• 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.

• BMB Reports
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• 제53권10호
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• pp.521-526
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• 2020

Endogenous retroviruses (ERVs) are retrotransposons present in various metazoan genomes and have been implicated in metazoan evolution as well as in nematodes and humans. The long terminal repeat (LTR) retrotransposons contain several regulatory sequences including promoters and enhancers that regulate endogenous gene expression and thereby control organismal development and response to environmental change. ERVs including the LTR retrotransposons constitute 8% of the human genome and less than 0.6% of the Caenorhabditis elegans (C. elegans) genome, a nematode genetic model system. To investigate the evolutionarily conserved mechanism behind the transcriptional activity of retrotransposons, we generated a transgenic worm model driving green fluorescent protein (GFP) expression using Human endogenous retroviruses (HERV)-K LTR as a promoter. The promoter activity of HERV-K LTR was robust and fluorescence was observed in various tissues throughout the developmental process. Interestingly, persistent GFP expression was specifically detected in the adult vulva muscle. Using deletion constructs, we found that the region from positions 675 to 868 containing the TATA box was necessary for promoter activity driving gene expression in the vulva. Interestingly, we found that the promoter activity of the LTR was dependent on che-1 transcription factor, a sensory neuron driver, and lin-15b, a negative regulator of RNAi and germline gene expression. These results suggest evolutionary conservation of the LTR retrotransposon activity in transcriptional regulation as well as the possibility of che-1 function in non-neuronal tissues.

• Journal of Plant Biotechnology
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• 제36권1호
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• pp.68-74
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• 2009

B. rapa로부터 분리한 BrMT3 유전자를 도입시킨 효모와 애기장대가 카드뮴을 비롯한 중금속에 저항성을 보이는 것이 확인되었고 이 결과를 토대로 이 유전자가 중금속 흡착을 통한 환경 정화 및 스트레스에 내성을 갖는 형질전환 식물체를 개발하는데 유용하게 사용될 것으로 기대된다.

• Journal of Microbiology
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• 제45권1호
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• pp.11-14
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• 2007

Over the last few years, the endophytic bacterial community associated with citrus has been studied as an important component interacting with Xylella fastidiosa, the causal agent of citrus variegated chlorosis(CVC). This bacterium may also colonize some model plants, such as Catharanthus roseus and Nicotiana clevelandii. In the present study, we compared the endophytic colonization of Citrus sinensis and Catharanthus rose us using the endophytic bacteria Klebsiella pneumoniae. We chose an appropriate strain, K. pneumoniae 342 (Kp342), labeled with the GFP gene. This strain was inoculated onto seedlings of C. sinensis and C. roseus. The isolation frequency was determined one week after the inoculation and the endophytic colonization of K. pneumoniae was observed using fluorescence microscopy. Although the endophytic bacterium was more frequently isolated from C. roseus than from C. sinensis, the colonization profiles for both host plants were similar, suggesting that C. roseus could be used as a model plant to study the interaction between endophytic bacteria and X. fastidiosa.

• 대한의생명과학회지
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• 제11권2호
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• pp.135-141
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• 2005

A Jopock (Jpk), a trans-acting factor associating with the position-specific regulatory element of murine Hoxa-7, has shown to have a toxicity to both prokaryotic and eukaryotic cells when overexpressed. Since Jpk protein harbors a transmembrane domain and a putative endoplasmic reticulum (ER)-retention signal at the N-terminus, a subcellular localization of the protein was analyzed after fusing it into the green fluorescent protein (GFP): Both N-term (Jpk-EGFP) and C-term tagged-Jpk (EGFP-Jpk) showed to be localized in the ER when analyzed under the fluorescence microscopy after staining the cells with ER- and MitoTracker. Since ER stress triggers the ER-stress mediated apoptosis to eliminate the damaged cells, we analyzed the expression pattern of Jpk under ER-stress condition. When MCF7 cells were treated with the ER-stress inducer such as DTT and EGTA, the expression of Jpk was upregulated at the transcriptional level like that of Grp78, a molecular chaperone well known to be overexpressed under ER-stress condition. In the presence of high concentration of ER-sterss inducer (10 mM), about 70 (DTT) to $95\%$ (EGTA) of cells died stronly expressing ($10\~12$ fold) Jpk. Whereas at the low concentration ($0.001\~1.0\;mM$) of the inducer, the expression of Jpk was increased about 2.5 (EGTA) to 5 fold (DTT), which is rather similar to those of ER chaperone protein Grp78. These results altogether indicate that the ER-stress upregulated the expression of Jpk and the excess stress induces the ER-stress induced apoptosis and the concomitant expression of Jpk.

