• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.48-48
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• 2003

본 실험은 spermatogonia 단계에 발현하는 유전자를 찾기 위하여 suppression subtractive hybridization를 수행하였다. 기존에 mouse에서는 spermatogonia 특이적인 유전자들이 밝혀져 있기 때문에 pig에 특이적인 유전자를 찾기 위하여 pig 250days testis와 pig 60days testis를 재료로 하여 실험하였다. SSH를 통하여 254days testis에 특이적으로 발현되는 후보유전자를 7개 찾았고 25days testis와 60days testis 의 Northern blot을 통하여 25days에 과발현하고 60days에 발현의 양이 대폭 줄어드는 spermatogonia 유전자로 생각되는 후보유전자 2개를 선택하여 pig tissue northern blot, genomic DNA southern blot, RT-PCR 그리고 In-situ hybridization을 수행하였다. Tissue northern blot과 RT-PCR을 통하여 후보자 1번은 간과 폐, 난소, 정소에서 발현하고, 후보유전자 15번은 난소와 정소에서만 특이적으로 발현함을 알았다. DNA sequence analysis와 NCBI Blast search를 통하여 후보자 1번은 다른 종에서 밝혀진 유전자였고 후보유전자 15번은 어느 종에서도 밝혀지지 않은 새로운 유전자였다. Degenerated primer를 통하여 후보자 1번의 pig full sequence를 밝히고 NCBI에 등록하였다. 그리고 In-situ hybridization을 통하여 후보유전자득이 20일째 testis의 Leydic cell에서 많이 발현되고 adult testis에서는 발현이 감소하는 결과를 얻었다. 이것으로 보아 위의 두 후보유전자는 spermatogonia에 직접 관련된 유전자이기 보다는 spermatogonia의 발달에 영향을 주는 leydic cell 특이발현을 가진 유전자로 사료되어진다.

• Korean Journal of Plant Resources
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• v.19 no.3
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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.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.2
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• pp.111-118
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• 2008

Objective: The purpose of the present study was to examine the hormonal regulation of RGS-2 in the rat ovary. Methods: Immature rats were injected with 10 IU of PMSG to induce multiple growth of preovulatory follicles and 10 IU of hCG to induce ovulation. Northern blot analysis performed for gene expression and in situ hybridization performed for mRNA localization. Results: Northern blot analysis revealed that pregnant mare's serum gonadotropin (PMSG) treatment did not affect RGS-2 mRNA levels. In contrast, human chorionic gonadotropin (hCG) treatment of PMSG-primed rats resulted in an increase in RGS-2 expression within $1{\sim}3\;h$. The major cell-types expressing RGS-2 mRNA were oocytes regardless of follicle size. Interestingly, hCG treatment caused the stimulation of RGS-2 gene expression in granulosa cells of preovulatory and growing follicles. In contrast, cell types expressing RGS-2 protein were theca cells regardless of hCG treatment. Like in vivo, treatment of preovulatory granulosa cells with LH in vitro stimulated RGS-2 levels within 1 h. Interestingly, GnRH antagonist II enhanced the stimulatory action of LH. Conclusion: The present study demonstrates the LH/hCG induction of RGS-2 in preovulatory granulosa cells and suggests a role of RGS-2 in Gq protein signaling pathway during ovulation.

• Applied Biological Chemistry
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• v.40 no.4
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• pp.271-276
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• 1997

We have prepared several chimeric constructs containing the bacteriophage T7 RNA polymerase gene under control of the wound-inducible potato proteinase inhibitor II (pin2) promoter and have transformed Nicotiana tabacum plants with these constructs. Southern blot analyses indicate that either one or two copies of the gene constructs are present in the transgenic plants. Northern blot analyses indicate that mRNA encoding T7 RNA polymerase is expressed in a wound-inducible manner. We purified T7 RNA polymerase and prepared antiserum. This antiserum was used for Western blot analyses to demonstrate that a protein which is cross reactive with T7 RNA polymerase is produced. The molecular mass of this protein is 80 kDa, a size which is consistant with the nicked form of the polymerase as is often seen when expressed in E. coli. RNA polymerase assays were used to indicate that the nicked form of T7 RNA polymerase is active and capable of incorporating labeled nucleotides into transcripts in vitro. Analysis of transgenic plants did indeed show that wound-inducible activation of the T7 RNA polymerase permits the establishment of a genetic system to overexpress genes in plants using T7 RNA polymerase(Received March 20, 1997; accepted May 2, 1997)

• Journal of Plant Biotechnology
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• v.43 no.3
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• pp.341-346
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• 2016

The objectives of this study were to 1) evaluate the efficiency of the protocol of Agrobacterium-mediated transformation of cucumber to introduce phytoene synthase-2a carotene desaturase (PAC genes); 2) demonstrate the integration of PAC genes into the genome of putative transgenic cucumber based on growth on selection medium, PCR and Southern analysis; 3) evaluate the expression of PAC genes in transgenic cucumber based on the analysis of RT-PCR and Northern blot hybridization. Out of 5,945 cotyledonary-node explants inoculated with Agrobacterium, 65 (1.1%) explants produced 238 shoots. Integration of PAC genes into the genome of the cucumber was demonstrated based on the analysis of gDNA-PCR, 21 out of the 238 plants regenerated; while 6 plants proved positive for Southern blot hybridization. Transgene expression was demonstrated based on analysis of RT-PCR, 6 plants proved positive out of the 6 plants analyzed; while 4 plants out of 6 proved positive during Northern blot hybridization. This study successfully demonstrated the production of transgenic cucumber, integration, and expression of the PAC gene in cucumber.

