• Genomics & Informatics
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• 제12권4호
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• pp.240-246
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• 2014

Mutation in HNF1B, the hepatocyte nuclear factor-$1{\beta}$ (HNF-$1{\beta}$) gene, results in maturity-onset diabetes of the young (MODY) 5, which is characterized by gradual impairment of insulin secretion. However, the functional role of HNF-$1{\beta}$ in insulin secretion and glucose metabolism is not fully understood. We identified a family with early-onset diabetes that fulfilled the criteria of MODY. Sanger sequencing revealed that a heterozygous P159L (CCT to CTT in codon 159 in the DNA-binding domain) mutation in HNF1B was segregated according to the affected status. To investigate the functional consequences of this HNF1B mutation, we generated a P159L HNF1B construct. The wild-type and mutant HNF1B constructs were transfected into COS-7 cells in the presence of the promoter sequence of human glucose transporter type 2 (GLUT2). The luciferase reporter assay revealed that P159L HNF1B had decreased transcriptional activity compared to wild-type (p < 0.05). Electrophoretic mobility shift assay showed reduced DNA binding activity of P159L HNF1B. In the MIN6 pancreatic ${\beta}$-cell line, overexpression of the P159L mutant was significantly associated with decreased mRNA levels of GLUT2 compared to wild-type (p < 0.05). However, INS expression was not different between the wild-type and mutant HNF1B constructs. These findings suggests that the impaired insulin secretion in this family with the P159L HNF1B mutation may be related to altered GLUT2 expression in ${\beta}$-cells rather than decreased insulin gene expression. In conclusion, we have identified a Korean family with an HNF1B mutation and characterized its effect on the pathogenesis of diabetes.

• Molecules and Cells
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• 제40권3호
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• pp.202-210
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• 2017

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor $(TNF)-{\alpha}$. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of $NF-{\kappa}B$ and miR-503. We found that overexpression of NS5A inhibited $TNF-{\alpha}$-induced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the $TNF-{\alpha}$ induced Hep-mock cells was significantly less than the viability of the $TNF-{\alpha}$ induced Hep-NS5A cells, which demonstrates that NS5A inhibited $TNF-{\alpha}$-induced HepG2 cell apoptosis. Under $TNF-{\alpha}$ treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited $TNF{\alpha}$-induced $NF-{\kappa}B$ activation and $NF-{\kappa}B$ regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse $TNF-{\alpha}$-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits $NF-{\kappa}B$ activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.

• 대한약침학회지
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• 제9권2호
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• pp.5-16
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• 2006

Objective : It has long been known about the osteogenic effect of CPC-HAS on bone tissues. However, it has not been determined the effect of CPC-HAS on cancer cells. The purpose of this study is to screen the CPC-HAS mediated differentially expressed genes in cancer cells such as HepG2 hepatoma cells. Oligonucleotide microarray and proteomics approaches were employed to screen the differential expression genes. Methods : CPC-HAS was prepared by boiling and stored at $-70^{\circ}C$ until use. Cells were treated with various concentrations of CPC-HAS (0.1, 0.5, 1.5, 10, 20mg/ml) for 24 h. Cell toxicity was tested by MTT assay. To screen the differentially expressed genes in cancer cells, cells were treated with 1.5mg/ml of CPC-HAS. For oligonucleotide microarray assay, total RNA was used for gene expression analysis using oligonucleotide Genechip(Human genome Ul33 Plus 2.0., Affimatrix Co.). For proteomic analysis, total protein was analyzed by 2D gel electrophoresis and Q-TOF mass spectrometer. Results : It has no cytotoxic effects on both HepG2 cell in all concentrations(0.l, 0.5, 1.5, 10, 20mg/ml). In oligonucleotide microarray assay, the number of more than twofold differentially regulated known genes was 23 with 5 up-regulated and 18 down-regulated genes in HepG2 cells. In proteomic analysis, three spots were identified by 2D-gel electrophoresis and Q-TOF analysis. Two down-regulated proteins were aldehyde dehydrogenase 1 and enolase 1, and up-regulated protein was fatty acid binding protein 1 by 1.5mg/ml of CPC-HAS. Discussion : This study showed the screening of CPC-HAS mediated differentially regulated genes using combined approaches of oligonucleotide microarray and proteomic analysis. The screened genes will be used for the better understanding of the therapeutic effects of CPC-HAS on cancer fields.

