• Journal of Life Science
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• v.12 no.1
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• pp.96-105
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• 2002

To investigate expression of the artificial gene encoding a repeated tripeptide lysyl-glutamyl-tryptophan in tobacco plant, the plant binary vector, pART404 has been constructed, which contains the duplicated CaMV 35S promoter, an artificial gene coding for repetitive polymer (Lys-Glu-Trp)$_{64}$, and nopaline synthase (nos) terminator. The recombinant expression vector was introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated trans-formation. The transgenic calli selected by kanamycin containing medium were then regenerated to whole plants. Southern blot analysis indicated that five transgenic plants (No. 1, 7, 9, 43, 45) showed the hybridizing signals at 1.1 kb of the expected size on EcoRI digestion and each of the transgenic plants contained 1 or 3 copies of the artificial gene inserted into its genome. By northern blot analysis, the size of the hybridized total RNA was estimated to be approximately 1.2 kb and the RNA appeared generally to have the integrity. Western blot indicated that the protein was detected at the position of 33 kDa and the expression level of the polypeptide in the transgenic plant (No. 45) was measured to approximately 0.1% of the total protein.

• Radiation Oncology Journal
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• v.19 no.4
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• pp.389-396
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• 2001

Purpose : To detect differentially expressed genes in the patients with uterine cervical cancer during the radiation therapy. Materials and Methods : In patients with biopsy proven uterine cervical cancer, we took tumor tissue just before radiation therapy and at 40 minutes after external irradiation of 1.8 Gy. Total RNAs isolated from non-irradiated and irradiated tumor tissue samples were analyzed using the differential-display reverse transcription-polymerase chain reaction (DDRT-PCR). Complementary DNA (cDNA) fragments corresponding to differentially expressed messenger RNAs(mRNAs) were eluted, and cloned. The differential expression of the corresponding mRNAs was confirmed by reverse northern blot. Differentially expressed cDNA bands were sequenced. Nucleotide sequence data were analyzed in the Gene Bank and EMBL databases via the BLAST network sewer to identify homologies to known genes or cDNA fragments. Expression pattern of down-regulated clone was examined using RT-PCR in S patients undergoing radiotherapy. Results : We identified 18 differentially expressed bands by DDRT-PCR, which were eluted and cloned. There were 10 up-regulated clones and 1 down-regulated clone in reverse northern blot. One cDNA fragment had homology to chemokine receptor CXCR4, four were identified as Human ESTs in the EMBL database in EST clones. Down-regulated CxCa-11 was also down regulated in all patients. Conclusion : Using the DDRT-PCR, we have identified 10 up-regulated and 1 down-regulated clone(s) in the patients with uterine cervical cancer during the radiation therapy. The clinical relevance and the functions of these genes will be further investigated.

• BMB Reports
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• v.40 no.2
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• pp.270-276
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• 2007

We have cloned a novel mouse zinc finger protein gene Znf313 by rapid amplification of cDNA ends (RACE) according to the homologue of human ZNF313 gene. The cDNA is 2,163 base pairs (bp) in length and encodes a 229 amino acids (aa) protein with a $C_3HC_4$ ring finger domain and three $C_2H_2$ domains. 89% and 93% nucleotide (nt) and aa sequence identity is observed with its human homologue. Revealed by Northern blot and RT-PCR, full mRNA consists of 2.16 kb and widely expresses in tissues as a single transcript, most abundantly in heart, liver, kidney and testis. The expression of Znf313 in testis is detected in all development stages. Western blot analysis also reveals that Znf313 is expressed in the tissues. Immunohistochemical staining and subcellular localization demonstrate that Znf313 is expressed both in the cytoplasm and nucleus whereas predominantly localized in the nucleus. Present data suggests that Znf313 gene might play a fundamental role in gene transcription and regulation in organism and relates to spermatogenesis.

• 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.

• Journal of Plant Biotechnology
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• v.37 no.1
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• pp.89-95
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• 2010

Dihydroflavonol 4-reductase (DFR) is a key enzyme of the flavonoid biosynthesis pathway which catalyses the NADPH-dependent reduction of 2R,3R-trans-dihydroflavonols to leucoanthocyanidins. In this study we describe cloning and expression of the genes encoding the flavonoid-biosynthetic enzyme DFR in Gypsophila paniculata L. Inspection of the 1279 bp long sequence revealed an open reading frame 1063 bp, including a 36 bp 5' leader region and 181 bp 3' untranslated region. Comparison of the coding region of this DFR cDNA sequence including the sequences of Arabidopsis thaliana, Citrus sinensis, Dianthus caryophyllus, Ipomoea batatas, Matthiola incana, Nierembergia sp, Petunia hybrida, Solanum tuberosum, Vitis vinifera reveals an identity higher than 69% at the nucleotide level. The function of this nucleotide sequences was verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRNA from wild type and mutant plants, by in vitro expression yielding and enzymatically active reductase, as indicated by the small leucopelargonidin peak. Genomic southern blot analysis showed the presence of only one gene for DFR in Gypsophila paniculata.

• The Korean Journal of Zoology
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• v.33 no.4
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• pp.446-453
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• 1990

In order to investigate the retinoic acid indticed-differentiation of F9 teratocarcinoma stem cell, we have analyzed the change of cell morphology and laminin expression after exposure to retinoic add and cyclic AMP. It is shown that undifferentiated F9 stem cells grow as closely packed colonies, and it is difficult to distinguish cell-cell boundaries. After retinoic add and dibutyryl cyclic AMP treatment, F9 cells assume a flat morphology characterized by perinuclear granules and arrest growth. According to Northern blot analysis, laminin expression was increased markedly after retinoic acid treatment. Laminin Bi gene expression was increased at least 30-fold and laminin B2 gene expression was increased approximately 20-fold during differentiation process. Employing immunofluoresence analysis, it was proved that the synthesis of laminin protein was low level in F9 stem cell whereas it became high level in retinoic acid treated F9 cell and the laminin protein was largely accumulated in the cell surface. Our results suggest that induction of laminin Bi and B2 genes m F9 cells is retinoic acid-mediated control, and morphological change and differentiation of F9 cells might be associated with laminin gene expression.

• 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.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.186-189
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• 2003

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a potent immunogen as well as major virulence factor. In order to express the recombinant urease in tobacco plants, a DNA fragment containing the minimal H. pylori urease gene cluster was subcloned into a plant expression vector. The recombinant vector was transformed to tobacco plants. The integration of the recombinant plasmids into tobacco chromosomal genome was verified by genomic PCR. Expression to mRNA was confirmed by Northern blot analysis, and expression to recombinant urease protein was observed by Western blot analysis. These results showed that the recombinant urease can be produced in tobacco plants and will be tested for immune response to use as an edible vaccine.

• BMB Reports
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• v.32 no.2
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• pp.189-195
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• 1999

From the Panax ginseng chloroplast, the psbE and psbF genes, encoding the $\alpha$- and $\beta$-subunits of cytochrome b-559 of the photosystem II reaction center, respectively, were cloned and characterized. The psbE and psbF genes were composed of 252 and 117 nucleotides, respectively. The deduced amino acid sequence of the $\alpha$-subunits showed 95%, 93%, and 91% homology to monocots, dicots, and liverwort, respectively, whereas the $\beta$-subunits showed approximately 98% to 95% homology to the same species. Southern blot analysis revealed that a single copy of the psbEF gene exists in the chloroplast plastid. Northern blot analysis indicated that the psbE and psbF genes are cotranscribed as a polycistron.