• 한국잠사곤충학회지
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• 제52권2호
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• pp.102-109
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• 2014

본 연구의 목적은 누에형질전환 기술과 피브로인 재조합 단백질 발현시스템을 이용하여 황색형광실크를 개발하는 것으로서, 본 실험에서는 피브로인 H-chain의 N-말단과 C-말단을 이용하여 피브로인 재조합 단백질 발현 시스템을 제작하였고, 종결코돈이 없는 EYFP 유전자를 위의 발현 시스템에 클로닝하여 황색형광실크를 제작하였다. 누에형질전환체 선발을 위해서는 3xP3 promoter와 EGFP 유전자를 이용하여 선발하였고, 3,060개의 누에알에 microinjection 하여 F1 세대에서 8 bloods의 누에형질전환체를 선발하였다. 선발된 누에형질전환체는 초기배 단계의 눈과 신경조직, 유충과 번데기 그리고 성충의 눈에서 EGFP 형광단백질이 발현되는 것을 확인할 수 있었다. 또한 실크의 피브로인에서 EYFP 단백질이 발현되는 것을 확인하기 위해, F2세대의 누에형질전환체중에서 5령 3일 유충의 견사선을 형광현미경으로 관찰하였고, 중부 견사선에서 황색형광단백질이 발현되는 것을 확인할 수 있었다. 또한 F2 세대의 고치와 저온에서 정련한 실크에서도 황색형광단백질의 발현을 확인할 수 있었고, Western blot 분석에서도 EYFP 재조합 단백질이 피브로인 H-chain과 융합된 형태로 존재하는 것이 확인되었다. 이상의 결과에서 황색형광실크를 생산하는 누에형질전환체가 성공적으로 제작되었음을 확인할 수 있었다.

• Parasites, Hosts and Diseases
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• 제55권2호
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• pp.109-114
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• 2017

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.

• Parasites, Hosts and Diseases
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• 제54권2호
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• pp.133-138
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• 2016

Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.

• Parasites, Hosts and Diseases
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• 제49권2호
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• pp.103-108
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• 2011

Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.

• 미생물학회지
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• 제45권3호
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• pp.246-250
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• 2009

레트로바이러스는 바이러스 외막과 숙주세포막의 융합에 의해 세포내로 들어간다. Moloney 마우스레트로바이러스(murine leukemia virus)의 외막 단백질은 표면단백질(SU)과 막단백질(TM)을 포함하는 85 kDa의 전구체로 합성되는데 바이러스의 성숙과정에 막단백질의 카르복시 말단 16개의 아미노산(R-peptide)이 바이러스의 프로티아제에 의해 절단된다. R 펩타이드가 절단된 막단백질은 Moloney 마우스레트로바이러스에 대한 수용체를 가진 NIH3T3 세포주에서 합포체(syncytium)를 형성한다. R 펩타이드가 절단된 막단백질의 합포체 형성 기작 연구를 위해 R 펩타이드가 절단되어 있으며 표면단백질의 PRR (proline rich region) 부위가 EGFP로 삽입되어진 Moloney full length molecular clone을 만들었다. 이 clone은 NIH3T3 세포에서 합포체를 형성하였으며 형광이 세포질과 세포막에서 관찰되었으나 핵은 염색이 되지 않고 검게 보여 신속 정확하게 합포체 관찰이 가능하였다. 흥미롭게도 절단된 막단백질을 가진 비리온이 NIH3T3 세포에서 광학현미경으로 관찰하였을때는 합포체를 형성하였으나 형광현미경에서는 형광이 관찰되지 않아서 비리온이 세포감염 없이 바이러스-세포 융합 방식으로 합포체를 형성한 것으로 생각되었다. 본 연구에서는 형광의 발현 여부로 합포체 형성을 신속 정확하게 관찰할 수 있는 방법을 개발하였으며 R 펩타이드가 절단된 비리온이 세포 감염 없이 세포와 세포 사이의 융합을 매개할 수 있음을 밝혔다.

• 한국잠사곤충학회지
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• 제52권1호
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• pp.25-32
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• 2014

