• KSBB Journal
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• 제16권5호
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• pp.480-485
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• 2001

국화과에 속하는 다년생 초본식물의 민들레가 Agrobacteria 에 대한 숙주로서의 가능성을 조사하고 여러 가지 유용한 유전자를 민들레로 도입시키기 위해, 민들레잎 절편을 pBI121으로 형질전환된 Agrobacterium tumefaciens LBA4404와 10분동안 공동배양하여 형질전환시킨 후, 1$\mu$M IAA, 1$\mu$M BA. 50 $\mu$g/ML Km과 100$\mu$g/ML Cb이 함유된 MS 배지에서 약 2주후에 multiple shoot를 유가시켰다. 유기된 shoot로 부터 유식식물체를 얻었으며, 형질전환을 확인하기 위해 GUS활성을 측정한 결과 양성 반응을 보였다.

• The Plant Pathology Journal
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• 제21권1호
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• pp.13-20
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• 2005

Chlorella viruses are large icosahedral, plaque-forming, dsDNA viruses that infect certain unicellular, chlorellalike green algae. The genomic DNA of over 300 kb contains many useful genes and promoters. Over 40 chlorella viruses have been isolated from fresh water in Korea since 1998. The viruses were amplified initially in chlorella strain NC64A, and pure isolates were obtained by repeated plaque isolation. SDS-PAGE analysis revealed similar but distinct protein patterns, both among the group of purified viruses and in comparison with the prototype chlorella virus PBCV-1. Digestions of the 330- to 350-kb genomic DNAs with 10 restriction enzymes revealed different restriction fragment patterns among the isolates. The tRNA-coding regions of 8 chlorella viruses were cloned and sequenced. These viruses contain 14-16 tRNA genes within a 1.2- to 2-kb region, except for the SS-1 isolate, which has a 1039-bp spacer in a cluster of 11 tRNA genes. Promoter regions of several early genes were isolated and their activities were analyzed in transformed chlorella. Some promoters showed stronger activity than commonly used CaMV 35S promoter and chlorella transformation vectors for heterologous protein are beings constructed using these promoters.

• Biomolecules & Therapeutics
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• 제14권3호
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• pp.148-151
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• 2006

We have previously reported that 2.4 kb of L-plastin promoter (LP) could regulate the expression of adenoviral vector (AV) exogenous genes in a tumor cell specific manner. In the present study, we tested if the replication competent AdLPE1A vector results in a direct cytotoxic effect in hepatocelluar carcinoma (HCC) cells. In vitro cytotoxicity tests were carried out with replication-competent (AdLPE1A) and -incompetent (AdLPCD) LP-driven vectors. AdLPE1A is an AV in which LP was inserted 5' to the E1A and E1B genes. The AdLPCD vector contains LP and the E. coli cytosine deaminase (CD) gene in transcription unit. Exposure of cells to AdLPE1A generated a significant cytotoxic effect as compared to the control. Almost 90% of the cell had manifested the characteristic cytopatic effect on day 9 after infection of cells with 10 MOI of AdLPE1A. On the other hand, almost 35% of the cells were left when the cells had been treated with 100 MOI of AdLPCD together with 5-FC on day 9 when compared with the cells which had never been exposed neither 5-FC nor AdLPCD. These results showed that the replication competent AdLPE1A vector could kill the HepG2 cells directly by the oncolytic effect of the virus. The replication competent AV vector carrying viral E1A generated greater cytotoxic effect than the replication incompetent AV, which contains the CD prodrug activation transcription unit without E1A, in HepG2 cells.

• Journal of Microbiology
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• 제45권6호
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• pp.534-540
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• 2007

The Streptomyces coelicolor A3(2) genome contains six operons (rrnA to F) for ribosomal RNA synthesis. Transcription from rrnD occurs from four promoters (p1 to p4). We found that transcripts from the p1 and p3 promoters were most abundant in vivo in the early exponential phase. However, at later phases of exponential and stationary growth, transcripts from the p1 promoter decreased drastically, with the p3 and p4 transcripts constituting the major forms. Partially purified RNA polymerase supported transcription from the p3 and p4 promoters, whereas pure reconstituted RNA polymerase with core enzyme (E) and the major vegetative sigma factor ${\sigma}^{HrdB}$ ($E{\cdot}{\sigma}^{HrdB}$) did not. In order to assess any potential requirement for additional factor(s) that allow transcription from the p3 and p4 promoters, we fractionated a partially purified RNA polymerase preparation by denaturing gel filtration chromatography. We found that transcription from the p3 and p4 promoters required factor(s) of about 30-35 kDa in addition to RNAP holoenzyme ($E{\cdot}{\sigma}^{HrdB}$). Therefore, transcription from the p3 and p4 promoters, which contain a consensus -10 region but no -35 for ${\sigma}^{HrdB}$ recognition, are likely to be regulated by transcription factor(s) that modulate RNA polymerase holoenzyme activity in S. coelicolor.

