• 한국잠사곤충학회지
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• 제42권2호
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• pp.126-141
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• 2000

A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in target genome. The nucleotide sequence encoded in the genome contains the information that specifies the amino acid sequence of every protein and functional RNA molecule. In principle, it will be possible to identify every protein resposible for the structure and function of the body of the target organism. The pattern of expression in different cell types will specify where and when each protein is used. The amino acid sequence of the proteins encoded by each gene will be derived from the conceptional translation of the nucleotide sequence. Comparison of these sequences with those of known proteins, whose sequences are sorted in database, will suggest an approximate function for many proteins. This mini review describes the development of new sequencing methods and the optimization of sequencing strategies for whole genome, various cDNA and genomic analysis.

• Ocean and Polar Research
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• 제27권2호
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• pp.205-214
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• 2005

From ancient Antarctic glacier ice, we extracted total genomic DNA that was suitable for prokaryotic 16S rDNA gene cloning and sequencing, and bacterial artificial chromosome (BAC) library and end-sequencing. The ice samples were from the Dry Valley region. Age dating by $^{40}Ar/^{39}Ar$ analysis on the volcanic ashes deposited in situ indicated the ice samples are minimum 100,000-300,000 yr (sample DLE) and 8 million years (sample EME) old. Further assay proved the ice survived freeze-thaw cycles or other re-working processes. EME, which was from a small lobe of the basal Taylor glacier, is the oldest known ice on Earth. Microorganisms, preserved frozen in glacier ice and isolated from the rest of the world over a geological time scale, can provide valuable data or insight for the diversity, distribution, survival strategy, and evolutionary relationships to the extant relatives. From the 16S gene cloning study, we detected no PCR amplicons with Archaea-specific primers, however we found many phylotypes belonging to Bacteria divisions, such as Actinobacteria, Acidobacteria, Proteobacteria $({\alpha},\;{\beta},\;and\;{\gamma})$, Firmicutes, and Cytophaga-Flavobacterium-Bacteroid$. BAC cloning and sequencing revealed protein codings highly identical to phenylacetic acid degradation protein paaA, chromosome segregation ATPases, or cold shock protein B of present day bacteria. Throughput sequencing of the BAC clones is underway. Viable and culturable cells were recovered from the DLE sample, and characterized by their 16S rDNA sequences. Further investigation on the survivorship and functional genes from the past should help unveil the evolution of life on Earth, or elsewhere, if any.

• 한국축산식품학회지
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• 제41권4호
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• pp.701-714
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• 2021

In this study, the microbial community succession and the protein hydrolysis of donkey meat during refrigerated (4℃) storage were investigated. 16S rDNA sequencing method was used to analyze the bacteria community structure and succession in the level of genome. Meanwhile, the volatile base nitrogen (TVB-N) was measured to evaluate the degradation level of protein. After sorting out the sequencing results, 1,274,604 clean data were obtained, which were clustered into 2,064 into operational taxonomic units (OTUs), annotated to 32 phyla and 527 genus. With the prolonging of storage time, the composition of microorganism changed greatly. At the same time, the diversity and richness of microorganism decreased and then increased. During the whole storage period, Proteobacteria was the dominant phyla, and the Photobacterium, Pseudompnas, and Acinetobacter were the dominant genus. According to correlation analysis, it was found that the abundance of these dominant bacteria was significantly positively correlated with the variation of TVB-N. And Pseudomonas might play an important role in the production of TVB-N during refrigerated storage of donkey meat. The predicted metabolic pathways, based on PICRUSt analysis, indicated that amino metabolism in refrigerated donkey meat was the main metabolic pathways. This study provides insight into the process involved in refrigerated donkey meat spoilage, which provides a foundation for the development of antibacterial preservative for donkey meat.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.291-297
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• 1998

Cop protein has been overexpressed in Escherichia coli using a T7 RNA polymerase system. Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system. The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCI buffer (pH 7.5) containing 100 mM NaCl. Cop protein Was calculated to contain $39.1% {\alpha}-helix, 16.8% {\beta}-sheet$, 17.4% turn, and 26.8% random structure. The DNA binding property of Cop protein expressed in E. coli Was preserved during the expression and purification process. The isoelectric point of Cop was determined to be 9.0. The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase in E. coli.