본 연구의 목적은 누에형질전환 기술과 피브로인 재조합단백질 발현시스템을 이용하여 청색형광실크를 개발하는 것으로서, 본 실험에서는 피브로인 H-chain의 N-말단과 C-말단을 이용하여 피브로인 재조합단백질 발현 시스템을 제작하였고, 종결코돈이 없는 EBFP 유전자를 위의 발현 시스템에 클로닝하여 청색형광실크를 제작하였다. 누에형질전환체 선발을 위해서는 $3{\times}P3$ promoter와 DsRed2를 이용하여 선발하였고, 300개의 누에알에 microinjection하여 F1 세대에서 5 bloods의 형질전환체를 선발하였다. 선발된 누에형질전환체는 초기배 단계의 눈과 신경조직, 유충과 번데기 그리고 성충의 눈에서 DsRed2 형광단백질이 발현되는 것을 확인할 수 있었다. 또한 실크의 피브로인에서 EGFP 단백질이 발현되는 것을 확인하기 위해, F2 세대의 누에형질전환체 중에서 5령 3일 유충을 해부하여 견사선을 형광현미경으로 관찰하였고, 중부 견사선에서 청색형광단백질이 발현되는 것을 확인할 수 있었다. 또한 F2 세대의 고치와 저온에서 정련한 실크에서도 청색형광단백질의 발현을 확인할 수 있었고, Western blot 분석에서도 EBFP 재조합 단백질이 피브로인 H-chain과 융합된 형태로 존재하는 것이 확인되었다. 이상의 결과에서 청색형광실크를 생산하는 누에형질전환체가 성공적으로 제작되었음을 확인할 수 있었고 이러한 결과를 토대로 새로운 산업소재로서 실크를 활용할 수 있을 것으로 기대한다.

• International Journal of Industrial Entomology and Biomaterials
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• 제15권2호
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• pp.131-135
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• 2007

Open reading frame 4 of Bombyx mori nucleopolyhedrovirus (BmNPV), designated as Bm4, is a gene whose function is completely unknown. With the recently developed BmNPV bacmid and a modified pFastBac1 whose polyhedrin promoter was replaced with ie1 promoter, a recombinant bacmid expressing Bm4-EGFP fusion protein under the control of ie1 promoter in BmN cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced efficiently with other promoters to direct the expression of foreign gene in BmN cells by using Bac-to-Bac/BmNPV baculovirus expression system but also laid the foundation for rescue experiment of Bm4 deletion mutant due to the ability of ie1 promoter to direct gene expression throughout the infection cycle.

• BMB Reports
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• 제44권1호
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• pp.58-63
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• 2011

Zinc finger (ZNF) proteins play a critical role in cell growth, proliferation, apoptosis, and intracellular signal transduction. In this paper, we cloned and characterized a novel human KRAB-related zinc finger gene, ZNF425, which encodes a protein of 752 amino acids. ZNF425 is strongly expressed in the three month old human embryos and then is almost undetectable in six month old embryos and in adult tissues. An EGFP-ZNF425 fusion protein can be found in both the nucleus and the cytoplasm. ZNF425 appears to act as a transcription repressor. Over-expression of ZNF425 inhibits the transcriptional activities of SRE, AP-1, and SRF. Deletion analysis indicates that the C2H2 domain is the main region responsible for the repression. Our results suggest that the ZNF425 gene is a new transcriptional inhibitor that functions in the MAPK signaling pathway.

• Asian-Australasian Journal of Animal Sciences
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• 제21권5호
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• pp.648-656
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• 2008

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-N1) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for $30{\mu}s$ in $Ca^{2+}$ and $Mg^{2+}-containing$ 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

• 대한의생명과학회지
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• 제13권4호
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• pp.263-272
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• 2007

Nebulin is a giant actin binding protein (600-900 kDa) which is specific to skeletal muscle. This protein is known to regulate thin filaments length in sarcomere as a molecular template. The C-terminus of nebulin is located in the Z-disc of muscle sarcomere and is bound to other proteins such like myopalladin, titin, archvillin, and desmin. The N-terminus of nebulin binds to tropomodulin at the pointed ends of the thin filaments. In recent research, nebulin not only found in brain but also expressed in heart, stomach, and liver. So, the roles of nebulin in non-muscle tissue have been studied. However, lack of information or studies on nebulin binding proteins and nebulin function in brain are available so far. Therefore, the current study have investigated a novel binding partner of Nebulin C-terminus by using yeast two-hybrid screening with human brain cDNA library. Nebulin C-terminus, containing simple repeats, serine rich and SH3 domain, interacts with osteonectin C-terminal region. The specific interaction of nebulin and osteonectin were confirmed in vitro by using GST pull-down assay and reconfirmed in vivo by using transfected COS-7 cells with EGFP-tagged nebulin and DsRed-tagged osteonectin. Consequently, this study identified SH3 domain in nebulin C-terminus specifically binds to extracellular Ca-binding (EeC domain in osteonectin. Also, nebulin C-terminus fusion protein colocalized with osteonectin EC domain fusion protein in transfected COS-7 cells. The current study found the interaction between nebulin and osteonectin in human brain for the first time and suggested the nebulin in brain may be associated with osteonectin, as a regulator of cell cycle progression and mitosis.