• Environmental Analysis Health and Toxicology
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• 제24권4호
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• pp.321-332
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• 2009

본 연구의 목적은 다이아지논(Diazinon; O, O-diethyl O-[6-methyl-2 (1-methylethyl)-4-pyrimidinyl] phosphorothioate)에 노출된 모델 생물체(송사리)의 행동변화와 관련된 분자생물학적 기전 규명을 통하여 비정상적 행동의 모니터링을 위한 생물지표(biomarker)를 개발하는데 있다. 이를 위해 우선 suppression subtractive hybridization (SSH) 및 DNA microarray 기법을 활용하여 다양한 유전자를 스크리닝하였다. 다이아지논에 노출시킨 송사리에서 발현의 차이가 나는 상향 조절된 유전자 97개 (알려지지 않은 유전자 27개 포함)와 하향 조절된 유전자 99개 (알려지지 않은 유전자 60개 포함)를 동정 하였고 이들 중 이상행동과 관련되는 것으로 보이는 유전자 10개 (상향조절 5개, 하향조절 5개)를 선발하였다. 이들 중에서 primer 제작이 잘된 beta-1, Orla C3-1, parvalbumin 및 apolipoprotein E을 선발하여 그 유전자 발현을 real-time PCR 기법을 사용하여 정량적으로 모니터링 하였다. Orla C3-1, parvalbumin 및 apolipoprotein E는 고농도의 다이아지논 처리(1000 ppb; 24 h)에서 그 발현이 억제됨이 관찰되었다. 다이아지논 처리 시 신경질환 (알츠하이머 병 및 다운신드롬)에 관련된 apolipoprotein E와 근육세포의 유연화에 작용하는 parvalbumin 등의 발현억제는 송사리의 인지능력 교란 및 근육세포의 경직 등을 각각 유도하여 송사리의 비정상적 행동을 야기하는 것으로 판단되었다. 따라서 이들 생물지표는 신경독성물질에 의한 송사리 및 기타 어류의 이상행동의 변화의 감지에 활용될 수 있을 것으로 사료된다.

• BMB Reports
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• 제38권4호
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• pp.447-456
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• 2005

TNF-$\alpha$ plays a pivotal role in inflammation processes which are mainly regulated by endothelial cells. While TNF-$\alpha$ induces apoptosis of several cell types like tumor cells, endothelial cells are resistant to TNFa mediated cell death. The cytotoxic effects of TNF-$\alpha$ on most cells are only evident if RNA or protein synthesis is inhibited, suggesting that de novo RNA or protein synthesis protect cells from TNF-$\alpha$ cytotoxicity, presumably by NF-${\kappa}B$ mediated induction of protective genes. However, the cytoprotective genes involved in NF-${\kappa}B$ dependent endothelial cell survival have not been sufficiently identified. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF-$\alpha$ inducible genes in human arterial endothelial cells related to cell survival and cell cycle. The TNF-$\alpha$-induced expression of the RNA binding protein $p54^{nrb}$ and the 14-3-3 protein HS1 as shown here for the first time may contribute to the TNF-$\alpha$ mediated cell protection of endothelial cells. These genes have been shown to play pivotal roles in cell survival and cell cycle control in different experimental settings. The concerted expression of these genes together with other genes related to cell protection and cell cycle like DnaJ, $p21^{cip1}$ and the ubiquitin activating enzyme E1 demonstrates the identification of new genes in the context of TNF-$\alpha$ induced gene expression patterns mediating the prosurvival effect of TNF-$\alpha$ in endothelial cells.

• 한국발생생물학회지:발생과생식
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• 제6권2호
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• pp.89-96
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• 2002

성장을 멈추고 있는 원시난포(primordial follicle)에서 난포발달이 개시되어 1차난포(primary follicle)로 발달하는 조절기전은 잘 알려져 있지 않다. 이 초기 난포발달 과정에 관여하는 유전자를 알아내기 위해 suppression subtractive hybridization(SSH)을 사용하였다. 생후 1일과 5일째의 생쥐 난소로부터 얻은 cDNA로 forward와 reverse subtraction을 수행하여 각각 day1과 day5-subtracted cDNA library를 얻었다. 이를 cloning한 결과, 357개 clone의 염기 서열을 BLAST와 RIKEN을 이용해 분석하여 27개의 clone은 novel gene으로 330개의 clone은 데이터 베이스와 일치함을 알았다. 이 중에 기능이 알려진 유전자는 day1에서는 42종, day5에서는 47종이 각각 차이 나게 발현하고 있는 것으로 나타났다. Day1-subtracted cDNA library에서는 GDF8, lats2, septin2, wee1등 4개 유전자를, day5-subtracted cDNA library에서는 HSP84, laminin2, MATER, MTi7, PTP 및 wrn등 6개 유전자를 선택하여 LCM-RT-PCR방법으로 실제로 원시난포와 1차난포에서 차이 나게 발현되고 있는 것을 확인하였다. 본 연구에서 얻은 유전자 발현 양상의 결과는 앞으로 생쥐뿐만 아니라 사람 난소에서 primordial-primary follicle transition에 관여하는 기전을 연구하는데 중요한 정보를 제공할 수 있을 것으로 사료된다.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2002년도 추계국제학술대회
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• pp.167-167
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• 2002

