• The Plant Pathology Journal
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• 제19권3호
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• pp.162-165
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• 2003

Transformation of Fuji apple (Malus domestica 'Fuji') was performed using Agrobacterium tumefaciens harboring a coat protein (CP) gene of Cucumber mosaic virus (CMV). A plasmid DNA containing the virus CP and NPT II genes was introduced into the loaves of apple by th e Agrobacterium - mediated transformation procedure. Regenerated transformants of the apple were obtained by kanamycin resistance conferred by the introduced NPT II gene. PCR analysis showed that 3 out of 20 putatively selected R0 plant lines contain the CMV-CP gene. Nine putative transgenic lines out of 20 lines were investigated with the PCR analysis; 5 regenerants produced a 450 bp DNA band and 3 regenerants showed a 671 bp DNA band for the NPT II and CMV-CP genes, respectively. Southern hybyidization results demonstrate the successful integration of the CMV-CP gene into the genome of the apple. This is the first report on the generation of useful vius resistance source of transgenic apple for molecular breeding program.

• BMB Reports
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• 제43권3호
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• pp.164-169
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• 2010

CpG-DNA, which contains unmethylated CpG dinucleotides in the context of specific sequences, has remarkable and diverse immunological effects, including induction of proinflammatory cytokine expression and regulation of the Th1/Th2 immune response. Here, we examined the immunostimulatory activities of double-stranded (ds) CpG-DNA in the human B cell line RPMI8226. To investigate whether dsCpG-DNA stimulates immune cells, we constructed a plasmid containing repeated dsCpG-DNA and produced dsCpG-DNA by PCR amplification and EcoR I digestion. PCR-amplified dsCpG-DNA alone did not have immmunostimulatory activity. However, dsCpGDNA encapsulated with lipofectin induced IL-8 promoter activation, HLA-DRA expression, and IL-8 expression in a CG sequence-independent manner. The effects of encapsulated dsCpGDNA were independent of minor endotoxin contamination. These findings suggest the potential use of dsCpG-DNA as a therapy for immune response regulation.

• Journal of Microbiology
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• 제40권3호
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• pp.211-218
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• 2002

A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D$\_$3/ into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450$\_$Sca2/ protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Steptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D$\_$3/ to its hydroxylated forms.

• KSBB Journal
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• 제4권3호
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• pp.246-253
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• 1989

Agorobacterium rhizogenes에 의하여 유도된 당근(Daucus carota L. )의 hairy root를 배양하였으며 opine 유무에 대한 형질전환 확인 및 배지조성을 달리하여 성장률에 따른 색소함량을 비교하였고, 재분화된 식물체의 형태적 차이를 관찰하여 몇가지 결론을 얻었다. Hairy root는 균 접종 2-4주 후에 형성층 부위를 중심으로 유도되었다. 유도된 hairy root의 초기 배양에는 R.C.M 배지가 적합하였으며 MSO(2, 4-D $10^{-4} ml/ l, pH6, sucrose 5%, 질소원 0.03M 등)에서 최대 성장을 보여주었고 성장의 증가에 따른 색소의 형성은 비교적 안정하였다. 재분화된 식물체는 정상 식물체에 비하여 형태적으로 차이를 나타내었으며 형질전환된 hairy root 및 재분화된 식물체에서 mannopine 분석으로 Ri-plasmid에 의한 형질전환이 이루어졌음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2192-2198
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• 2016

The myeloid-specific IgA Fc receptor ($Fc{\alpha}R$) is a cell surface molecule on immunocytes that provides a fundamental connection between humoral and cellular immunity. In this study, the full-length cDNA sequence of swine $Fc{\alpha}RI$ ($swFc{\alpha}RI$) was isolated and characterized and found to contain a 792-base-pair open reading frame, encoding a 264-amino-acid transmembrane glycoprotein with a predicted molecular mass of 29.4 kDa. The $swFc{\alpha}RI$ shares high amino acid sequence homology (>50%) with its counterparts from cattle, seal, and horse. Rosetting analysis confirmed that COS-7 cells transfected with an $swFc{\alpha}RI$ expression plasmid was able to combine with chicken erythrocytes sensitized with porcine IgA, but not IgG.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2002년도 추계국제학술대회
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• pp.170-170
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• 2002

College of Pharmacy, Ewha womans University, Seoul, 120-750, Korea 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent halogenated aromatic hydrocarbon congener that induces expression of several genes including CYP1A1. Exposure to TCDD results in many toxic actions such as carcinogenesis, hepatotoxicity, immune suppression, and reproductive and developmental toxicity. Dramatic differences in dioxin toxicity have been observed between the sexes of some animal species, suggesting hormonal modulation of dioxin action. Many studies have been reported and propose several mechanisms of anti-estrogenic effects of TCDD. In contrast, the effect of estrogen on the regulation of CYP1A1 are not clear at present. There are several reports showing conflicting results. It seems that induction/inhibition of CYP1A1 may be dependent on cell-type and concentration. The purpose of this study was to investigate the regulation of TCDD-induced CYP1A1 gene expression by estradiol and its metabolites. We examined whether estradiol and its metabolites altered TCDD-mediated induction of CYP1A1 enzyme activity. 17 ${\beta}$ estradiol and 16 ${\alpha}$ estriol at non cytotoxic concentrations caused a significant concentration dependent decline of TCDD-induced EROD activity To determine whether reduced EROD activity reflected altered CYP1A1 mRNA expression, we measured CYP1A1 mRNA level by RT-PCR. And to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level, we also peformed transient transfection with an AhR responsive reporter plasmid containing the 5' flanking region of the human CYP1A1 gene to examine whether estradiol and its metabolites have effects on TCDD-induced CYP1A1 gene expression at the transcription level.

