• Molecular & Cellular Toxicology
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• 제2권2호
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• pp.114-119
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• 2006

Benzo[a]pyrene is a representative ecotoxicant in marine environment and a model compound of polycyclic aromatic hydrocarbons, which has an ability to bioaccumulate in aquatic organisms. This study aimed to identify molecular biomarkers suitable for assessing environmental pollution using a microarray technique. We examined the effects of benzo[a]pyrene on gene expressions in the rockfish, Sebastes schlegeli. We constructed the subtractive cDNA library with hepatic RNA from benzo[a]pyrene-exposed and non-exposed control fish. From the library 10,000 candidate clones were selected randomly and cDNA microarray was constructed. We determined benzo[a]pyrene-responsive genes using a high-density microarray. Statistical analysis showed that approximately 400 genes are significantly induced or reduced by benzo[a]pyrene treatment ($2\;{\mu}m$). Especially gene expression changes of 4 candidate clones among the up- or down-regulated genes were investigated in 6, 12 and 24 hr BaP-exposed fish groups. Many methods have been developed to monitor marine environmental status, which depend on quantifying the levels of the toxic components in polluted seawater or on ecological accessing, such as species diversity or richness. However, those methods could not provide information on physiological or genetic changes induced by such environmental stresses. Comparing with the conventional methods, these data will propose that benzo[a]pyrene-responsive genes can be useful for biological risk assessment of polycyclic aromatic hydrocarbons on marine organism at molecular level.

• Radiation Oncology Journal
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• 제23권1호
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• pp.43-50
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• 2005

목적 : 자경경부암세포에서 polymeric chain reaction (PCR)원리를 이용한 suppression subtractive hybridization (SSH) 방법으로 방사선조사 시 차등 발현되는 유전자를 동정하고자 하였다. 대상 및 방법 : 자궁경부암세포주인 HeLa세포주에 방사선조사 전과 후 총 RNA와 poly $(A)^+$ mRNA를 분리였다. SSH방법으로 forward 및 reverse-subtracted cDNA libraries를 만들었다. 차등 발현된 유전자를 screening하기 위해 reverse Northern blotting (dot blot analysis)을 이용하여 각각의 library에서 88개의 클론을 선택하였고 Nothern blotting으로 확인 후 sequencing하였다. 결과 : screening상 176개 클론 중 forward-subtracted library에서 10개의 유전자가 reverse-subtracted library에서 9개의 유전자가 동정되었다. forward-subtracted library로부터 3개의 유전자가 Northern blotting에 의하여 확인되었고 이중 telomerase catalytic subunit and sodium channel-like protein 유전자와 1개의 ESTs (expressed sequence tags) 유전자가 방사선선량에 따라 증가하쳐다. 결론 : 본 연구를 통해 자궁경부암세포주에서 방사선에 의해 유도되는 유전자를 SSH 방법을 통해 동정할 수 있었다. 그러나 이러한 유전자가 어떤 생물학적인 기능을 갖고 있는지에 대한 계속적인 연구가 필요하다

• 한국육종학회지
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• 제40권3호
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• pp.234-242
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• 2008

To identify low temperature-induced genes of wheat chromosome substitution line 5D, suppression subtractive hybridization (SSH) was performed with mRNAs from leaf samples that treated with low temperature ($4^{\circ}C$). A cDNA library was constructed using mRNA isolated from wheat chromosome substitution line 5D leaves treated with low temperature ($4^{\circ}C$). The nucleotide and deduced amino acid sequences of the putative gene products were compared. wfr-9 and wfr-32 showed identity over 90% related to vernalization gene. Other two genes, wfr-77 and wfr-83 which is related to freezing-resistant gene have also identity over 90%. This result suggest that those genes may be transcribed into antifreeze proteins which are accumulated within leaf apoplasts, when wheat chromosome substitution line 5D is acclimated during low temperature treatment.

• BMB Reports
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• 제36권6호
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• pp.580-585
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• 2003

Subtractive library hybridization was used to isolate the cDNA clones that corresponded to the transcripts that were specifically up-regulated during wheat embryo germination. The clones with numbers 5, 6, 7, 8, 24, and 26 appeared to be more abundant in germinating wheat embryos. Among the isolated clones, we identified four new members of the wheat "germin" gene family. We also identified two novel sequences which exhibited distinct germination up-regulation, and displayed characteristic spatial patterns of expression. One of these, represented by clone pSB10, was principally expressed in the root tissue of germinating embryos. The second was represented by the pSB7 clone and was expressed in both the root and shoot primordia of the embryonic axis, as well as within the coleoptile.

• 한국어병학회지
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• 제19권3호
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• pp.189-195
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• 2006

A subtracted cDNA library of a marine scuticociliate, Philasterides dicentrarchii, in response to 17β-estradiol exposure was constructed using suppression subtractive hybridization (SSH). As a result of SSH, 275 clones were isolated, and among them, only glutathione peroxidase (GPX) gene was isolated as an antioxidative enzyme responding to 17β-estradiol. The semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis revealed that the transcription of GPX gene of P. dicentrarchii was clearly increased by exposure to 17β-estradiol. The GPX transcription was also clearly increased by exposure to xenoestrogens such as bisphenol A (BPA) and genistein.

