• Journal of Microbiology and Biotechnology
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• 제16권6호
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• pp.952-960
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• 2006

As a unicellular green alga that possesses many of the metabolic pathways present in higher plants, Chlorelia offers many advantages for expression of heterologous proteins. Since strong and constitutive promoters are necessary for efficient expression in heterologous expression systems, the development of such promoters for use in the Chlorella system was the aim of this study. Proteins encoded by the early genes of algal viruses are expressed before viral replication, probably by the host transcriptional machinery, and the promoters of these genes might be useful for heterologous expression in Chlorella. In this study, putative promoter regions of DNA polymerase, ATP-dependent DNA ligase, and chitinase genes were amplified from eight Korean Chlorella virus isolates by using primer sets designed based on the sequence of the genome of PBCV-1, the prototype of the Phycodnaviridae. These putative promoter regions were found to contain several cis-acting elements for transcription factors, including the TATA, CAAT, NTBBF1, GATA, and CCAAT boxes. The amplified promoter regions were placed into Chlorella transformation vectors containing a green fluorescence protein (GFP) reporter gene and the Sh ble gene for phleomycin resistance. C. vulgaris protoplasts were transformed and then selected with phleomycin. The GFP fluorescence intensities of cells transformed with chitinase, DNA polymerase, and DNA ligase gene promoter-GFP fusion constructs were 101.5, 100.8, and 95.8%, respectively, of that of CaMV 35S-GFP-transformed Chlorella cells. These results demonstrate that these viral promoters are active in transformed Chlorella.

• 공업화학
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• 제31권2호
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• pp.220-225
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• 2020

모발은 여러 가지 아미노산들을 포함하는 단백질로 이루어져 있다. 자외선(UV)은 태양광선중에서 모발손상에 가장 큰 영향을 미치며 모발 노화에 주된 역할을 한다. 본 연구의 목적은 전자현미경(SEM), 공초점현미경(CLSM) 및 적외선 현미경분광법(IR micro spectroscopy)을 이용하여 정상모발에 UVB를 조사한 후 특징적인 형태학적 및 화학적 구조변화를 알아보는 것이다. 에너지 분산형 X선 분광기가 부착된 전자현미경은 자외선 조사모발의 표면이 정상모발과 비교했을 때 거칠고 높은 산소원소의 함량을 보였다. 형광 및 3차원 위상 이미지를 CLSM으로 분석한 결과 정상모발의 초록색 형광방출이 UVB 조사모발에 비해 매우 높았다. 또한 fluorescamine 형광 염색법을 통해 UVB 조사모발은 정상모발에 비해 펩타이드 결합의 파괴로 생성된 자유 아미노기가 많음을 확인할 수 있었다. UVB 조사모발의 강한 푸른색 형광은 아미노기의 함량이 높다는 것을 의미하며, 이는 CLSM에서도 관찰되었다. 따라서 fluorescamine은 UVB 조사모발에서 펩타이드 결합의 파괴를 관찰하는데 유용한 도구가 될 수 있다. 정상모발과 UVB 조사모발의 단면을 IR micro-spectroscopy를 통해 이미지 맵핑(mapping)한 결과, UVB 조사모발은 정상 모발에 비해 모발의 표면은 물론 내부에 걸쳐 디설파이드 결합(disulfide bond)의 산화가 일어나고 있음을 확인할 수 있었다. 이러한 분광학적 방법은 단독 또는 다른 분석법과 함께 모발화장품의 개발에 응용될 수 있을 것이다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권2호
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• pp.137-142
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• 2004

In this study, a simple procedure is described for patterning biotin on a glass substrate and then selectively immobilizing proteins of interest onto the biotin-patterned surface. Microcontact printing (CP) was used to generate the micropattern of biotin and to demonstrate the selective immobilization of proteins by using enhanced green fluorescent protein (EGFP) as a model protein, of which the C-terminus was fused to a core streptavidin (cSA) gene of Streptomyces avidinii. Confocal fluorescence microscopy was used to visualize the pattern of the immobilized protein (EGFP-cSA), and surface plasmon resonance was used to characterize biological activity of the immobilized EGFP-cSA. The results suggest that this strategy, which consists of a combination of $\mu$CP and cSA-fused proteins. is an effective way for fabricating biologically active substrates that are suitable for a wide variety of applications. one such being the use in protein-protein assays.

