx 유전자를 과발현시키는 세포주를 만들고 Psx에 의해 조절되는 유전자 탐색, 6) 새로 밝히 PLP-C members 유전자들의 조절기전을 Rcho-1 세포주를 이용하여 여러 거지 성장인자와 다른 호르몬에 대한 반응을 탐색, 7) Psx와 PLP-C${\gamma}$ 유전자의 chromosomal mapping 등을 밝힐 것이다.

• Korean journal of applied entomology
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• v.46 no.2
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• pp.229-234
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• 2007

Pure Bombus ignitus venom samples were submitted to two-dimensional gel electrophoresis. A total of 64 excised spots were analyzed by mass spectrometry. Three main proteins resulted in the identification have not been described in other bee venoms before. Dose-dependence against human carcinoma (Hep3B, BT-20, A549 and AGS) were observed from 1ng/ml to 100ng/ml. Expecially, the treatment of 100ng/ml B. ignitus venoms showed the highest cytotoxicity with 55% against hepatocellular carcinoma (Hep3B). The B. ignitus venoms showed strong antimicrobial activities against Enterococcus faecium and Shigella sonnei, and practically antimicrobial activity against the other microorganisms tested. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of E. faecium and S. sonnei, were 0.256ug/ml, respectively.

• Horticultural Science & Technology
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• v.28 no.6
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• pp.1039-1050
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• 2010

Carrot is one of the useful crops used abundantly in cooking in Western as well as Asia regions such as China and Korea. However, seed coats have hairs which should be removed to increase germination rate. Furthermore, because of seed hairs, farmers face several additional losses, such as time consumption, manpower, capital and so on, for seed handling. To prevent these problems, study of gene related hair formation using short-hair seed lines is required. We analyzed genes related to hair formation from seed through expressed sequenced tag (EST) profiling, based on the fact that the development of carrot seed hair is related to cellulose synthesis pathway in secondary cell wall synthesis stage. To study the gene expression related to hair formation of the carrot seed, a cDNA library was constructed by using the early maturation stage of the short-hair line (659-1) and hairy seed line (677-14). In short-hair (659-1) and hairy seed (677-14) lines, results from of EST profiling through BLASTX search analysis using the NCBI database showed that 172 and 224 unigenes had significant homology with known protein sequences, whereas 233 and 192 unigenes were not, respectively. All ESTs were grouped into 16 categories according to their putative functions. Twenty nine unigenes among all ESTs were considered to be genes regulating seed hair development from cellulose synthesis pathway during secondary cell wall synthesis stage; in results, 14 unigenes related to seed hair development were found only in hairy seed line.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.246-251
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• 2006

A lot of mutants which cannot initiate sexual development were screened and several loci including nsdA, nsdB, nsdC, and nsdD were identified in homothallic ascomycetes Aspergillus nidulans. The NSD206, which has nsdC6 allele, showed typical phenotype of NSD (Never in sexual development) mutants. The nsdC gene was cloned by transforming NSDP697 ($nsdC^-$, $pryG^-$) with AMA1-NotI genomic library. The transforming library DNA recovered from several transformants showing wild phenotype carried about 10 kb genomic DNA insert. The DNA sequence of nsdC was analysed using GPS (Genome priming system). The nsdC gene has an open reading frame (ORF) of 1,929 bp encoding a putative polypeptide of 643 amino acids. The NsdC carries $C_2H_2C_2H_2C_2HC$ type zinc finger DNA binding domains in the middle of the polypeptide. A coiled-coil domain at its C terminus were also found. In nsdC6 allele, a single T insertion was occurred between 407-408 bp leading to the frameshift mutation and early termination of translation producing the truncated protein which has only 139 amino acids.

• Korean Journal of Microbiology
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• v.55 no.1
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• pp.69-73
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• 2019

During screening of microbes for compounds having cosmetic benefits, we isolated Marinobacter salarius HL2708#2 from lava seawater on Jeju Island, Republic of Korea. The complete genome sequence was determined. Strain HL27080#2 features a circular chromosome of 4,304,603 bp with 57.21% G+C content and a 244,163 bp plasmid with 53.14% G+C. There were 4,180 protein coding sequences identified, along with 49 transfer RNA and 18 ribosomal RNA noncoding genes. The genome harbored genes for the utilization of alcohol, maltose/starch, and monosaccharide as sole carbon sources. Genes responsible for halophilic characteristics and heavy metal resistance could be annotated, as well as aromatic and alkane hydrocarbons. Contrary to the prior report that M. salarius is negative for nitrate and nitrite reduction, nitrate/nitrite reductase along with nitrate/nitrate transporters and nitronate monooxygenase were evident, suggesting that strain HL2708#2 may be able to denitrify extracellular nitroalkenes to ammonia.

• Korean Journal of Food Science and Technology
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• v.36 no.1
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• pp.152-157
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• 2004

Gluten was extracted from domestic wheat flour using UTH buffer (4 M urea in 0.1 M Tris-HCl, pH 8.6) and validated by SDS-PAGE analysis for production of wheat flour products with reduced gluten content.. Anti-gluten polyclonal antibody was made by administering extracted gluten fraction on animal model. Anti-gluten serum titer of extracted gluten fraction was evaluated by ELISA, and that of antibody titer according to administration period. Anti-gluten sera were used for ELISA and immunoblot analysis before and after hydrolysis of gluten fraction at optimal pH and temperature condition for each protease. Gluten fraction separated by SDS-PAGE showed several bands covering 75 to 10 kDa, in which anti-gluten sera were 25, 34, and 45 kDa. Enzyme hydrolysis of gluten fraction revealed protein band sizes to be lower than 15 kDa. Content of pretense from bovine pancreas (b.p. protease) for gluten hydrolysis was estimated as 1 mg in 10 mL gluten fraction extracted for 4 hr.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.420-427
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• 2018

The orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase (Sc YRS) and a variant of E. coli initiator tRNA, fMam $tRNA_{CUA}$ which recognizes the amber stop codon is an effective tool for site-specific incorporation of unnatural amino acids into the protein in E. coli. To evolve the amber suppression activity of the orthogonal pair, we generated a mutant library of Sc YRS by randomizing two amino acids at 320 and 321 which involve recognition of the first base of anticodon in fMam $tRNA_{CUA}$. Two positive clones are selected from the library screening with chloramphenicol resistance mediated by amber suppression. They showed growth resistance against high concentration of chloramphenicol and their $IC_{50}$ values were approximately 1.7~2.3 fold higher than the wild type YRS. In vivo amber suppression assay reveals that mutant YRS-3 (mYRS-3) clone containing amino acid substitutions of P320A and D321A showed 6.5-fold higher activity of amber suppression compared with the wild type. In addition, in vitro aminoacylation kinetics of mYRS-3 also showed approximately 7-fold higher activity than the wild type, and the enhancement was mainly due to the increase of tRNA binding affinity. These results demonstrate that optimization of anticodon recognition by engineered aminoacyl tRNA synthetase improves the efficiency of unnatural amino acid incorporation in response to nonsense codon.