• 한국어병학회지
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• 제22권3호
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• pp.375-382
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• 2009

목적 유전자를 숙주 세포의 게놈에 삽입하거나 발현하는데 있어서, retrovirus 매개의 유전자 전달 시스템을 사용하게 되면, 복잡하고 힘든 절차를 거치게 된다. 본 연구에서는 목적 유전자의 BF-2 세포 게놈 내 삽입을 하기 위해, UV로 불활화한 어류 레트로바이러스인 SnRV를 사용한 간단한 방법에 대해 조사하였다. 우선, BF-2 세포를 사용한 transfection을 위해 Lipofectamine 2000과 Transome을 사용하여 최적 조건을 결정하였다. 0.5 $\mu\ell$ Lipofectamine 2000을 사용한 경우 0.5, 1 그리고 2 $\mu{g}$ DNA 사용에 대해 33.8, 40.6 그리고 40.2%의 transfection 효율을 보인 동시에 최소 80 % 이상의 높은 세포 생존율을 나타낸 반면, Transome을 사용한 transfection 효율은 모두 5% 이하였다. UV 처리 시간에 있어서는 5분간의 UV 처리로 SnRV의 감염성이 불활성화되는 것을 확인하였다. 다음으로 GFP 유전자의 양측에 SnRV에서 유래된 LTR 서열을 접하고 있는 cassette를 구축한 뒤 BF-2 세포에 transfection 하고, cassette 유전자의 삽입과 발현을 위해 UV로 불활화한 SnRV를 처리하였다. 그 결과 UV로 불활화한 SnRV를 1회 처리 또는 SnRV 무처리 BF-2 세포에서는 형광이 관찰되지 않았던 반면, 3회와 5회 처리한 BF-2 세포에서 형광발현이 확인되었다. 이러한 결과로, GFP 유전자가 불활화한 SnRV를 이용하여 BF-2 세포 게놈에 삽입되는 것을 확인하였다.

• Clinical and Experimental Pediatrics
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• 제50권10호
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• pp.1011-1017
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• 2007

