• Journal of Plant Biotechnology
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• 제31권4호
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• pp.285-293
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• 2004

FMDV는 동물에서 구제역을 일으키는 병원체이며, VP1은 이 바이러스의 주요 capsid단백질이므로 구제역의 진단과 단백질 백신의 개발에 가장 많이 사용되는 재료 중 하나이다. 본 연구는 FMDV taiwan O형과 베트남에서 분리된 FMDV의 VP1 sequence를 기반으로 식물에서 VP1 유전자의 발현을 위하여 633 bp의 VP1유전자로 재편집하였으며, 이를 long-nucleotide를 사용한 multiple fragment extension 방법을 사용하여 인공적인 DNA 단편을 합성하였다. 또한 새로운 식물 형질전환 벡터로 pBI121 과 pCAMBIA1390의 장점을 수용하여, hygromycin 저항성과 CaMV 35S promoter를 포함하는 pCAMBIA II를 제작하였다. 제작된 벡터와 VP1 유전자 및 GFP유전자를 사용하여 담배를 형질 전환시켰고, 각각의 형질전환식물체내에서 전체길이의 target gene(VPl)의 성공적인 삽입을 확인하였다. 각 유전자의 발현은 RT-PCR과 Real-Time PCR의 결과로 측정하였으며, VP1 유전자의 전사가 담배 내에서 이루어졌음과 고효율의 전사체를 만드는 형질전환체 VP1-4를 선별하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7875-7882
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• 2015

Background: The objectives of this study were to evaluate the expression of brain and acute leukemia, cytoplasmic (BAALC) gene and erythroblast transformation-specific related gene (ERG) in de novo cases of acute myeloid leukemia (AML) and identify roles in disease progression and outcome. Materials and Methods: This study included 50 newly diagnosed AML patients, along with 10 apparently healthy normal controls. BAALC and ERG expression was detected in the bone marrow of both patients and controls using real-time RT-PCR. Results: BAALC and ERG expression was detected in 52% of cases but not in any controls. There was a statistically significant correlation between BAALC and ERG gene expression and age (p-value=0.004 and 0.019, respectively). No statistical significance was noted for sex, lymphadenopathy, hepatomegaly, splenomegaly, other hematological findings, immunophenotyping and FAB sub-classification except for ERG gene and FAB (p-value=0.058). A statistical significant correlation was found between response to treatment with ERG expression (p-value=0.028) and age (p-value=0.014). A statistically significant variation in overall survival was evident with patient age, BM blast cells, FAB subgroups, BAALC and ERG expression (p-value=<0.001, 0.045, 0.041, <0.008 and 0.025 respectively). Conclusions: Our results suggest that BAALC and ERG genes are specific significant molecular markers in AML disease progression, response to treatment and survival.

• Tuberculosis and Respiratory Diseases
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• 제41권4호
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• pp.339-353
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• 1994