• 한국식물학회:학술대회논문집
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• 한국식물학회 2002년도 춘계학술발표대회:발표눈문요지록
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• pp.73-82
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• 2002

The committing step in Met and S-adenosyl-L-methionine (SAM) synthesis is catalyzed by cystathionine ${\gamma}$ -synthase (CGS). Transgenic Arabidopsis thaliana overexpressing CGS under control of 35S promoter show increased soluble Met and its metabolite S-methylmethionine, but only at specific stages of development. CGS-overexpressing seedlings are resistant to ethionine. Similar results were obtained with transgenic potato plants overexpressing Arabidopsis CGS. Several of the transgenic lines show silencing of CGS resulting in deformed p]ants with a reduced capacity for reproductive growth similar as transgenic plants by antisense RNA (CGS[-]). Exogenous feeding of Met to the CGS[-] and CGS[+] silenced plants partially restores their growth. Similar morphological deformities are observed in plants cosuppressed for SAM synthetase, even though such plants accumulate 250 fold more soluble Met than wild type and they overexpress CGS. The results suggest that the abnormalities associated with CGS and SAM synthetase silencing are due in part to a reduced ability to produce SAM, and that SAM may be a regulator of CGS expression.

• Molecules and Cells
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• 제40권8호
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• pp.577-586
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• 2017

Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors that are implicated in protein turnover and stress responses. Here, we characterized a PhyCYS from Arabidopsis thaliana, which was designated AtCYS5. RT-qPCR analysis showed that the expression of AtCYS5 in germinating seeds was induced by heat stress (HS) and exogenous abscisic acid (ABA) treatment. Analysis of the expression of the ${\beta}-glucuronidase$ reporter gene under the control of the AtCYS5 promoter showed that AtCYS5 expression during seed germination was induced by HS and ABA. Constitutive overexpression of AtCYS5 driven by the cauliflower mosaic virus 35S promoter led to enhanced HS tolerance in transgenic Arabidopsis, which was characterized by higher fresh weight and root length compared to wild-type (WT) and knockout (cys5) plants grown under HS conditions. The HS tolerance of AtCYS5-overexpressing transgenic plants was associated with increased insensitivity to exogenous ABA during both seed germination and post-germination compared to WT and cys5. Although no HS elements were identified in the 5'-flanking region of AtCYS5, canonical ABA-responsive elements (ABREs) were detected. AtCYS5 was upregulated in ABAtreated protoplasts transiently co-expressing this gene and genes encoding bZIP ABRE-binding factors (ABFs and AREB3). In the absence of ABA, ABF1 and ABF3 directly bound to the ABREs in the AtCYS5 promoter, which activated the transcription of this gene in the presence of ABA. These results suggest that an ABA-dependent pathway plays a positive role in the HS-responsive expression of AtCYS5 during seed germination and post-germination growth.

• Plant Biotechnology Reports
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• 제5권3호
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• pp.273-281
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• 2011

Diverse epigenetic phenotypes are frequently found during research on transgenic plants. To understand the factors underlying such diversity, hundreds of independent 35S-GFP transgenic N. benthamiana plants were analyzed. The diverse GFP-expression phenotypes of the transgenic plants were classified into three major types based on the GFP expression patterns and their response to 35S-GFP agroinfiltration: steady-green, silenced and non-uniform phenotype. The non-uniform phenotype was further sub-divided into five minor phenotypes: variegated, red-dropped, on-silencing, partitioned and misty, according to the distribution of GFP expression on the leaves. Many of transgenic plants continuously generated diverse phenotypes over several generations despite the transgene identity. Such epigenetic GFP phenotyping was found to be the result of spontaneous transgene silencing mediated by either or both of post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). This finding was verified by the detection of 21- and 24-nt small interfering RNA (siRNA) molecules, and DNA methylation in the transgenic plants that showed repeated epigenetic variation. Agroinfiltration demonstrated that irregular distribution of GFP on a leaf was the result of erratic transgene silencing, and the technique also proved to be a rapid and effective method for selecting fully silenced plants within 3 days. Furthermore, two novel phenotypes described are potential materials for in-depth investigations into the genes and mechanisms responsible for spontaneous transgene silencing.