• 한국잠사곤충학회지
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• 제50권2호
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• pp.122-125
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• 2012

본 연구는 누에 배자 발생 초기 특이 발현 유전자 프로모터를 개발하기 위한 연구의 일환으로 추진하였다. 누에 초기 및 후기 배자로 부터 분리한 mRNA를 사용하여 subtractive hybridization 분석법으로 누에 배자 발생 초기 특이 발현 유전자 4종을 선발할 수 있었다. 선발된 4종 유전자는 각각 BmNanos protein mRNA, BmNanos-P protein mRNA, BmNanos-O protein mRNA 및 BmVasa protein mRNA 유전자와 매우 높은 상동성을 보였다. 또한, 본 연구에서는 Northern hybridization 분석 및 real time PCR 분석을 통하여 배자 초기에 특이적으로 고발현하는 BmNanos-like 등 4개 선발 유전자의 발현 특성을 확인하였다. 이러한 결과는 추후 추진 할 누에 형질전환용 전이벡터의 효율성 제고를 위한 연구에 활용될 것으로 기대된다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.80.1-80
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• 2003

Several plant and microbial genes that could confer disease resistance in transgenic rice plants are being cloned and characterized. We are currently constructing transgenic rice lines that overexpress the gene products, such as a galactinol synthase, a defensin, and a bacterial ACC deaminase. Subtractive hybridization of a rice cDNA library constructed from the Xanthomonas oryzae-infected ice leaves resulted in isolation of many inducible cDNA clones including a elongation factor EF2, a oryzain alpha, a catalase, a aldehyde dehydrogenase, a S-adenosylmethionine synthetase, a caffeic acid O-methyltransferase, a glyceraldehyde-3-phosphate dehydrogenase, a light-regulated protein, nKY transcription factors, and a nucleotide diphosphate kinase. Some genes among those may be useful genetic sources for construction of disease resistant transgenic rice. Full lengths of the rice OsFIERG and a rice oryzain genomic clones were cloned, and serial deletion fragments of the promoter regions of these genes were fused with GUS reporter gene in pCAMBIA1201, respectively. Promoter activities of these constructs will be examined upon various stresses and Pathogen infections to obtain the pathogen specific inducible-promoter. This work was supported by a grant from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.90.1-90
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• 2003

Colonization of Pseudomonas chlororaphis O6, a nonpathogenic rhizobacterium, on the roots induced systemic resistance in cucumber plants against tai-get leaf spot, a foliar disease caused by Corynespora cassiicola. A cDNA library was constructed using mRNA extracted from the cucumber leaves 12 h after inoculation with C. cassiicola, which roots had been previously treated with O6. To identify the genes involved in the O6-mediated induced systemic resistance (ISR), we employed a subtractive hybridization method using mRNAs extracted from C cassiicola-inoculated cucumber leaves with and without previous O6 treatment on the plant roots. Differential screening of the cDNA library led to the isolation of 5 distinct genesencoding a GTP-binding protein, a putative senescence-associated protein, a galactinol synthase, a hypersensitive-induced reaction protein, and a putative aquaporin. Expressions of these genes are not induced by O6 colonization alone. Before challenge inoculation, no increase in the gene transcriptions could be detected in previously O6-treated and untreated plants but, upon subsequent inoculation with the pathogenic fungus, transcription levels in O6-treated plants rose significantly faster and stronger than in untreated plants. Therefore, the O6-mediated ISR may be associated with an enhanced capacity for the rapid and effective activation of cellular defense responses which becomes apparent only after challenge inoculation on the distal, untreated plant parts, as suggested by Conrath et al. (2002). This work was supported by a grant R11-2001-092-02006-0 from the Korea Science and Engineering Foundation through the Agricultural Plant Stress Research Center at Chonnam National University.

• 한국어류학회지
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• 제23권1호
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• pp.21-29
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• 2011

볼락의 성장단계에 따른 차등발현 유전자를 탐색하기 위하여 6개월령 및 18개월령 근육조직을 사용하여 subtracted cDNA library를 제작하였고, 각각의 연령에서 발현량 차이를 나타낸 202개의 cDNA 단편을 확보하였으며, 발현량 차이가 뚜렷한 32개의 cDNA 클론은 성장단계별 특이발현 후 보유전자로 선발하여 염기서열을 분석하였다. Myosin, adenylate kinase, calsequestrin, dystrobrevin beta, diphosphate kinase 유전자는 6개월령 근육조직에서 발현량이 많았으며, desmin, TGFBR2 (transforming growth factor-beta receptor), creatine kinase (muscle type), cathepsin D 유전자는 18개월령 근육조직에서 발현량이 많았다. 볼락의 성장초기와 성장절정기에서 차등발현 양상을 나타낸 유전자는 6, 18, 30, 42개월령 근육조직에서 연령 증가에 따른 발현양상을 분석하였으며, dystrobrevin beta와 diphosphate kinase-Z1은 6개월령 이후에는 발현량이 급격히 감소하여 18개월령, 30개월령 및 42개월령에서는 발현량이 극히 적었으며, creatine kinase (muscle type)와 cathepsin D 유전자는 연령 이 증가함에 따라 발현량이 증가되어 18개월령 이후, 30개월령과 42개월령 근육조직에서도 발현량이 많았다. 이와 같이 성장단계에 따른 차등발현 유전자를 탐색하고 연령 증가에 따른 발현양상을 비교 분석한 결과로부터 본 연구에서는 어류의 성장 초기단계 근육조직에서는 근육수축 관련 유전자가 많이 발현되고, 성장 절정기에는 근육 내 에너지 양 조절 관련 유전자가 많이 발현되는 것을 확인하였다.

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2003년도 International Symposium of Silkworm/Insect Biotechnology and Annual Meeting of Korea Society of Sericultural Science
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• pp.91-92
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• 2003

For studies of unfolded protein response (UPR), we isolated differentially expressed genes in Bm5 cell line induced with treatment of tunicamycin, the synthesis inhibitor of N-linked oligosaccharides in cells and constructed the subtractive cDNA library enriching UPR-related genes. (omitted)