목 적 : 쥐의 섬유아세포인 NIH 3T3와 eGFP 유전자를 표지로 하는 레트로바이러스 벡터를 이용하여 유전자 전달효율을 향상시킬 수 있는 조건들을 살펴보고자 하였다. 방 법 : 표적세포에 대한 벡터의 비율과(1:1-1:8) 전달감염 횟수를 변화시켰을 때(1회, 2회), 양이온 복합체인 polybrene($4{\mu}g/mL$)을 첨가하였을 때 유전자 전달효율의 변화를 분석하였다. eGFP 유전자의 발현을 확인하기 위하여 형광 현미경 하에서 녹색빛을 내는 세포들을 관찰하고 FACscan으로 eGFP 양성세포의 비율을 측정하였다. 결 과 : 유전자 전달효율은 벡터와 표적세포의 비율 1:1에서 7%, 1:4에서 38%로 표적세포에 대한 레트로바이러스 벡터의 비율이 높을수록 상승하였지만 비율 1:4와 1:8사이에서는 차이가 없었다. 전달감염을 두 번 시행하는 것이 벡터와 표적세포의 비율 1:4까지는 유전자 전달효율에 영향을 미치지 않았지만 비율 1:8에서는 유전자 전달효율을 증가시켰다. 전달감염 후 eGFP 유전자의 발현은 3회 계대배양까지 약 3배 가량 증가하였지만 이후에는 감소하였는데 이와 같은 감소 정도는 전달감염을 한 번 시행한 경우가 두 번 시행한 경우보다 더 커서 전달감염을 반복하는 것이 유전자 전달효율의 증가효과보다는 주입된 유전자의 지속발현에 더 영향을 미치는 것으로 판단되었다. Polybrene을 첨가하였을 때 유전자 전달효율은 5.8%에서 38.8%로 대폭 상승하였으며 독성반응은 관찰되지 않았다. 배양접시의 크기에 따른 유전자 전달효율을 비교하였을 때 NIH 3T3세포의 증식정도는 6-well plate가 더 컸지만 eGFP 양성세포의 비율은 24-well plate에서 더 높았다. 결 론 : 이번 연구결과를 기초로 삼아 유전자 치료의 연구를 발전시키고 특히 전달된 유전자의 안정적인 발현과 바이러스 벡터들의 독성 등에 대하여 향후 연구의 초점을 두어야 할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제30권9호
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• pp.1430-1435
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• 2020

Bacterial cellulose (BC) has outstanding physical and chemical properties, including high crystallinity, moisture retention, and tensile strength. Currently, the major producer of BC is Komagataeibacter xylinus. However, due to limited tools of expression, this host is difficult to engineer metabolically to improve BC productivity. In this study, a regulated expression system for K. xylinus with synthetic ribosome binding site (RBS) was developed and used to engineer a BC biosynthesis pathway. A synthetic RBS library was constructed using green fluorescent protein (GFP) as a reporter, and three synthetic RBSs (R4, R15, and R6) with different strengths were successfully isolated by fluorescence-activated cell sorting (FACS). Using synthetic RBS, we optimized the expression of three homologous genes responsible for BC production, pgm, galU, and ndp, and thereby greatly increased it under both static and shaking culture conditions. The final titer of BC under static and shaking conditions was 5.28 and 3.67 g/l, respectively. Our findings demonstrate that reinforced metabolic flux towards BC through quantitative gene expression represents a practical strategy for the improvement of BC productivity.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.181-186
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• 2005

본 연구에서는 배양 체계(혈청 첨가 TCM199, TALP, CRlaa 및 혈청 미첨가; IVMD101, IVF100, IVMD101)가 체외수정 또는 미세주입된 수정란의 체외 발달에 미치는 효과를 검토하였다. 또한 미세주입에 사용하는 GFP유전자의 양 및 미세주입 수정란에서 형광발현 양상을 검토하였다. 체외수정된 수정란의 $\geq$ 2세포기, 8세포기 및 배반포 도달율이 미세주입 수정란에 비하여 유의하게 높았다. 혈청 미첨가 배지에서의 8세포기 발달율이 수정란의 종류에 관계없이 유의하게 높았으나(p<0.05; $ 3.3\%\;vs.\;15.5\%$ 및 $21.4\%\;vs.\;39.4\%$, respectively), 배반포 도달율은 유사한 경향이었다($2.7\%\;vs.\;2.3\%$ 및 $23.0\%\;vs.\;23.6\%$, respectively). 한편, 2ng/uL 유전자를 미세주입한 수정란의 $\geq$ 2세포기, 8세포기 및 배반포 도달율이 4 및 8ng/uL의 것에 비하여 높은 경향이었다. 미세주입 수정란의 형광발현율은 1세포기가2및 8세포기에 비하여 유의하게 높았으나(p<0.05), 4세포기 및 배반포 단계와는 차이가 없었다.

• The Plant Pathology Journal
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• 제29권1호
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• pp.17-30
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• 2013

Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB) treat-ments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW). BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3.