• Journal of Plant Biotechnology
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• 제2권2호
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• pp.55-60
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• 2000

Experiments initiated 30 years ago to obtain selectable markers have led to a series of studies of Trp biosynthesis and anthranilate synthase (AS) the control enzyme using largely plant tissue cultures since they have experimental properties that can be readily exploited. Enzymological and compound feeding studies provided evidence that AS is the control point in the Trp biosynthesis branch and that altering the AS feedback control by the selection of mutants resistant to the Trp analog 5-methyl-tryptophan (5MT) can lead to the overproduction of this important amino acid. Plants regenerated from these Trp overproducing lines of most species also had high free Trp levels but Nicotiana tabaum (tobacco) plants expressed the feedback altered AS only in cultured cells and not in the regenerated plants. further tests by transient and stable expression of the cloned promoter for the naturally occurring tobacco feedback-insensitive AS, denoted ASA2, confirmed the tissue culture specific nature of the expression control. The 5MT caused by the expression of a feedback-insensitive AS from tobacco has been used to select protoplast fusion hybrids with several species since the resistance is expressed dominantly. Recently the ASA2 gene has been used successfully as a selectable marker to select transformed Astragalus sinicus and Glycine max hairy roots induced by Agrobactetium rhizogenes. These results show that the ASA2y-subunit can interact with the y-subunit of another species to form active feedback-insensitive enzyme that may be useful for selecting transformed cells. Plastid DNA transformation of tobacco has also effectively expressed ASA2 in the compartment in which Trp biosynthesis is localized in the cell.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.47-47
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• 2001

The neuropeptide vasopressin (VP) is a nine- amino acid hormone synthesized as preprohormone in the cell bodies of hypothalamic magnocellular neurons. The tumor in magnocellular neurons of the hypothalamus is associated with disfunctions of the cell bodies, leading to the diabetes insipidus. In order to study with the diabetes insipidus caused by a defect in VP synthesis and its secretion, we have produced the transgenic mice regulated by vasopressin promoter inserted to SV40 T antigen coding sequence (pVPSV.IGR2.1). One transgenic line expressing high levels of SV40 T antigen was propagated. The founder and all transgene positive adult animals have appeared with shorten mortality or apparent phenotypic abnormalities, including immune complex disease, and eventually die between 4 and 8 months of age. The mRNA and protein of SV40T antigen transgene were detected in brain of fetus as well as in brain, spleen, lung and lymph node in moribund at the age of 20 weeks. Histological analysis of transgenic mice showed that tumor developed in brain similar to primitive neuroectodermal tumors (PNET) in man. We also detected lymphomas in spleen and lymph node, and consequent tumor formation in various tissues of the transgenic mice. In pVPSV.IGR2.1, 21% mice showed brain tumor (PNET) at 5 weeks and 100% mice showed brain tumor after 15 weeks. In addition, Expression of apoptosis related genes (Bcl-28 & Bax) was increased over their age in mice with PNET as compared to control mice. Apoptosis related gene expression might be deregulated in mice with brain tumor. However, transgenic mice were not developed with the diabetes insipidus. These mice represent the first disease model to exhibit primitive neuroectodermal tumor in brain, as well as a unique model system for exploring the cellular pathogenesis of lymphomas.

• Journal of Microbiology
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• 제42권2호
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• pp.103-110
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• 2004