• Asian-Australasian Journal of Animal Sciences
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• 제31권3호
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• pp.449-456
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• 2018

Objective: Nanog, a homeodomain protein, has been investigated in humans and mice using embryonic stem cells (ESCs). Because of the limited availability of ESCs, few studies have reported the function and role of Nanog in porcine ESCs. Therefore, in this study, we investigated the location of the porcine Nanog chromosome and its basal promoter activity, which might have potential applications in development of ESCs specific marker as well as understanding its operating systems in the porcine. Methods: To characterize the porcine Nanog promoter, the 5'-flanking region of Nanog was isolated from cells of mini-pig ears. BLAST database search showed that there are two porcine Nanog genomic loci, chromosome 1 and 5, both of which contain an exon with a start codon. Deletion mutants from the 5'-flanking region of both loci were measured using the Dual-Luciferase Reporter Assay System, and a fluorescence marker, green fluorescence protein. Results: Promoter activity was detected in the sequences of chromosome 5, but not in those of chromosome 1. We identified the sequences from -99 to +194 that possessed promoter activity and contained transcription factor binding sites from deletion fragment analysis. Among the transcription factor binding sites, a Sp1 was found to play a crucial role in basal promoter activity, and point mutation of this site abolished its activity, confirming its role in promoter activity. Furthermore, gel shift analysis and chromatin immunoprecipitation analysis confirmed that Sp1 transcription factor binds to the Sp1 binding site in the porcine Nanog promoter. Taken together, these results show that Sp1 transcription factor is an essential element for porcine Nanog basal activity the same as in human and mouse. Conclusion: We showed that the porcine Nanog gene is located on porcine chromosome 5 and its basal transcriptional activity is controlled by Sp1 transcription factor.

• 대한바이러스학회지
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• 제28권3호
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• pp.225-232
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• 1998

We have expressed GFP in Sf9 and Bm5 cells or Bombyx mori larvae by using Ac-Bm hybrid virus capable of replicating in both Bm5 and Sf9 cells. Genomic DNA of Ac-Bm hybrid virus expressing ${\beta}$-galactosidase was cotransfected with baculovirus transfer vector containing GFP gene, pBacPAK-GFP in Sf9 cells. The Ac-Bm hybrid virus harboring GFP was named as Ac-Bm hybrid virus-GFP. The Ac-Bm hybrid virus-GFP-infected insect cells were easily selected by detecting the emission of GFP from each well of cell culture dish on the UV illuminator. GFP produced by Ac-Bm hybrid virus-GFP in Sf9 and Bm5 cells or B. mori larvae was confirmed by SDS-PAGE and Western blot analysis using GFP antibody. In addition, B. mori larvae infected with Ac-Bm hybrid virus-GFP was apparently appeared fluorescence from the whole body at S days postinoculation. The fluorescence of GFP from the hemolymph and fat body of B. mori larvae infected with Ac-Bm hybrid virus-GFP was also observed by fluorescence microscope. In conclusion, our results demonstrated that in baculovirus expression vector system, use of Ac-Bm hybrid virus have an additional advantage of expanded host range for producing recombinant proteins.

• 생명과학회지
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• 제29권9호
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• pp.949-954
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• 2019

세포막 단백질의 topology는 막단백질의 중요한 특징이다. 우리는 이전에 C4orf32단백질을 클로닝 하였으나, 이 단백질의 세포내 위치나 topology는 알지 못했다. 이번 연구를 통해 C4orf32는 세포내에서 소포체에 위치되는 막단백질임을 알게 되었다. C4orf32의 topology를 알기 위해 protease protection assay, fluorescence protease protection (FPP) assay, FRB/rapamycin/FKBP system을 활용하였다. Protease protection assay와 FPP assay를 적용한 결과 C-말단에 GFP를 붙인 C4orf32-GFP의 경우 GFP가 소포체의 세포질 표면에 위치함을 확인할 수 있었다. 또한, FRB/rapamycin/FKBP시스템을 이용한 실험에서 rapamycin이 처리되지 않은 경우는 mRFP-FKBP가 세포질에 위치하다가 rapamycin이 처리되면 C4orf32-GFP-FR가 위치하는 소포체로 이동함을 확인할 수 있었다. 이러한 사실은 C4orf32의 C-말단이 소포체의 세포질쪽 면에 위치한다는 사실을 말해준다. 이러한 연구를 통해 C4orf32는 Type I 소포체 막단백질에 속한다는 사실을 확인할 수 있었다.