연구배경 : p53 유전자는 염색체 17p에 존재하는 종양억제유전자인데, 돌연변이가 있는 경우의 mutant protein은 그 반감기가 4~8시간으로 wild type protein의 반감기 6~20분에 비하여 현저한 증가를 보이게 된다. 결과적으로 돌연변이가 있는 경우 p53 단백질이 과축적되어 과발현이 유발되는데, 이러한 기전을 이용하여 p53 면역조직화학염색은 p53유전자의 돌연변이의 검색 방법으로 많이 이용되고 있다. 유방암에서는 p53 핵단백질의 과발현이 있는 경우에 없을 때보다 무병생존기간 및 전체 생존기간 모두에서 유의하게 나쁜 임상 경과를 취함이 보고되고 있지만 폐암에서는 p53 유전자의 돌연변이 유무, 또는 p53단백의 과발현 유무가 예후에 미치는 영향은 아직까지 확실하지 않다. 저자 등은 한국인의 폐암에서 p53 종양억제 유전자의 돌연변이 빈도를 확인하고 이들이 폐암 환자의 임상 경과에 미치는 영향을 분석하며, 면역조직화학염색 및 PCR-SSCP 분석의 감수성과 특이성을 염기서열분석과 비교하여 확인하고자 본 연구를 시행하였다. 방법 : 근치적 폐절제술 후 임상경과를 추적중인 원발성폐암환자 75예를 대상으로 하였으며, 면역조직화학검사 및 분자생물학적 연구를 위하여는 이들의 종양조직(paraffin block)을 이용하였다. p53 단백질의 면역조직화학검사를 위한 일차항체로는 DO7(Novocastra, U.K.)을 사용하였고, PCR-SSCP 및 염기서열분석 등을 위하여는 파라핀 포매조직에서 암조직을 선택적으로 박절하여 DNA를 추출하여 이용하였다. 결과: 1) 전체 75예 폐암환자의 면역조직화학염색 결과 27예(36%)에서 p53단백질의 과발현을 보였다(Table 2.3, Fig 1). 2) 전체 환자의 p53 핵단백질 과발현 여부에 따른 중앙전체생존기간은 p53 음성군과 양성군 모두 25개월, 무병생존기간은 두군 모두 13개월로 동일하였다(Fig. 2). 3) PCR-SSCP 분석에 의한 p53 유전자의 mobility shift는 시험한 58예중 16예(27.6%)에서 확인되었다(Fig 3). Mobility shift 유무에 따른 중앙전체생존기간은 shift가 있는 군과 없는 군에서 각각 27개월과 20개월, 무병생존기간은 각각 8개월, 10개월로 유의한 차이가 없었다. 4) 염기서열분석을 통한 p53 유전자의 돌연변이는 시험한 29예중 10예(34.5%)에서 확인되었다(Fig 4). 염기서열분석 결과 돌연변이 유무에 따른 중앙전체생존기간은 돌연변이가 있었던 군과 없었던 군에서 각각 27개월, 22개월이었으며, 무병생존기간은 각각 20개월과 10개월로 유의한 차이는 없었으나 돌연변이가 있는 경우 조기재발을 하는 경향을 보였다(Fig 5, 6). 5) 면역조직화학염색은 PCR-SSCP 결과를 기준으로 할 때 민감도 67.0%, 특이도 74.0%, 그리고 일치도는 62.5% 이었다. PCR-SSCP의 결과는 염기서열분석 결과를 기준으로 할 때 민감도 91.8%, 특이도 96.2%, 일치도는 95.3% 이었다. 결론 : p53 핵단백질의 과발현 정도, PCR-SSCP 및 염기서열분석상 돌연변이 유무는 비소세포폐암환자의 근치적수술후 예후의 예측지표로서는 그 효용성이 적었으나, 돌연변이가 있는 경우 조기재발을 하는 경향을 보였다. 또한 p53유전자의 돌연변이 검색법으로는 면역조직화학염색보다는 PCR-SSCP법이 우수하였다.

• 미생물학회지
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• 제26권3호
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• pp.167-172
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• 1988

A genetic map of the IncP-1 group plasmid pKU10 has been prepared through the construction of recombinant plasmids containing various fragments of pKU10. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to ampicillin, tetracyclin, and chloramphenicol. The region involved in conferring resistance to ampicillin was located around two PstI sites that are 1.0Kb apart. The tetracyclin resistance gene was mapped on the region of HindIII E fragment and a part of HindIII D fragment, and the determinant for chloramphenicol resistance gene was localized on HindIII D fragment.

• BMB Reports
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• 제36권3호
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• pp.326-331
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• 2003

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSB) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, $\gamma$-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multi copy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride ($10\;{\mu}M$) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal8 amino acid-coding region were fused into the promoteriess $\beta$-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of $\beta$-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of $\gamma$-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.