In order to understand the molecular basis of selective expression of stationary-phase genes by RNA polymerase containing$\sigma$$\^$38/ (E$\sigma$$\^$38/) in Escherichia coli, we examined transcription from the stationary-phase promoters, katEP, bo1AP, hdeABP, csgBAP, and mcbP, in vivo and in vitro. Although these pro-moters are preferentially recognized in vivo by E$\sigma$$\^$38/, they are transcribed in vitro by both E$\sigma$$\^$38/ and E$\sigma$$\^$70/ containing the major exponential $\sigma$, $\sigma$$\^$70/. In the presence of high concentrations of glutamate salts, how-ever, oldy E$\sigma$$\^$38/ was able to efficiently transcribe from these promoters, which supports the concept that the promoter selectivity of $\sigma$$\^$38/-containing RNA polymerase is observed only under specific reaction con-ditions. The examination of 6S RNA, which is encoded by the ssr1 gene in vivo, showed that it reduced E$\sigma$$\^$70/ activity during the stationary phase, but this reduction of activity did not result in the elevation of E$\sigma$$\^$38/ activity. Thus, the preferential expression of stationary-phase genes by E$\sigma$$\^$38/ is unlikely the con-sequence of selective inhibition of E$\sigma$$\^$70/ by 6S RNA.

• The Korean Journal of Physiology and Pharmacology
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• 제28권3호
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• pp.265-273
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• 2024

This study aims to explore possible effect of RNA polymerase I subunit D (POLR1D) on proliferation and angiogenesis ability of colorectal cancer (CRC) cells and mechanism herein. The correlation of POLR1D and Yin Yang 1 (YY1) expressions with prognosis of CRC patients in TCGA database was analyzed. Quantitative realtime polymerase chain reaction (qRT-PCR) and Western blot were applied to detect expression levels of POLR1D and YY1 in CRC cell lines and CRC tissues. SW480 and HT-29 cells were transfected with si-POLR1D or pcDNA3.1-POLR1D to achieve POLR1D suppression or overexpression before cell migration, angiogenesis of human umbilical vein endothelial cells were assessed. Western blot was used to detect expressions of p38 MAPK signal pathway related proteins and interaction of YY1 with POLR1D was confirmed by dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP). TCGA data showed that both POLR1D and YY1 expressions were up-regulated in CRC patients. High expression of POLR1D was associated with poor prognosis of CRC patients. The results showed that POLR1D and YY1 were highly expressed in CRC cell lines. Inhibition or overexpression of POLR1D can respectively suppress or enhance proliferation and angiogenesis of CRC cells. YY1 inhibition can suppress CRC progression and deactivate p38 MAPK signal pathway, which can be counteracted by POLR1D overexpression. JASPAR predicted YY1 can bind with POLR1D promoter, which was confirmed by dual luciferase reporter gene assay and ChIP. YY1 transcription can up-regulate POLR1D expression to activate p38 MAPK signal pathway, thus promoting proliferation and angiogenesis ability of CRC cells.

• 생명과학회지
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• 제20권9호
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• pp.1314-1323
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• 2010

유전자를 이소성으로 발현하고 억제하는 것은 유전자의 기능 연구에 있어서 매우 유용하다. 본 연구에서는 표적 유전자의 동시 과발현, 조직/발달 단계 특이적 발현 및 스트레스 유도성 발현을 위해 pPZP를 골격으로 다양한 binary 벡터를 제작하고 그 유용성을 검증하였다. 변형된 CaMV 35S 프로모터를 이용하여, 다른 두 개의 유전자를 동시 과발현시키는 binary 벡터를 제작하였고, 이 벡터가 동시에 그리고 같은 장소에서 다른 두 개의 표적 유전자를 과발현 하는데 효과적임을 확인하였다. At2S3, KNAT1 및 LFY 프로모터를 포함하는 조직 또는 발달 단계 특이적 발현 binary 벡터들을 제작하고 분석한 결과, 이 벡터들은 각각 배/유식물 시기, 새싹 끝의 분열조직 및 잎 원기 특이적 발현에 유용하였다. RD29A와 AtNCED3 프로모터를 포함하는 스트레스 유도성 발현 binary 벡터들은 고염, ABA, MV 또는 저온과 같은 비생물성 스트레스에 의한 유전자의 이소성 발현에 유용하였다. 본 연구에서 제작된 binary 벡터들은 표적 유전자의 이소성 발현을 통해 유전자의 생물학적 기능연구, 분자생물학적작용 기작 연구에 유용하게 사용될 것으로 사료된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.46-46
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• 2003