• Journal of Genetic Medicine
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• 제7권2호
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• pp.119-124
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• 2010

약 7,000 여개의 단일유전자질환이 보고되어 있지만 보고된 질환의 절반도 아직 원인 유전자가 밝혀지지 못한 상황이다. 그리고 기존에 밝혀진 원인 유전자의 돌연변이형들은 대부분 단백질을 코딩하는 부위의 돌연변이에 의하여 발생하고 있어서 인간 유전체에서 단백질을 코딩하는 엑손 부위만을 선별적으로 분리하여 염기서열을 분석하는 엑솜 염기서열 분석 방법은 희귀한 유전질환의 신규 원인 유전자 발굴을 위한 매우 효과적인 유전 분석법이 될 것이다. 엑솜은 전체 유전체의 약 1.5% 정도를 차지하고 있어서 매우 경제적으로 분석이 가능하다. 그리고 엑솜 염기서열 분석 방법은 엑솜 부위를 선별하는 기술과 대용량 염기서열 분석기술로 수행된다. Freeman-Sheldon 증후군의 원인 유전자를 엑솜 염기서열 분석 방법으로 발굴한 이후로 단일유전자질환의 원인 유전자 발굴을 위한 표준 분석법으로 엑솜 염기서열 분석방법이 사용되고 있다. 향후에는 엑솜 염기서열 분석 방법이 다양한 복합질병의 유전분석에도 활용되어 개인 맞춤의학의 실현을 앞당기는데 크게 기여할 것으로 기대된다.

• 한국식물병리학회지
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• 제11권1호
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• pp.73-79
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• 1995

Complementary DNAs (cDNAs) to the coat protein gene of an ordinary Korean strain of potato virus Y (PVY-OK) isolated from potato (cv. Superior) were synthesized and cloned into a plasmid pUC119 and sequenced. The RNA of the virus propagated in tobacco (Nicotinaa sylvestris) was extracted by the method of phenol extraction. The first strand of cDNAs to the coat protein penomic RNA of the virus was made by Moloney murine leukemia virus reverse transcriptase. The cDNA were synthesized and amplified by the method of polymerase chain reaction (PCR) using a pair of oligonucleotide primers. PVYCP3P and PVYCP3M. The size of cDNAs inserted in pUC119 plasmid was estimated as about 840 bp upon agarose gel electrophoresis. Double stranded cDNAs were transformed into the competent cell of E. coli JM109. Sequence analysis of cDNAs was conducted by the dideoxynucleotide chain termination method. Homology of cDNAs of the PVY-OK coat protein genomic RNA with those of PVY-O (Japan), PVY-T (Japan), PVY-TH (Japan), PVYN (The Netherlands),and PVYY (France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. Homology at the amino acid level turned out to the be 97.4%, 92.5%, 92.9%, 92.9% and 98.5%, respectively.

• Asian-Australasian Journal of Animal Sciences
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• 제26권8호
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• pp.1144-1151
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• 2013

In this study, protein domains with cellulase activity in goat rumen microbes were investigated using metagenomic and bioinformatic analyses. After the complete genome of goat rumen microbes was obtained using a shotgun sequencing method, 217,892,109 pair reads were filtered, including only those with 70% identity, 100-bp matches, and thresholds below $E^{-10}$ using METAIDBA. These filtered contigs were assembled and annotated using blastN against the NCBI nucleotide database. As a result, a microbial community structure with 1431 species was analyzed, among which Prevotella ruminicola 23 bacteria and Butyrivibrio proteoclasticus B316 were the dominant groups. In parallel, 201 sequences related with cellulase activities (EC.3.2.1.4) were obtained through blast searches using the enzyme.dat file provided by the NCBI database. After translating the nucleotide sequence into a protein sequence using Interproscan, 28 protein domains with cellulase activity were identified using the HMMER package with threshold E values below $10^{-5}$. Cellulase activity protein domain profiling showed that the major protein domains such as lipase GDSL, cellulase, and Glyco hydro 10 were present in bacterial species with strong cellulase activities. Furthermore, correlation plots clearly displayed the strong positive correlation between some protein domain groups, which was indicative of microbial adaption in the goat rumen based on feeding habits. This is the first metagenomic analysis of cellulase activity protein domains using bioinformatics from the goat rumen.