• 한국잠사곤충학회지
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• 제52권1호
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• pp.39-44
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• 2014

효율적인 형질전환 누에 시스템 구축을 위해서는 새로운 전이인자의 개발과 함께 선발을 위한 마커 유전자 및 transposase 발현을 효과적으로 조절할 수 있는 다양한 유전자 프로모터 개발이 필수적이다. 이와 관련하여 선행연구를 통해 누에 후부실샘으로부터 고발현하는 RNA binding protein-1 homologue(RBP-1) 유전자를 선발한 바 있다. 본 연구에서는 RBP-1유전자의 누에 발육시기별 및 유충 조직별 발현양상을 Northen blot hybridization 방법으로 분석한 결과, RBP-1 유전자는 유충기로부터 번데기 후기까지의 전기간에 걸쳐 발현하였으며, 두부, 표피, 중장, 지방체 및 견사선 등 실험한 모든 유충 조직에서 고발현 하는 것으로 관찰되었다. 또한, 누에 게놈 유전자은행을 제작한 후 RBP-1 cDNA 유전자를 탐침으로 5'-UTR 영역을 클로닝하고 luciferase assay 방법으로 RBP-1 유전자 프로모터의 활성을 분석하였다. 실험 결과, RBP-1 cDNA를 탐침으로 RBP-1 유전자 ORF와 5'-UTR이 포함된 약 1,660 bp 영역의 게놈 유전자를 클로닝하였다. RBP-1 유전자 프로모터 활성검정을 위해 전사 개시점(+ 30)으로부터 상류의 -740 bp 영역을 PCR로 분리한 후 pGL3 basic vector에 도입하여 luciferase 활성 측정을 위한 전이벡터, pGL-RBP1를 제작하였다. 제작된 pGL-RBP1는 곤충 세포주(Sf9)에 transfection 한 후 luciferase 발현량을 측정한 결과, 기존의 BmA3 유전자 프로모터 대비 10% 가량 높은 발현 효율을 확인할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.331-335
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• 1996

The second $\beta$-Xylosidase gene (xylB) from Bacillus stearothermophilus was isolated from the genomic library, cloned into pBR322, and subsequently transferred into Escherichia coli HB101. Six out of 10, 000 transformants were selected from the selective LB medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf) and ampicillin ($50\mu g$/ml) based on their ability to form a yellow ring around the colony. One of the clones was found to harbor the recombinant plasmid with 5.0 kb foreign DNA, which was identical to the $\alpha$-L-arabinofuranosidase gene (arfI) previously cloned in this lab, while the other five had 3.5 kb of the foreign DNA. Southern blotting experiments confirmed that the 3.5 kb insert DNA was from B. stearothermophilus chromosomal DNA. A zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate revealed that the cloned gene product was one of the mutiple $\alpha$-L-arabinofuranosidases produced by B. stearothermophilus. Unlike the arfI gene product, the product of the gene on the insert DNA (xylB) showed an activity not only on pNPAf but also on oNPX suggesting that the cloned gene product could be a bifunctional enzyme having both $\alpha$-L-arabinofuranosidase and $\beta$-xylosidase activities.

• 한국축산식품학회지
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• 제35권6호
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• pp.867-873
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• 2015

1,4-Dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2), has an effect on growth stimulation of bifidobacteria and prevention of osteoporosis, making it a promising functional food material. Therefore, we tried to clone the menB gene encoding DHNA synthase from Leuconostoc mesenteroides CJNU 0147. Based on the genome sequence of Leu. mesenteroides ATCC 8293 (GenBank accession no., CP000414), a primer set (Leu_menBfull_F and Leu_menBfull_R) was designed for the PCR amplification of menB gene of CJNU 0147. A DNA fragment (1,190 bp), including the menB gene, was amplified, cloned into pGEM-T Easy vector, and sequenced. The deduced amino acid sequence of MenB (DHNA synthase) protein of CJNU 0147 had a 98% similarity to the corresponding protein of ATCC 8293. The menB gene was subcloned into pCW4, a lactic acid bacteria - E. coli shuttle vector, and transferred to CJNU 0147. The transcription of menB gene of CJNU 0147 (pCW4::menB) was increased, when compared with those of CJNU 0147 (pCW4) and CJNU 0147 (−). The DHNA was produced from it at a detectable level, indicating that the cloned menB gene of CJNU 0147 encoded a DHNA synthase which is responsible for the production of DHNA, resulting in an increase of bifidogenic growth stimulation activity.