• Animal cells and systems
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• 제16권4호
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• pp.280-288
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• 2012

Targeted degradation of proteins through ubiquitin-mediated proteolysis is an important control mechanism in various cellular processes. The process of ubiquitin conjugation is achieved by three enzyme complexes, among which the ubiquitin ligase complex (E3) is in charge of substrate specificity. The SCF (SKP1-CUL1-F-box) family portrays the largest and the most characterized member of the E3 ligases. For each SCF complex, the ubiquitination target is recognized by the F-box protein subunit, which interacts with the substrate through a unique C-terminal domain. We have characterized a novel F-box protein CFL-1 that represents a single LRR-type F-box (FBXL) in the Caenorhabditis elegans genome. CFL-1 is highly homologous to FBXL20 and FBXL2 of mammals, which are known to regulate synaptic vesicle release and cell cycle, respectively. A green fluorescence protein (GFP)-reporter gene fused to the cfl-1 promoter showed restricted expression around the amphid and the anus. Modulation of CFL-1 activity by RNAi affected the time interval between defecations. RNAi-treated worms also exhibited reduced tendency to form dauer when exposed to daumone. The potential involvement of CFL-1 in the control of defecation and pheromone response adds to the ever expanding list of cellular processes controlled by ubiquitin-mediated proteolysis in C. elegans. We suggest that CFL-1, as a single LRR-type F-box protein in C. elegans, may portray a prototype gene exerting diverse functions that are allocated among multiple FBXLs in higher organisms.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2004년도 제21차 정기총회 및 학술발표회
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• pp.19-20
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• 2004

본 연구는 1세포기 닭 수정란에서 외래 표지유전자(EGFP)와 리포좀(liposome)을 사용하여 외래유전자의 핵 전이의 효율성을 검증하고자 하였다. 실험은 리포좀과 혼합된 표지유전자와 naked 유전자 두 그룹으로 나누고, 유전자 미세 주입방법을 이용하여 배반엽 단계(stage X)와 1세포기 수정란의 세포질에 미세 주입하고 지속적으로 배양하면서 GFP의 발현 양상들을 관찰하였다. 실험결과, 배반엽 단계와 1세포기 수정란 모두에서 리포좀과 외래 유전자의 혼합물을 미세 주입한 경우 일주일 정도 지속적으로 GFP가 발현되었으나, 외래 유전자만을 주입한 경우 GFP의 발현이 관찰되지 않았다. 본 연구결과는 리포좀이 효율적으로 외래 유전자를 닭 수정란의 핵으로 이동시킴을 보여주고 있다.

• The Plant Pathology Journal
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• 제32권4호
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• pp.371-376
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• 2016

Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

• 한국잠사곤충학회지
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• 제52권2호
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• pp.117-122
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• 2014

선행연구를 통하여, 형질전환 누에를 제작하는 원천기술을 개발하고 이를 통해 녹색 형광실크를 생산하는 형질전환 누에를 제작한 바 있다. 본 연구에서는 선행연구에서 개발된 녹색 형광실크 형질전환 누에와 국립농업과 학원이 보유하고 있는 누에 유전자원 중 유색견(絹) 누에품종을 이용하여 형질전환 누에와 유색견 누에품종 간 교차교배를 통한 잡종강세를 이용하여 고치 생산량 및 고치의 색채 등이 향상된 녹색 형광실크 형질전환 누에 1대 잡종(F1) 계통을 개발하였다. 본 연구를 통해 선발된 실용형질이 우수한 농가보급형 녹색 형광실크 형질전환 누에 1대 잡종(F1) 계통은 기존 형질전환 누에의 녹색 형광단백질 뿐 아니라 유색견 누에 특유의 천연 연록색도 함께 갖고 있어 별도의 염색이 필요 없는 차별화된 고급 패션의류나 벽지 등의 고품질 소재로 활용이 가능할 것으로 기대된다.