• 정보처리학회논문지D
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• 제16D권5호
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• pp.661-672
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• 2009

인간의 유전체 서열에는 유전체 단위반복변위(copy number variation, CNV)를 포함하는 다양한 유전적 구조 변이(genetic structural variation)가 존재하며, 이는 기능적으로 질병에 대한 감수성, 치료에 대한 반응, 유전적 특성 등과 밀접한 관련이 있다. 본 논문에서는 기가 시퀀싱(giga sequencing)의 결과 산출되는 대량의 짧은 길이의 DNA 서열 데이터를 이용한 새로운 CNV 검색 방식을 제안한다. 제안하는 알고리즘에서는 레퍼런스 시퀀스에 DNA 서열 데이터를 서열 정렬시켜 각 레퍼런스 시퀀스의 위치에 대한 서열 데이터의 출현 빈도 정보를 얻은 후, 출현 빈도 정보의 패턴을 분석하여 통계적 유의성을 갖는 1kbp 이상의 연속 영역을 CNV 후보 영역으로 추출한다. 또한 제안된 알고리즘을 효율적으로 지원하기 위한 서열 정렬 방식에 대한 비교 및 분석을 수행한다. 제안된 기법의 유용성을 규명하기 위하여 다양한 실험을 수행하였다. 실험 결과에 의하면, 제안된 기법은 비교적 낮은 커버리지의 기가 시퀀싱 데이터를 이용하여 반복되거나 결실되는 다양한 형태의 CNV 영역을 효율적으로 검출하며, 또한 작은 사이즈의 CNV 영역에서부터 큰 사이즈의 CNV 영역까지 다양한 크기의 CNV 영역을 효율적으로 검출 할 수 있는 것으로 나타났다.

• Molecules and Cells
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• 제40권2호
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• pp.100-108
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• 2017

Cathepsin F, which is encoded by CTSF, is a cysteine proteinase ubiquitously expressed in several tissues. In a previous study, novel transcripts of the CTSF gene were identified in the crab-eating monkey deriving from the integration of an Alu element-AluYRa1. The occurrence of AluYRa1-derived alternative transcripts and the mechanism of exonization events in the CTSF gene of human, rhesus monkey, and crabeating monkey were investigated using PCR and reverse transcription PCR on the genomic DNA and cDNA isolated from several tissues. Results demonstrated that AluYRa1 was only integrated into the genome of Macaca species and this lineage-specific integration led to exonization events by producing a conserved 3' splice site. Six transcript variants (V1-V6) were generated by alternative splicing (AS) events, including intron retention and alternative 5' splice sites in the 5' and 3' flanking regions of CTSF_AluYRa1. Among them, V3-V5 transcripts were ubiquitously expressed in all tissues of rhesus monkey and crab-eating monkey, whereas AluYRa1-exonized V1 was dominantly expressed in the testis of the crab-eating monkey, and V2 was only expressed in the testis of the two monkeys. These five transcript variants also had different amino acid sequences in the C-terminal region of CTSF, as compared to reference sequences. Thus, species-specific Alu-derived exonization by lineage-specific integration of Alu elements and AS events seems to have played an important role during primate evolution by producing transcript variants and gene diversification.

• Laboraroty Animal Research
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• 제34권4호
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• pp.166-175
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• 2018

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. Rag-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of Rag-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of Rag-2, we recently established a new Rag-2 knockout FVB mouse line ($Rag-2^{-/-}$) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. $Rag-2^{-/-}$ mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in $Rag-2^{-/-}$ mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in $Rag-2^{-/-}$ mice. These findings indicate that our $Rag-2^{-/-}$ mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

• 한국육종학회지
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• 제42권5호
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• pp.502-506
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• 2010

콩 알레르기에 민감한 사람들은 15가지가 넘는 콩 알레르기 단백질을 인지한다. 이러한 알레르기 단백질 때문에 콩의 광범위한 사용이 제한적이다. 시스테인 프로테아제에 속하는 P34 단백질은 콩의 주된 알러젠이다. 미국 국무부에서 16,226개의 유전자원에서 P34 단백질이 결핍된 PI567476 유전자원을 찾아냈다. P34 유전자 염기서열과 P34 유전자가 결실된 염기서열을 NCBI 데이터베이스에서 확인한 결과 P34 유전자가 결실된 염기서열에서 4 bp 가 삽입되었다는 것을 확인하였고, 그 부위에서 SSLP 마커를 개발하였다. 본 연구에서는 태광콩과 PI567476을 이용한 교배조합 $F_2$ 339개체를 개발한 분자표지마커로 확인하였다. 실험결과 태광콩 유형과 heterozygous 유형 및 PI567476 유형의 분리비는 85: 187: 67로 $X^2{_{0.05}}=5.99$, df=2에서 1:2:1의 분리비로 하나의 유전자가 관여한다는 것으로 나타났다. 앞으로 P34 단백질 관련 분자마커가 단백질 수준에서 정확히 일치하는지 확인 할 것이다.