• Asian Pacific Journal of Cancer Prevention
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• 제16권6호
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• pp.2555-2559
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• 2015

Purpose: To investigate effects of the TESTIN (TES) gene on proliferation and migration of highly metastatic nasopharyngeal carcinoma cell line 5-8F and the related mechanisms. Materials and Methods: The target gene of human nasopharyngeal carcinoma cell line 5-8F was amplified by PCR and cloned into the empty plasmid pEGFP-N1 to construct a eukaryotic expression vector pEGFP-N1-TES. This was then transfected into 5-8F cells. MTT assays, flow cytometry and scratch wound tests were used to detect the proliferation and migration of transfected 5-8F cells. Results: A cell model with stable and high expression of TES gene was successfully established. MTT assays showed that the OD value of 5-8F/TES cells was markedly lower than that of 5-8F/GFP cells and 5-8F cells (p<0.05). Flow cytometry showed that the apoptosis rate of 5-8F/TES cells was prominently increased compared with 5-8F/GFP cells and 5-8F cells (p<0.05). In vitro scratch wound assays showed that, the width of the wound area of 5-8F/TES cells narrowed slightly, while the width of the wound area of 5-8F/ GFP cells and 5-8F cells narrowed sharply, suggesting that the TES overexpression could inhibit the migration ability. Conclusions: TES gene expression remarkably inhibits the proliferation of human nasopharyngeal carcinoma cell line 5-8F and reduces its migration in vitro. Thus, it may be a potential tumor suppressor gene for nasopharyngeal carcinoma.

• Asian-Australasian Journal of Animal Sciences
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• 제19권2호
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• pp.171-175
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• 2006

The aim of this in vitro study was to test the effect of microinjection (Mi) of foreign gene into the rabbit egg pronucleus and epidermal growth factor (EGF) addition on the blastocyst rate, the cell number and the diameter of embryos, and to determine possible relationships between embryo cell number and embryo diameter. Blastocyst rate was significantly decreased in gene- Mi (G-Mi/E0) group (63.1%) comparing to intact ones (83.5%, $p_1$<0.05). The addition of EGF at 20ng/ml (G-Mi/E20) or 200 ng/ml (GMi/ E200) to gene-Mi embryos did not affect blastocyst rate (65.6 and 55.2% resp.). As a control for Mi, the eggs were microinjected with the same volume of phosphate-buffered solution (PBS-Mi) instead of the gene construct solution. Cell numbers and embryo diameters were measured from embryo images obtained on confocal laser scanning microscope. Bonferroni-modified LSD test showed that the embryo cell number in PBS-Mi group was significantly lower ($p_1$<0.05) and in gene-Mi group was tended to decrease compared with intact embryos. Embryo diameter was not different among experimental groups. No effect of EGF given at any doses both on the cell number and embryo diameter was found. A positive correlation between cell number and embryo diameter was observed in all groups of embryos. Since embryo diameter was not changed under the influence of Mi or EGF addition in this study, this seems to be more conservative characteristics of the embryo morphology. These results suggest that the pronuclear microinjection compromises developmental potential of embryos, decreasing blastocyst rate and embryo cell number, whilst embryo diameter is not affected. No effects of EGF on studied parameters were confirmed. Declined quality of Mi-derived embryos is caused by the microinjection procedure itself, rather than by the gene construct used.