• Horticultural Science & Technology
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• v.32 no.1
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• pp.91-99
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• 2014

The objective of this study is to identify cold-tolerance genes in Brassica rapa. In order to acheive this goal, we analyzed a KBGP-24K oligo chip data [BrEMD (B. rapa EST and Microarray Database)] using B. rapa ssp. pekinensis inbred line 'Chiifu' under cold stress condition ($4^{\circ}C$). Among 23,929 unigenes of B. rapa, 417 genes (1.7%) were primarily identified as cold responsive genes that were expressed over 5-fold higher than those of wild type control, and then a gene which has unknown function and has full length sequence was selected. It was named BrCSR (B. rapa Cold Stress Resistance). BrCSR was transformed using expression vector pSL101 to confirm whether BrCSR can enhance cold tolerance in tobacco plants. $T_1$ transgenic tobacco plants expressing BrCSR were selected by PCR and Southern hybridization analyses, and the function of BrCSR was characterized by expression level analysis and phenotype observation under cold stress condition. The expression level of BrCSR in transgenic tobacco plants increased up to about two folds in quantitative real-time RT-PCR assay and this was very similar to Northern blot hybridization analysis. Analysis of phenotypic characteristics clearly elucidated that transgenic tobaccos expressing BrCSR were more cold tolerant than wild type control under $4^{\circ}C$ treatment. Based on these results, we conclude that the over-expression of BrCSR might be closely related to the enhancement of cold tolerance.

• Radiation Oncology Journal
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• v.23 no.1
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• pp.43-50
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• 2005

Purpose : A number of genes and their products are Induced early or late following exposure of cells to ionizing radiation. These radiation-Induced genes have various effects on irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to Identify the differentially expressed genes by radiation in cervix carcinoma cells. Materials and Methods : Total RNA and poly $(A)^+$ mRNA were Isolated from Irradiated and non-irradiated HeLa cells. Forward- and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Results : Of the 17t clones, 10 genes in the forward-subtracted library and 9 genes In the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. Conclusion : We Identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.15-20
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• 2002

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid and it may play a role in signal transduction in Escherichia coli as well as in eukaryotic cells. In addition, DGK is important for microorganisms to adapt to several physiological stimuli. In Bacillus subtilis, the effect of stress on dgk transcription was examined by northern hybridization. The high level of dgk transcription was induced against high osmolarity, low pH value and low temperature. Transcriptional analysis revealed that the dgk gene and dgk upstream locus (ORF2, ORF3 and ORF4) were transcribed as a polycistronic mRNA to form an approximately 2.5 kb transcript.

• Molecules and Cells
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• v.23 no.1
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• pp.100-107
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• 2007

The moss Physcomitrella patens has two life cycles, filamentous protonema and leafy gametophore. A modified from of suppression subtractive hybridization (SSH), mirror orientation selection (MOS), was applied to screen genes differentially expressed in the P. patens protonema. Using reverse Northern blot analysis, differentially expressed clones were identified. The identified genes were involved mainly in metal binding and detoxification. One of these genes was an AP2 (APETALA2) domain-containing protein (PpACP1), which was highly up-regulated in the protonema. Alignment with other AP2/EREBPs (Ethylene Responsive Element Binding Proteins) revealed significant sequence homology of the deduced amino acid sequence in the AP2/EREBP DNA binding domain. Northern analysis under various stress conditions showed that PpACP1 was induced by ethephon, cadmium, copper, ABA, IAA, and cold. In addition, it was highly expressed in suspension-cultured protonema. We suggest that PpACP1 is involved in responses to metals, and that suspension culture enhance the expression of genes responding to metals.

• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.2
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• pp.223-228
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• 2008

A novel beetle antimicrobial protein from stimulated Copris tripartitus and the corresponding gene were isolated in parallel through differential display-PCR and expression in Escherichia coli. To find cDNA clones responsible for bacteria resistance, the suppression subtractive hybridization and GeneFishing differentially expressed genes system were employed in the dung beetle, Copris tripartitus immunized with lipopolysaccaride. One cDNA clone from eight subtracted clones was selected through dot blot analysis and confirmed by northern blot analysis. The 516-bp, selected cDNA clone was determined by 5' and 3' rapid amplication of cDNA ends and cloned into the GST fusion expression vector pGEX-4T-1 for expression of the protein. The expressed protein was predicted 14.7 kDa and inhibited the growth of gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. These results implied that the expressed protein is related to immune defense mechanism against microorganism.