• Environmental Analysis Health and Toxicology
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• 제24권2호
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• pp.107-117
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• 2009

Metal pollution of aquatic ecosystems is a problem of economic and health importance. Information regarding molecular responses to metal exposure is sorely needed in order to identify potential biomarkers. To determine the effects of heavy metals on chironomids, the full-length cDNA of alcohol dehydrogenase (ADH3) from Chironomus riparius was determined through molecular cloning and rapid amplification of cDNA ends (RACE). The expression of ADH3 was analyzed under various cadmium and copper concentrations. A comparative and phylogenetic study among different orders of insects and vertebrates was carried out through analysis of sequence databases. The complete cDNA sequence of the ADH3 gene was 1134 bp in length. The sequence of C. riparius ADH3 shows a low degree of amino acid identity (around 70%) with homologous sequences in other insects. After exposure of C. riparius to various concentrations of copper, ADH3 gene expression significantly decreased within 1 hour. The ADH3 gene expression was also suppressed in C. riparius after cadmium exposure for 24 hour. However, the effect of cadmium on ADH3 gene expression was transient in C. riparius. The results show that the suppression of ADH3 gene by copper exposure could be used as a possible biomarker in aquatic environmental monitoring and imply differential toxicity to copper and cadmium in C. riparius larvae.

• Plant Biotechnology Reports
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• 제2권1호
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• pp.1-6
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• 2008

Cotyledonary leaves of tomato cv. Megha were transformed with the hepatitis B virus 's' gene, which encodes surface antigen. Six plant expression cassettes (pHBS, pHER, pEFEHBS, pEFEHER, pSHER and pEFESHER) were used to assay the possible expression levels by agroinfiltration. The maximum transient expression level of 489.5 ng/g D.W. was noted in pEFEHER-infiltrated cotyledonary leaves. Transgenic tomato plants with pEFEHBS and pEFEHER expression cassettes were regenerated and characterized by molecular analysis. The expression of the antigen in the fruits was confirmed by RT-PCR and ELISA analysis. This is the first report on the expression of hepatitis B surface antigen in tomato.

• 한국양식학회지
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• 제13권1호
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• pp.21-27
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• 2000

The successful gene transfer and transient expression was demonstrated in olive flounder embryos using lipofected sperm. Olive flounder sperm interacted with foregn plasmid DNA encapsidated by positively charged liposome. The maximum plasmid copy number that associated with the sperm was 5 copies/sperm based on the examination of DNA blot assay. The foreign DNA was transferred into fertilized eggs without any adverse effect on fertilization and survival of embryos (P>0.05) and retained in embryos until at least 42 hours with successful expression. The maximal expression was detected in 18 hours after fertilization at 18$^{\cird}C$ and gradually decreased with development of embryo. Most of DNA transferred into embryos persisted extrachromosomally without significant sign of integration or replication.

• 미생물학회지
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• 제27권2호
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• pp.85-91
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• 1989

조직특이적 및 일반적 유전자 발현에 미치는 SV40와 murine cytomegalovirus (MCMV) enhancer의 영향을 조사하였다. 이를 위하여 chloramphenicol acetyl transferase (CAT) 유전자의 아래쪽에 이들 enhancer들을 삽입한 재조합 플라스미드 들을 제조하였다. 원숭이세포(CV1PO)와 HeLa 세포에 이들 플라스미드들을 이입시킨 후, CAT 유전자가 발현되는 정도를 조사하였다. Enhancer가 없는 플라스미드에 비해 SV40와 MCMV enhancer는 CAT의 발현을 각각 20배와 150배로 가중시켰다. CAT 유전자의 앞에 있는 SV40 프로모터를 2.2kbp의 소성장호르몬(bGH) 유전자의 조절부위로 치환한 경우는 enhancer가 있어도 전혀 CAT 의 말현이 검출되지 않았다. 조절부위을 230 bp 로 짧게 하여 치환한 경우는, SV 40 enhancer 가 있을 때, CAT의 말현이 매우 증가하였다. 이와는 내조적으로 더 강한 MCMV enhancer는 bGH 특이적인 발현을 별로 증가시키지 못하였다.