Interleukin-10 (IL-10) is a homodimeric protein with a wide spectrum of anti-inflammatory and immune activities. It inhibits cytokine production and expression of immune surface molecules in various cell types. The transgenic mice carrying the human IL-10 gene in conjunction with the bovine $\beta$-casein promoter produced the human IL-10 in milk during lactation. Transgenic mice were generated using a standard method as described previously. To screen transgenic mice, PCR was carried out using chromosomal DNA extracted from tail or toe tissues with a primer set. In this study, stability of germ line transmission and expression of IL-10 gene integrated into host chromosome were monitored up to generation F15 of a transgenic line. When female mouse of generation F9 was crossbred with normal male, generation F9 to F15 mice showed similar transmission rates (66.0$\pm$20.13%, 61.5$\pm$16.66%, 41.1$\pm$8.40%, 40.7$\pm$20.34%, 61.3$\pm$10.75%, 49.2$\pm$18.82%, and 43.8$\pm$25.91%, respectively), implying that the IL-10 gene can be transmitted stably up to long term generation in the transgenic mice. For ELISA analysis, IL-10 expression levels were determined with an hIL-10 ELISA and a mIL-10 ELISA kit in accordance with the supplier's protocol. Expression levels of human IL-10 from milk of generation F9 to F13 mice were 3.6$\pm$1.20 mg/ml, 4.2$\pm$0.93 mg/ml, 5.7$\pm$1.46 mg/ml, 6.3$\pm$3.46 mg/ml, and 6.8$\pm$4.52 mg/ml, respectively. These expression levels are higher than in generation F1 (1.6 mg/ml) mice. We concluded that transgenic mice faithfully passed the transgene on their progeny and successively secreted target proteins into their milk through several generations, although there was a little fluctuation in the transmission frequency and expression level between the generations.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.160-160
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• 2017

Drought stress is a major constraint of crop development and productivity. Plants have evolutionally developed several mechanisms at the molecular, cellular, and physiological levels to overcome drought stress. The basic Leucine zipper (bZIP) transcription factor (TF) family members are starting to be concerned about their roles in drought stress responses. In this study, we functionally characterized OsbZIP66, a rice group-E bZIP TF, to be associated with rice drought tolerance mechanisms. Expression of OsbZIP66 was significantly induced upon treatments of rice plants with drought, high salinity, and ABA. These observations and the fact that the OsbZIP66 promoter contains ten ABA-responsive elements suggest that OsbZIP66 is up-regulated by drought stress in an ABA-dependent manner. Overexpression of both OsbZIP66 in a whole plant body and specifically in roots enhanced drought tolerance of rice plants, indicating that the rice drought tolerance positively correlates with the expression levels of OsbZIP66. Thus, our results demonstrated that OsbZIP66 has a potential for use in biotechnological development of high-yielding rice plants under drought conditions.

• Journal of Microbiology and Biotechnology
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• 제20권11호
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• pp.1491-1499
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• 2010

Rhizosphere microorganisms possessing phytase activity are considered important for rendering phytate-phosphorus (P) available to plants. In the present study, the Citrobacter braakii phytase gene (appA) was overexpressed in rhizobacteria possessing plant growth promoting (PGP) traits, for increasing their potential as bioinoculants. AppA was cloned under the lac promoter in the broadhost-range expression vector pBBR1MCS-2. Transformation of the recombinant construct pCBappA resulted in high constitutive phytase activity in all of the eight rhizobacterial strains belonging to genera Pantoea, Citrobacter, Enterobacter, Pseudomonas (two strains), Rhizobium (two strains), and Ensifer that were studied. Transgenic rhizobacterial strains were found to display varying levels of phytase activity, ranging from 10-folds to 538-folds higher than the corresponding control strains. The transgenic derivative of Pseudomonas fluorescens CHA0, a well-characterized plant growth promoting rhizobacterium, showed the highest expression of phytase (~8 U/mg) activity in crude extracts. Although all transformants showed high phytase activity, rhizobacteria having the ability to secrete organic acid showed significantly higher release of P from Ca-phytate in buffered minimal media. AppA overexpressing rhizobacteria showed increased P content, and dry weight (shoot) or shoot/ root ratio of mung bean (Vigna radiata) plants, to different extents, when grown in semisolid agar (SSA) medium containing Na-phytate or Ca-phytate as the P sources. This is the first report of the overexpression of phytase in rhizobacterial strains and its exploitation for plant growth enhancement.