• Biomolecules & Therapeutics
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• 제8권2호
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• pp.153-159
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• 2000

A novel serine/threonine protein phosphatase with EF-hand motif, which belongs to PPEF family was partially cloned from rat brain cDNA by employing RT-PCR method. The size of the amplified clone was 1.6kbp. The amplified DNA was subcloned into pGEM-T-Easy vector and the resulting plasmid was maned as pGEM-rPPEF2. The nucleuotide sequence is shared by 88% with that of mouse PPEF-2 cDNA, and the deduced amino acid sequence reveal 92% homology with that of mouse PPEF-2 cDNA. The N-terminal region of the cloned rat brain PPEF contains a putative phosphatase catalytic domain (PP domain) and the C-terminal region contains multiple $Ca^{2+}$ binding sites (EF region). The putative catalytic domin (PP) and the EF-hand motif (EF) regions were subcloned into pGEX4T-1 and were overexpressed in E. coli DH5 as glutathione-S-transferase (GST) fusion proteins. Expression of the desired fusion protein was identified by SDS-PAGE and also by immunoblot analysis using monoclonal antibody against GST. The recombinant proteins were purified by glutathione-agarose chromatography. This report is first to demonstrate the cloning of PPEF family from rat brain tissues. The clone reported here would be invaluable for the investigation of the role of this new type-phosphatase in rat brain.

• 식물병연구
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• 제13권2호
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• pp.88-92
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• 2007

우리나라 참다래 과수원에서 참다래 꽃썩음병을 일으키는 원인 세균인 Pseudomonas syringae pv. syringae를 분리하였다. 총 41개 균주 중 스트렙토마이신 저항성을 보이는 2개의 균주를 대상으로 PCR과 염기서열 결정을 통해 저항성 유전자 구조를 조사하였다. PCR결과 스트렙토마이신 저항성유전자는 Tn5359a에 들어 있는 strA-strB 구조인 것으로 밝혀졌다. Xanthomonas campestris와 Erwinia amylovora에서 알려진 IS6100과 IS1133은 발견되지 않았다. strA-strB의 염기서열은 이미 밝혀진 Tn5393a와 동일하였다. 두 스트렙토마이신 저항성 균주는 각각 3개의 플라스미드를 가지고 있었으며 저항성 유전자는 그 중 100kb의 플라스미드에 들어 있었다.

• 약학회지
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• 제39권2호
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• pp.161-168
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• 1995

Brain dopamine systems play a central role in the control of movement, hormone release, and many complex behavior. The action of dopamine at its synapse is terminated predominately by high affinity reuptake into presynaptic terminals by dopamine transporter (DAT). The dopamine transporter(DAT) is membrane protein localized to dopamine-containing nerve terminals and closely related with cocaine abuse, Parkinsonism, and schizophrenia. In present study, the recombinant plasmid pRc/CMV-DAT, constructed by subcloning of a cDNA encoding a bovine DAT into eukaryotic expression vector pRc/CMV, was stably transfected into CV-1 cells(monkey kidney cell line). The DAT activities in the cell lines selected by Geneticin$^{R}$ were determined by measuring the uptake of $[^3H]$-dopamine. The transfected cell lines showed 30-50 fold higher activities than untransfected CV-1 cell line, and this result implies that DAT is well expressed and localized in transfected cells. The transfected cells accumulated $[^3H]$-dopamine in a dose-dependent manner with a $K_{m}$ of 991.6nM. Even though high doses of norepinephrine, epinephrine, serotonin, and choline neurotransmitters inhibited the uptake of $[^3H]$-dopamine, DAT in transfected cell line was proven to be much more specific to dopamine. The psychotropic drugs such as GBR12909, CFT, normifensine, clomipramine, desipramine, and imipramine inhibited significantly the dopamine uptake in tissue culture cells stably transfected with DAT cDNA. Radioactive in situ hybridization was done to map the cellular localization of DAT mRNA-containing cells in the adult rat central nervous system. The strong hybridization signals were detected only in the substantia nigra pars compacta and ventral tegmental area. The restricted anatomical localization of DAT mRNA-containing cells confirms the DAT as a presynaptic marker of dopamine-containing cells in the rat brain.

• 한국자원식물학회지
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• 제20권5호
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• pp.437-441
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• 2007

Agrobacterium tumefaciens에 의해 일미벼 품종에 도입하여 많은 형질전환벼를 생산했다. 배발생 캘러스는 hygromycin 저항성 선발마커를 포함한 pCAMBIA1300 벡터를 이용하여 Agrobacterium strain AGL1으로 공동배양하였다. 공동배양 후 50mg/L hygromycin에 저항성을 보이는 형질전환체가 선발되었다. $T_0$ 세대 형질전환벼로부터 genomic DNA를 추출하여 PCR과 Southern blot을 통한 외래 유전자의 안정한 삽입을 검정하였다. 형질전환된 벼라인에서 650bp의 HPT 유전자 단편이 나타났다. 그리고 argE의 특이적 priomer를 이용하여 검정한 결과 230bp 단편이 검출되었다. SEM을 이용하여 형태학적 분석을 한 결과 기공 분포와 배열상의 특징에 있어서 형질전환체는 대조구와 비교하여 기공형태가 불규칙적인 차이를 보여 주었다.