• Journal of Animal Science and Technology
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• 제44권1호
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• pp.13-22
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• 2002

한우의 성장단계 특이발현 유전자를 탐색하기 위하여, 본 연구에서는 유전자의 발현 유무 및 발현정도의 차이를 나타내는 유전자를 분리하는데 있어 가장 강력한 수단으로 알려진 subtractive cDNA hybridization 기법을 이용하여 한우 등심조직으로부터 12개월령 및 24개월령 특이적인 subtractive cDNA library를 제작하였다. 성장단계 특이적인 유전자를 탐색하기 위하여, 6, 12 및 24개월령 cDNA를 사용하여 reverse northern blot 분석을 실시하였으며, 6개월령 cDNA probe에 대하여 특이적인 signal을 나타낸 3개의 clone은 EPV 20, Ca2+ ATPase, 및 TCTP 유전자와 유사성을 나타내었다. 12개월령 cDNA probe에 대하여 특이적인 signal을 나타낸 9개의 cDNA clone은 각각 VCP, HSP 70, aldolase A, MSSK1, GM-2 activator protein, ryanodine receptor, acidic ribosomal phosphoprotein p1, ADP/ATP translocase T1 및 UCP 2 유전자와 높은 homology를 가지고 있었다. 또한 2개의 clone이 각각 12개월령 및 24개월령 cDNA probe에 대하여 특이적인 signal을 나타내었는데, 12개월령 cDNA probe에 대해서만 signal을 나타낸 clone은 ferrochelatase 유전자와 유사하였으며, 24개월령 probe에 대해서만 signal을 나타낸 clone은 ADRP 유전자와 유사하였다. 이상에서와 같이, 본 연구에서 제작한 성장단계 특이적인 subtractive cDNA library를 분석하여 14종의 유전자를 한우 성장단계 특이 발현 후보 유전자로 선정하여 염기서열을 분석하였으며, 이외에도 성장단계에 있어 특이적으로 발현될 것으로 추정되는 cDNA 클론의 염기서열을 분석하였다.

• 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.

• The Plant Pathology Journal
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• 제19권1호
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• pp.34-42
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• 2003

The ascomycete Magnaporthe grisea is a pathogen of rice blast and is known to form specialized infection structures called appressoria for successful infection into host cells. To understand the molecular mechanism underlying infection process, appressorium-related genes were identified through global approaches including EST sequencing, differential hybridization, and sup-pression subtractive hybridization. EST database was generated on >2,000 cDNA clones randomly selected from appressorium stage cDNA library. Large number of ESTs showed homology to known proteins possibly involved in infection-related cellular development (attachment, germination, appressorium formation, and colonization) of rice blast fungus. The 1051 ESTs showing significant homology to known genes were assigned to 11 functional categories. Differential hybridization and suppression subtractive hybridization were applied to identify genes showing an appressorium stage specific expression pattern. A number of genes were selected as up-regulated during appressorium formation compared with the vegetative growing stage. Clones from various cDNA libraries constructed in different developmental stages were arrayed on slide glass for further expression profiling study. functional characterization of genes identified from these global approaches may lead to a better understand-ing of the infection process of this devastating plant disease, and the development of novel ways to protect host plant.

• 한국패류학회지
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• 제29권3호
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• pp.181-187
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• 2013

In order to investigate environmental stress inducible genes in abalone, we analyzed differentially expressed transcripts from a Pacific abalone, Haliotis discus hannai, after exposure to heat-, cold- or hyposalinity-shock by suppression subtractive hybridization (SSH) method. 1,074 unique sequences from SSH libraries were composed to 115 clusters and 986 singletons, the overall redundancy of the library was 16.3%. From the BLAST search, of the 1,316 ESTs, 998 ESTs (75.8%) were identified as known genes, but 318 clones (24.2%) did not match to any previously described genes. From the comparison results of ESTs pattern of three SSH cDNA libraries, the most abundant EST was different in each SSH library: small heat shock protein p26 (sHSP26) in heat-shock, trypsinogen 2 in cold-shock, and actin in hyposalinity SSH cDNA library. Based on sequence similarities, several response-to-stress genes such as heat shock proteins (HSPs) were identified commonly from the abalone SSH libraries. HSP70 gene was induced by environmental stress regardless of temperature-shock or salinity-stress, while the increase of sHSP26 mRNA expression was not detected in cold-shock but in heat-shock condition. These results suggest that the suppression subtractive hybridization method is an efficient way to isolate differentially expressed gene from the invertebrate environmental stress-response transcriptome.

• BMB Reports
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• 제37권5호
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• pp.527-532
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• 2004

A subtracted cDNA library specific to osmotic stress of Haloxylon ammodendron (Mey.) Bge seedlings was constructed by suppression subtractive hybridization (SSH) and T/A cloning. SSH was performed between two groups of H. ammodendron seedlings, one was cultivated in Hoagland (H) solution as a driver and the other group was treated with osmotic stress of the Hoagland solution by the addition of 400 mM mannitol (M), as a tester. The library consisted of about 400 recombinant clones, with the average size being of 500 bp, ranging from 300 bp to 1500 bp. Using a PCR-select differential screening kit, 100 recombinant clones were randomly chosen from the subtracted cDNA library and hybridized with forward,reverse subtracted and unsubtracted probes for two rounds. As a result, 21 positive clones specific to osmotic stress were obtained and some of them were verified by Northern blot analysis. The sequencing analysis of 6 positive clones and the following homology comparison to GenBank [blastx] non-redundant databases characterized that two sequences obtained in this experiment may contribute to novel drought-related genes.