Methylmercury (MeHg), one of the heavy metal compound, can cause severe damage to the central nervous system in humans. Many reports have contributed MeHg poisoning to contaminated foods and release into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established. To find genes differentially expressed by MeHg in neuronal cell, we peformed forward and reverse suppression subtractive hybridization (SSH) method on mRNA derived from neuroblastoma cell line, SH-SY5Y treated with solvent (DMSO) and 6.25 uM (IC$\sub$50/) MeHg. Differentially expressed CDNA clones were sequenced and the mRNAs were re-examined on Northern blots. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences has provided an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as common environmental pollutants.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2003년도 춘계학술대회
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• pp.187-187
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• 2003

Methylmercury (MeHg), one of the heavy metal compounds, can cause severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. In this study, two methods, cDNA Microarray and SSH, were performed to assess the expression profile against MeHg and to identify differentially expressed genes by MeHg in neuroblastoma cell line. TwinChip Human-8K (Digital Genomics) was used with total RNA from SH-SY5Y (human neuroblastoma cell line) treated with solvent (DMSO) and 6.25 uM (IC50) MeHg. And we performed forward and reverse SSH method on mRNA derived from SH-SY5Y treated with DMSO and MeHg (6.25 uM). Differentially expressed cDNA clones were sequenced and were screened by dot blot and ribonuclease protection assay to confirm that individual clones indeed represent differentially expressed genes. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences may provide an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as environmental pollutants.

• 생명과학회지
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• 제14권4호
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• pp.650-656
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• 2004

야생벼의 일종인 Oryza grandiglumis (CCDD, 2n=48)는 도열병, 잎집무늬마름병, 흰빛잎마름병, 그리고 벼멸구와 같은 병충해에 저항성을 가지는 것으로 알려져 있다. 이와 같은 곰팡이와 해충에 반응하여 차별 발현하는 유전자를 클로닝 하기 위하여 상처처리와 yeast extract를 Oryza grandiglumis에 0시간과 24시간 각각 처리하였다. 유전자의 클로닝을 위하여 희귀 발현유전자의 클로닝에 효율적인 것으로 알려진 Suppression Subtractive Hybridization (SSH) 방법이 처리 후 24시간 된 식물을 재료로 사용되었다. 그 결과, 776개의 cDNA clones이 확보되었으며, 유전자 발현의 진위여부를 빠르게 스크린하기 위하여 colony array가 수행되었다. 115개의 colony가 positive로 판명되었고, 이들의 평균 insert size는 400 bp에서 700 bp에 이르렀고, 이들에 대한 염기서열 분석이 수행되었다. 염기서열 분석 결과, 68개 clone들이 알려진 기능의 유전자와 homology를 나타냈으며, 이중에서 16개 clone이 일차대사에 관련된 것과 유사성을, 5개가 plant retrotransposon과 유사성을, 5개가 식물 방어기작 관련 metallothionein-like gene과 염기서열 유사성을 보였다. 이외에 다양한 유전자들이 아미노산 합성관련, membrane transport, signal transduction등에 관여하는 유전자들과 상동성을 나타내었다. 이들 유전자중에서 4 개의 클론(ogwfi-161, ogwfi-646, ogwfi-663, ogwfi-695)들이 선발되었고 이들에 대한 Northern 분석이 수행되었다. Northern 분석 결과 ogwfi-161, ogwfi-646, ogwfi-663, ogwfi-695는 wounding과 yeast extract처리 에 의한 차별 발현이 확인되었다. 이상의 결과를 종합하여 볼 때, SSH방법은 병충해등과 같은 조건에 의해 차별 발현되는 유전자들을 빠른 시간 내에 다량으로 발굴할 수 있는 매우 효율적인 방법이라고 생각된다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.69.1-69
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• 2003

RSH (relA/spoT homolog) has been known to determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotide of the prokaryotic stringent response and also play a role in antibiotic production and differentiation in Streptomyces species but not a little in eukaryotic organism, especially in plant. Salicylic acid (SA), a critical signal molecule of establishing systemic acquired resistance (SAR), could induce SAR in Pepper (Capcicum annuum) against Phytophthora capsici. And the extent of SAR induction was in proportion to the dosage of SA (or BTH). Suppression subtractive hybridization (SSH), a PCR-based method for cDNA subtraction, was carried out between SA-treated and non-SA-treated pepper leaves to isolate genes which may be responsible for defense signaling against pathogens. Early upregulated gene was selected from reverse northern and kinetics of SSH-genes transcripts in SA-treated pepper leaves upon SA treatment. Full-length cDNA of the gene (PepRSH； Pepper RelA / SpoT homolog) had an open reading frame (ORF) of 2166 bp encoding a protein of 722 amino acids and a significant homology with (p)ppGpp phosphohydrolase or synthetase. Genomic DNA gel blot analysis showed that pepper genome has at least single copy of PepRSH. PepRSH transcripts was very low in untreated pepper leaves but strongly induced by SA and methyljasmonic acid (MeJA), indicating that PepRSH may share common SA and MeJA-mediated signal transduction pathway Functional analysis in E. coli showed PepRSH confers phenotypes associated with (p)ppGpp synthesis through a complementation using active site mutagenesis.