• Journal of Plant Biotechnology
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• 제31권4호
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• pp.279-283
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• 2004

백합 (Lilium longiflorum cv. Georgia) 화분의 생장과 agro-infiltration에 의한 일시발현에 대한 물리적, 화학적, 생물적 요인의 영향을 분석하였다 화분을 배지에 섞기 위한 물리적 과정이나 agro-infiltration을 위한 진공작업과정은 정상적 화분생장을 위하여 최소화되는 것이 바람직한 것으로 나타났다. 비교적 넓은 범위에서의 온도 (19 to 27$^{\circ}C$)나 pH(5.0 to 8.0)에서 화분의 생장이 유사하게 진행되었으며 화학적 요인으로서의 cefotaxime (300mg/L), acetosyringone (800 $\mu$M), syringealdehyde (800 $\mu$M) 등의 처리는 화분의 생장에 영향을 나타내지 않았다. 그러나 kanamycin의 경우 매우 심한 생장저해현상을 보였는데 25mg/L의 농도에서도 저해현상을 보이는 경우도 있었다. GUS유전자의 화분발현시 acetosyringone(200-400$\mu$M)의 처리에 의하여 그 효율이 약간 향상되는 것으로 나타났으나 syringealdehyde의 경우에는 효과가 없었다. 짧은 시간 내의 agro-infiltration과정과 이어서 18 hr의 화분 및 박테리아의 동시배양으로서도 acetosyringone의 첨가에 상관없이 화분에서의 GUS 일시 발현결과를 얻을 수 있었다.

• Molecules and Cells
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• 제38권1호
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• pp.33-39
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• 2015

The correction of disease-causing mutations by single-strand oligonucleotide-templated DNA repair (ssOR) is an attractive approach to gene therapy, but major improvements in ssOR efficiency and consistency are needed. The mechanism of ssOR is poorly understood but may involve annealing of oligonucleotides to transiently exposed single-stranded regions in the target duplex. In bacteria and yeast it has been shown that ssOR is promoted by expression of $Red{\beta}$, a single-strand DNA annealing protein from bacteriophage lambda. Here we show that $Red{\beta}$ expression is well tolerated in a human cell line where it consistently promotes ssOR. By use of short interfering RNA, we also show that ssOR is stimulated by the transient depletion of the endogenous DNA mismatch repair protein MSH2. Furthermore, we find that the effects of $Red{\beta}$ expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity. These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.

• Molecules and Cells
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• 제42권2호
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• pp.123-134
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• 2019

Lysophosphatidic acid (LPA) is an endogenous lysophospholipid with signaling properties outside of the cell and it signals through specific G protein-coupled receptors, known as $LPA_{1-6}$. For one of its receptors, $LPA_1$ (gene name Lpar1), details on the cis-acting elements for transcriptional control have not been defined. Using 5'RACE analysis, we report the identification of an alternative transcription start site of mouse Lpar1 and characterize approximately 3,500 bp of non-coding flanking sequence 5' of mouse Lpar1 gene for promoter activity. Transient transfection of cells derived from mouse neocortical neuroblasts with constructs from the 5' regions of mouse Lpar1 gene revealed the region between -248 to +225 serving as the basal promoter for Lpar1. This region also lacks a TATA box. For the region between -761 to -248, a negative regulatory element affected the basal expression of Lpar1. This region has three E-box sequences and mutagenesis of these E-boxes, followed by transient expression, demonstrated that two of the E-boxes act as negative modulators of Lpar1. One of these E-box sequences bound the HeLa E-box binding protein (HEB), and modulation of HEB levels in the transfected cells regulated the transcription of the reporter gene. Based on our data, we propose that HEB may be required for a proper regulation of Lpar1 expression in the embryonic neocortical neuroblast cells and to affect its function in both normal brain development and disease settings.