• KSBB Journal
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• 제20권3호
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• pp.220-227
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• 2005

독소루비신 생합성 유전자의 발현을 촉진시키는 유전자인 dnrI와 다나루비신으로부터 독소루비신으로의 생전환에 관여하는 유전자인 doxA를 ermE 프로모터가 포함된 pSE34에 도입하였을 때 각각 5.5배, 2.5배의 독소루비신 생산성 증가가 이루어졌다. 독소루비신 생합성 유전자군의 발현패턴 분석을 위한 DNA microarray system을 구축하였고, 고생산 균주의 독소루비신 생합성 유전자 발현 패턴을 DNA microarray를 통해 확인하였다. 독소루비신 생합성 유전자군의 세포성장에 따른 발현패턴을 분석한 결과, 독소루비신 생산성 증가에 따라 생합성 유전자의 발현도 증가함을 확인할 수 있었고, pSE34를 통해 도입해준 donA, dnrI 유전자의 경우 전체 생합성 유전자의 평균보다 높은 수준의 발현량을 보여줌으로써, ermE 프로모터에 의해 발현이 극대화되었음을 확인할 수 있었다. 독소루비신 내성 유전자의 경우 다른 독소루비신 생합성 유전자들에 비해 발현정도가 크게 증가했고, DnrI 의해 조절을 받는 다른 유전자들의 발현 수준과 비교하였을 때 TDP-daunosamine을 생합성의 첫 번째 단계에 관여하는 dnmL 유전자는 그 발현양의 증가가 크지 않았다. 따라서 DNA microarray 시스템 분석 결과, 독소루비신 생산성 극대화를 위해서는 dnrI, doxA, drrA, drrB, drrC, dnmL 등의 유전자들의 안정적 발현이 매우 중요하고도 핵심적인 인자임이 확인되었다.

• 한국미생물·생명공학회지
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• 제35권4호
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• pp.272-277
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• 2007

P. pastoris는 대장균에 비해 정확한 접힘, 당화, 효율적인 분비기작등의 장점을 가지고 있어 재조합 단백질의 생산을 위한 균주로서 관심을 받고 있다. 또한 재조합 단백질의 효율적인 생산공정 개발을 위하여 배양공정 중 특정 시간이나 조건에서 발현될 수 있는 효율적인 유도성 프로모터의 개발도 중요 관심 분야이다. 본 연구에서는 연속배양을 이용하여 P. pastoris의 배양 중 특정 기질이 소모되었을 때 발현되는 자동 유도성 프로모터를 개발하기 위하여, 인산이 고갈되었을 때 과발현 되는 유전자들을 탐색하였다. 인산 제한 연속배양의 정상상태에서 얻어진 균체로 부터 total RNA와 mRNA를 분리하였고, 이로부터 cDN를 합성하여 인산제한조건에서 과발현되는 유전자들을 확보하였다. 그 중 빈도수가 높은 8종의 유전자 3-phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase(GAPDH), glucokinase, thiol-specific antioxidant protein, triosephosphate isomerase, sodium/phosphate symporter(NPS) 그리고 pyruvate decarboxylase를 선별하였고, Northern blot analysis를 수행한 결과 인산 섭취에 관련된 NPS 유전자가 인산제한조건에서 과발현 됨을 확인하였다. 본 연구실에서는 자동유도성 프로모터로서 NPS유래의 프로모터의 잠재성을 알아보기 위하여, 관련 유전자를 확보하여 외래 유전자를 이용한 발현연구를 진행 중이다.