• Food Science and Biotechnology
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• 제17권5호
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• pp.1092-1096
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• 2008

Commercial food products from major cities of Coahuila, Mexico were screened to identify residues of transgenic deoxyribonucleic acid (DNA) and/or proteins. After performed, an inventory on all products that contained a soybean-based ingredient in a commercial grocery store in the city of Saltillo, Coahuila, Mexico, 245 food products were identified and grouped in 15 classes according to the soybean ingredient as well as the manufacturing process used for their elaboration. Similar sampling was made for the different food classes in the cities of Monclova, Piedras Negras, and Torreon. A total of 88 samples were analyzed and DNA was extracted by the hexadecyltrimethyl-ammonium bromide (CTAB) technique with slight modification to obtain better DNA quality (1). In addition, segments of the transgenic genes one that codifies for 5-enolpyruvylshikimate-3-phosphate synthase (epsps), cry 1A, and the cauliflower mosaic virus (CaMV) promoter were amplified using polymerase chain reaction (PCR). The transgenic proteins 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) and insecticidal crystal protein (Cry 1Ab/Ac) were identified using double antibody sandwich-enzymatic linked immunoassay analysis (DAS-ELISA). Presence of transgenic genes and/or proteins was identified in 35.3% of the commercial products samples.

• Journal of Plant Biotechnology
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• 제35권4호
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• pp.275-280
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• 2008

Agrobacterium공동배양법으로 오이의 기관발생을 통한 형질전환에서 가장 문제점 중 하나는 chimeric 형질전환체의 발생빈도이다. 이러한 문제점을 극복하기 위하여 항생제로서 paromomycin이 첨가된 선발배지에서 "은성" 품종의 배축절편으로부터 체세포배발생을 통한 형질전환시스템을 개발하였다. 배축절편을 pPPTN290발현벡터가 도입된 Agrobacterium 균주 (EHA101)에 30분간 접종한 후 2일간 공동배양 하였고, 선발배지에서 2주 간격으로 5회 계대 배양하면서 항생제 저항성 캘러스 선발, 체세포배발생 및 식물체를 유도하였다. pPPTN290발현벡터의 T-DNA는 reporter유전자로서 Ubi 프로모터에 의해 gus유전자가 발현조절 되도록 그리고 항생제로서 paromomycin에 저항성을 갖는 nptII유전자가 35S 프로모터에 의해 발현되도록 제조하였다. 안정적 형질전환과 빈도는 캘러스의 paromomycin항생제 저항성과 GUS유전자의 발현 여부에 의해 조사하였다. Agrobacterium과 공동배양한 928개의 배축절편에서 paromomycin에 저항성을 갖는 56개의 캘러스 클론을 얻었고, 이중 48개 캘러스 클론 (5.2%)에서 GUS유전자가 안정적으로 발현되고 있음을 확인하였다. 48개의 캘러스 클론중에서 오직 5개의 캘러스 클론으로부터 식물체를 얻어 낮은 빈도 (0.5%)를 나타냈다. 수확한 $T_1$종자에서 GUS양성반응은 gus유전자가 오이 게놈에 안정적으로 도입 및 발현되고 있음을 확인하였다.

• Applied Biological Chemistry
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• 제38권3호
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• pp.224-231
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• 1995

형질전환 식물체에서 외래 단백질의 발현을 높이기 위하여, 외래 단백질 유전자를 TMV RNA 또는 Gy2 5'-untranslated leader 부위와 재조합시킨 plasmid들을 제조하였다. pGA643에서 유래된 이들 plasmid에는 cauliflower mosaic virus의 35S promoter와 Gy2 terminator를 포함하고 있고, 외래 유전자로는 옥수수의 10kDa zein (10kZ) 유전자를 사용하였다. Gy2 또는 TMV RNA의 leader 부위는 promoter 부위와 단백질 coding sequence 사이에 삽입하였다. Agrobacterium을 매개로 하는 leaf disc 형질전환법을 이용하여 재조합 단백질들을 담배에 도입하였다. Leader를 포함하지 않은 도입 유전자의 전사 효율이 leader를 포함하는 도입 유전자들 보다 높았지만, leader를 포함한 mRNA들이 보다 효율적으로 전사되었다. 이는 5'-untranslated sequence의 길이와 AUG codon 주위의 context에 의한 영향보다는 이들 leader sequence의 특이적 염기서열에 의한 영향이 큼을 의미한다. 이러한 결과는 형질전환된 담배에서 외래 유전자의 전이효율을 조절하는 데 있어서 Gy2와 TMV의 leader sequence들이 enhancer로서 역할을 하고 있음을 의미한다.