• Genes and Genomics
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• 제40권12호
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• pp.1339-1349
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• 2018

Cerebral palsy (CP) is a non-progressive neurological disease, of which susceptibility is linked to genetic and environmental risk factors. More and more studies have shown that CP might be caused by multiple genetic factors, similar to other neurodevelopmental disorders. Due to the high genetic heterogeneity of CP, we focused on investigating related molecular pathways. Ten children with CP were collected for whole-exome sequencing by next-generation sequencing (NGS) technology. Customized processes were used to identify potential pathogenic pathways and variants. Three pathways (axon guidance, transmission across chemical synapses, protein-protein interactions at synapses) with twenty-three genes were identified to be highly correlated with CP. This study showed that the three pathways associated with CP might be the molecular mechanism of pathogenesis. These findings could provide useful clues for developing pathway-based pharmacotherapies. Further studies are required to confirm potential roles for these pathways in the pathogenesis of CP.

• 한국미생물·생명공학회지
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• 제31권2호
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• pp.117-123
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• 2003

공시균인 Saccha. erythraea IFO 13426으로부터 VB-C에 의한 erythromycin 생산 유도능이 시사된 바 있으므로, 공시균으로부터 VB-C와 특이적으로 결합하는 autoregulators 및 receptor gene을 탐색하여, EM의 생산 조절 기구를 규명하고자 하였다. 탐색의 일환으로 기존의 Streptomyce속 receptor gene의 공통배열을 primer로 이용하여 PCR을 수행하였고, 예상 크기인 120bp의 단편을 pUC19 vector에 ligation하여 E. coli DH5$\alpha$에 형질전환한 후, plasmid를 분리하여 BamHI을 처리하여 2% agarose gel에 전기영동한 결과, pUC19 (2.7kbp)외에 receptor gene PCR 산물이 120bp위치에 존재하는 것을 확인하였다. 형질전환된 plasmid로 PCR을 수행하여 염기배열을 결정한 후 해석한 결과 Streptomyces sp. 유래의 receptor gene과 유사함을 확인하였다. 따라서 Saccha. erythraea IFO 13426에는 항생물질인 erythromycin의 생산에 관여한다고 추정되는 autoregulator receptor protein을 코드하는 유전자가 존재할 것으로 예상되어 120 bp의 PCR product를 probe로 이용하여 Southern 및 colony hybridization을 통하여 3.2 kbp의 SacI 단편을 가지는 plasmid(pESG)를 제작하였고, 이를 sequencing한 결과, autoregulator receptor protein 유전자가 KpnI과 SalI을 포함하는 영역에 존재한다는 것을 알 수 있었으며 이를 EsgR이라 명명하였다. 유전자 해석 결과, EsgR은 205개의 아미노산으로 구성되어 있으며, 이는 기존의 autoregulator receptor proteins과 비교시 30%이상의 상동성을 나타내었으며, 기존의 autoregulator receptor prorein들이 하부의 항생물질 생합성 유전자들의 제어를 위해 보유하고 있는 helix-turn-helix DNA binding motif를 EsgR이 보유하고 있는 점에서, EsgR은 Saccha. erythraea가 보유하는 autoregulator receptor protein을 code하는 유전자로 추정되었다.

• Genomics & Informatics
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• 제11권1호
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• pp.24-33
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• 2013

We had analyzed 10 exome sequencing data and single nucleotide polymorphism chips for blood cancer provided by the PGM21 (The National Project for Personalized Genomic Medicine) Award program. We had removed sample G06 because the pair is not correct and G10 because of possible contamination. In-house software somatic copy-number and heterozygosity alteration estimation (SCHALE) was used to detect one loss of heterozygosity region in G05. We had discovered 27 functionally important mutations. Network and pathway analyses gave us clues that NPM1, GATA2, and CEBPA were major driver genes. By comparing with previous somatic mutation profiles, we had concluded that the provided data originated from acute myeloid leukemia. Protein structure modeling showed that somatic mutations in IDH2, RASGEF1B, and MSH4 can affect protein structures.