• 생명과학회지
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• 제29권9호
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• pp.1047-1054
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• 2019

이동성 유전인자는 인간 유전체의 45%를 차지하며 기능성 유전자 내부로 자유롭게 들어갈 수 있다. 이들은 진화과정에서 중복현상으로 다수의 복사수로 생성되며, 생물종다양성 및 계통유전체학 분야에 기여한다. 이동성 유전인자의 대부분은 메틸화 또는 아세틸화 현상과 같은 후성유전학적 조절에 의해 제어된다. 다양한 생물종은 그들만의 고유의 이동성 유전인자를 가지고 있으며, 일반적으로 DNA트란스포존과 레트로트란스포존으로 나뉜다. 레트로트란스포존은 LTR의 유무에 따라 다시 HERV와 LINE으로 구분된다. 이동성 유전인자는 프로모터, 인핸서, 엑손화, 재배열 및 선택적 스플라이싱과 같은 다양한 생물학적 기능을 수행한다. 또한 이들은 유전체의 불안정성을 야기시켜 다양한 질병을 유발하기도 한다. 따라서, 암과 같은 질병을 진단하는 바이오 마커로 사용될 수 있다. 최근, 이동성 유전인자는 miRNA를 만들어 내는 것으로 밝혀졌으며, 이러한 miRNA는 타겟 유전자의 seed 영역에 결합함으로서 mRNA의 분해 및 번역을 억제하는 역할을 수행한다. 이동성 유전인자 유래의 miRNA는 기능성 유전자의 발현에 큰 영향을 미친다. 다양한 생물종과 조직에서 서로 다른 miRNA의 비교 분석 연구는 생물학적 기능과 관련하여 진화학과 계통학 영역에서 흥미 있는 연구 분야라 할 수 있겠다.

• 생명과학회지
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• 제29권5호
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• pp.524-529
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• 2019

엉겅퀴는 대표적인 다년생의 약용식물이다. 최근 국제적 추세에 따라 자국의 유전자원의 발굴, 보존 등이 강화 됨에 따라 인접국가와 국내 자생 엉겅퀴 계통을 판별 할 수 있는 기준 설정에 관한 연구의 필요성이 대두되고 있지만, 분자생물학적 판별 기술의 개발은 아직 미흡한 실정이다. 본 연구에서는 국내 토종과 해외 유래 엉겅퀴종의 기원을 판별하기 위해 엽록체에 존재하는 trnL-trnF와 MatK 유전자단편에서 SNP를 이용한 판별 프라이머를 확보하였으며 이를 보완하여 보다 신속하게 판별하기 위하여 HRM 분석 기술을 이용한 판별 마커와 그 조건을 확립하였다. 그러므로, 본 연구에서 개발된 SNP 마커는 다양한 지역 또는 국가에서 서식하는 엉겅퀴 종들의 신속한 확인을 위해 매우 유용하게 이용될 것으로 생각된다.

• BMB Reports
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• 제55권9호
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• pp.453-458
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• 2022

Diabetes mellitus (DM) is a serious disease in which blood sugar levels rise abnormally because of failed insulin production or decreased insulin sensitivity. Although many studies are being conducted for the treatment or early diagnosis of DM, it is not fully understood how mitochondrial genome (mtDNA) abnormalities appear in patients with DM. Here, we induced iPSCs from fibroblasts, PBMCs, or pancreatic cells of three patients with type 2 DM (T2D) and three patients with non-diabetes counterpart. The mtDNA mutations were detected randomly without any tendency among tissues or patients. In T2D patients, 62% (21/34) of iPSC clones harbored multiple mtDNA mutations, of which 37% were homoplasmy at the 100% mutation level compared to only 8% in non-diabetes. We next selected iPSC clones that were a wild type or carried mutations and differentiated into pancreatic cells. Oxygen consumption rates were significantly lower in cells carrying mutant mtDNA. Additionally, the mutant cells exhibited decreased production of insulin and reduced secretion of insulin in response to glucose. Overall, the results suggest that screening mtDNA mutations in iPSCs from patients with T2D is an essential step before pancreatic cell differentiation for disease modeling or autologous cell therapy.