• BMB Reports
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• 제38권4호
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• pp.420-431
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• 2005

The differentially virulent race T1 of common bunt (Tilletia tritici) was used to inoculate the wheat lines Neepawa (compatible) and its sib BW553 (incompatible) that are nearly isogenic for the Bt-10 resistance gene. Inoculated crown tissues were used to construct a suppression subtractive hybridization (SSH) cDNA library. Of the 1920 clones arrayed from the SSH cDNA library, approximately 10% were differentially regulated. A total of 168 differentially up-regulated and 25 down-regulated genes were identified and sequenced; 71% sequences had significant homology to genes of known function, of which 59% appeared to have roles in cellular metabolism and development, 24% in abiotic/biotic stress responses, 3% involved in transcription and signal transduction responses. Two putative resistance genes and a transcription factor were identified among the up regulated sequences. The expression of several candidate genes including a lipase, two non-specific lipid transfer proteins (ns-LTPs), and several wheat pathogenesis-related (PR)-proteins, was evaluated following 4 to 32 days post-inoculation in compatible and incompatible interactions. Results confirmed the higher overall expression of these genes in resistant BW553 compared to susceptible Neepawa, and the differential up-regulation of wheat lipase, chitinase and PR-1 proteins in the expression of the incompatible interaction.

• Reproductive and Developmental Biology
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• 제32권4호
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• pp.211-215
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• 2008

The HMG box containing protein (HBP) has a high mobility group domain and involved in the regulation of proliferation and differentiation of tissues. We screened HBP2 in glioblastoma using Suppression Subtractive Hybridization (SSH) and isolated human spermatogonial stem cell-like cells (hSSC-like cells) derived from patients of nonobstructive azoospermia (NOA). Expression of HBP2 was analyzed by RT-PCR in undifferentiated stem cells (human Embryonic Stem Cells, hSSC-like cells 2P) and spontaneous differentiated stem cells (hSSC-like cells 4P). It was overexpressed in hESC and hSSC-like cells 2P but not in hSSC-like cells 4P. Also, the expression level of HBP2 was downregulated in colon tumor tissues compared to normal tissues. Specifically in synchronized WI-38 cells, HBP2 was highly upregulated until the G1 phase of the cell cycle and gradually decreased during the S phase. Our results suggest that HBP2 was downregulated during the spontaneous differentiation of hSSC-like cells. HBP2 was differently expressed in colon tissues and was related to G1-progression in WI-38 cells. It may playa role in the maintenance of an undifferentiated hSSC-like cell state and transits from G1 to S in WI-38 cells. This research was important that it identified a biomarker for an undifferentiated state of hSSC-like cells and characterized its involvement to arrest during cell cycle in colon cancer.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2002년도 추계국제학술대회
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• pp.156-156
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• 2002

The goal of this study is to develop a biomarker used in monitoring abnormal behaviors of Japanese medaka (Oryzias latipes) as a model organism caused by hazardous chemicals that are toxic and persistent in the ecosystem. A widely used insecticide, diazinon (O, O-diethyl O- (2-isopropyl-4-methyl-6-pyrimidinyl) phosphorothioate), is highly neurotoxic to fish, and it is also well known that it causes vertebral malformation and behavioral changes of fish at relatively low concentrations. The fish behaviors were observed on a real time basis using an image processing and automatic data acquisition system. The genes potentially involved in the abnormal behaviors were cloned using suppression subtractive hybridization (SSH) technique. The untreated individuals showed common behavioral characteristics. When the test fish was affected by diazinon at a concentration of 0.1 and 1 ppm, some specific patterns were observed in its behavioral activity and locomotive tracks. The typical patterns were enhanced surfacing activity, opercular movement, erratic movement, tremors and convulsions as reported previously. The number of genes up-regulated tty diazinon treatment were 97 which includes 27 of unknown genes. The number of down-regulated genes were 99 including 60 of unknown genes. These gene expression patterns will be analyzed by the artificial neural networks such as self organization map (SOM) and multilayer perceptron (MLP), revealing the role of genes responsible for the behaviors. These results may provide molecular biological and neurobehavioral bases of a biomonitoring system for diazinon using a model organism such as fish.