• 한국자원식물학회지
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• 제19권3호
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• pp.440-446
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• 2006

To investigate flowering related genes in winter-type oilseed rape (Brassica napus L. cv. Tammi), differentially expressed genes were isolated from leaves of the plant after low temperature treatment which is requirements for floral induction. As a result of suppression subtractive hybridization (SSH), 288 clones were randomly selected from SSH library. Using reverse Northern blot analysis, 150 of 288 clones were identified to be differentially expressed. Out of these 150 clones, 45 clones showed very high identities with the known genes. Four clones showed very high identities over 90% with metallothionein-like gene that is related to flowering-induced genes. Of these 4 clones, the cDNA clone, rfs-13, revealed high identity with meotallothionein-like protein in Arabidopsis thaliana (98%) and Brassica compestris (89%). Furthermore, gene expressed in immature flower stages was confirmed by Northern blot analysis.

• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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• pp.17-24
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• 2010

In the antral follicle phase, several layers of cumulus cells surround the oocyte and play an important support and regulation role in oocyte development and maturation via intercellular communications and interactions between oocytes and cumulus cells. However, information on stage specific gene expression in swine during the phase is not well understood. To investigate the function of cumulus cells during in vitro maturation of porcine oocytes and gene expression, suppression subtractive hybridization (SSH) was performed to screen genes that were differentially expressed between cumulus-oocyte complexes (COCs) and naked oocytes (NOs). Utilizing mRNAs from in vitro maturation oocytes, a SSH cDNA library from COCs as the tester and NOs as the driver was constructed. The SSH cDNA library was then screened using dot blot analysis. Results showed that a total of 70 clones randomly selected from the library were differentially expressed. Among these, 41 exhibited high homology to known genes and 11 were novel expressed sequences tags (ESTs). Four differentially expressed genes, including bfgf, sprouty 2, egr and btc, were further studied by real time quantitative PCR; results confirmed an increased expression of respective mRNA in COCs compared with NOs, which suggests that these factors may play an important role in oocyte development and maturation.

• 한국해양바이오학회지
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• 제2권3호
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• pp.192-197
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• 2007

넙치의 생체방어 및 면역관련 유전자의 대량확보를 위하여, 지질다당질로 자극한 넙치의 말초백혈구 cDNA library를 suppression subtractive hybridization 방법으로 제작하여 발현유전자의 발현 양상 및 특성을 분석하였다. 총 470개의 SSH 클론 중 기존에 밝혀진 유전자와 상동성을 보인 218개의 클론을 분석한 결과, 대표적인 전염증사이토카인 IL-1의 유인수용체인 IL-1RII가 34번의 가장 높은 빈도로 발현되었으며, IL-$1{\beta}$가 14번의 높은 발현 빈도를 보였다. 그 중 펩티도글리칸 인식 단백질및 혈관활성 장 펩타이드 유전자는 어류에서는 처음으로 보고되는 것으로, 지질다당질로 자극한 넙치의 말초백혈구 SSH cDNA library는 다양한 생체방어 및 면역관련 유전자를 포함하고 있었다.

• Molecular & Cellular Toxicology
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• 제2권3호
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• pp.170-175
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• 2006

The extensive isolation of genes responsive to stressful conditions from a soft coral Scleronephthya gracillimum was described. Soft coral colonies were exposed to thermal and chemical stressors to induce the expression of stress related genes. Differentially expressed genes by natural or anthropogenic stressors were identified by construction of standard and stress exposed-paired subtractive cDNA library. Thirty-two and thirty-seven kinds of candidate genes were identified from thermal or benzo[a]pyrene stress exposed group, respectively, which are associated with cell cycle, cell signaling, transcription, translation, protein metabolism, and other cellular functions. The expected function of each gene was described. The isolated and identified differentially expressed genes have a great potential to identify environmental stressors in global environmental changes and could act as molecular biomarkers for biological responses against environmental changes. Finally, it may open a new paradigm on soft coral health assessment.

• Journal of Plant Biology
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• 제39권3호
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• pp.189-195
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• 1996

The major cuticular components have been shown to be synthesized in the epidermis. Therefore, cloning of epidermis-specific genes could yield information to be used to isolate and characterize the enzymes involved in the cuticle biosynthesis. A subtractive cDNA library was prepared from Senecio odorus in which epidermis-specific cDNAs were enriched. Differential screening of the library using epidermal and non-epidermal probes revealed two cDNAs. One of them designated epi425 was identified, based on the sequence homology, as a member of a new class in the LTP gene family and the other clone designated epi23 as a gene encoding an aldehyde decarbonylase. Northern blot analyses showed that epi425 and epi23 cDNAs hybridized with a transcript of about 600 and 2, 100 nucleotides, respectively, from the epidermis but not from the non-epidermal tissues. Further characterization of these clones will provide more information on the mechanism of the cuticle biosynthesis.