• Journal of Plant Biotechnology
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• 제29권4호
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• pp.223-228
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• 2002

본 연구실에서 개발된 particle inflow gun (PIG)은 조작이 간편하고, 사용비용도 저렴하며, 식물 세포 내의 유전자 도입효율이 높은 특징을 갖고 있다. PIG 장비를 이용하여 벼 캘러스 내로의 유전자 도입 조건을 검토하기 위해서 사용된 vector는 pIG121Hm으로서 T-DNA 내부에 intron GUS ($\beta$-glucuronidase)와 hygromycin 및 kanamycin 저항성 유전자를 포함하고 있다. 또한 벼 캘러스 내에 물리적으로 DNA를 도입할 때에 DNA 도입 효율과 관계가 높은 요인들을 GUS의 발현빈도를 통하여 조사하였다. 그 결과 gold particle에 DNA를 부착하는 과정에 사용되는 spermidine과 calcium chloride의 경우 무첨가구에 비해 16 mM의 spermidine과 1.5 M의 calcium chloride 첨가구에서 GUS 발현율이 각각 2배, 3배 증가하였다. 그리고 1회 분사되는 gold particles양이 2 mg의 경우 가장 높은 GUS 발현율을 보여주었으며, 또한 PIG장비의 분사거리와 헬륨의 압력은 벼의 배양세포의 경우 12cm의 분사거리에서 3.5 bar (50 psi)의 헬륨압력으로 분사하였을 때 GUS 발현율이 가장 높았다. 이상의 결과에서 PIG 장비를 이용한 유전자 도입은 본 연구에서 검토한 최적의 조건을 이용하였을 경우 기존에 많이 사용되고 있는 Biolistic Gun (Bio-Rad 사)과 거의 비슷한 유전자 도입효율을 보여 주었다. 특히 PIG 장비의 경우 조작이 매우 간편하고, 분사에 사용되는 일회용 부품이 필요하지 않기 때문에 대량의 반복실험을 필요로 하는 연구에서 손쉽게 사용되리라 기대된다.

• Journal of Microbiology
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• 제38권3호
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• pp.145-149
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• 2000

GroEL is a typical molecular chaperone. GroEL synthesis patterns at various culture temperatures in Escherichia coli were investigated in this study. No significant differences in the amount of GroEL produced from the chromosome were found at 30 and 37$^{\circ}C$. However, GroEL production increased 3.4-fold at 42$^{\circ}C$. GroEL synthesis was not transient but continuous at 42$^{\circ}C$, although most heat shock gene expression is known to be transient. To understand the role of the groEL structural gene, a groE promoter-lacZ fusion was constructed. Interestingly , while transcriptional fusion is not thermally inducible, it is inducible by ethanol, suggesting that the secondary structure of the groEL transcript is involved in thermal regulation of the groEL gene. Secondary structures of groE mRNA at 37 and 42$^{\circ}C$ were compared using the computer program RNAdraw. Distinct structures at the two temperatures were found, and these structures may be related to a high level of GroEL expression at 42$^{\circ}C$.

• KSBB Journal
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• 제5권4호
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• pp.323-327
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• 1990

Protoplasts isolated from embryogenic cell suspensions were electroporated in buffered solutions containing plasmid DNA of pBI121. Transient GUS (beta-glucuronidase) activity measurement and selection for kanamycin resistent showed that expression of foreign genes and stable loransformation were achieved. GUS transient gene expression was increased by increasing DNA concentration of pBI121 plasmid and affected by the level of the applied voltage. An optimal level of GUS activity was obtained after electroporation with a pulse of 200 voltage/1180 uF. Protoplast viability was up to the 60% at the optimal voltage. Cell colonies resistent to 200$\mu\textrm{g}$/ml kanamycin were selected in agar medium and identified by histochemical GUS assay.