• Journal of Plant Biotechnology
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• 제37권1호
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• pp.89-95
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• 2010

Dihydroflavonol 4-reductase(DFR)는 flavonoid 생합성 경로의 가장 중심부에 작용하는 효소로 2R,3R-trans-dihydroflavonols로부터 leucoanthocyanidins 으로의 변환을 촉매한다. 본 연구에서는 색소유전자의 전이를 통하여 새로운 색소발현체계를 가진 품종을 육종하기 위한 기초연구로 안개초 (Gypsophila paniculata L.)의 꽃봉오리로부터 cDNAlibrary를 합성하였고 카네이션의 DFR 유전자를 probe로 사용하여 anthocyanin 합성경로의 중요 효소의 하나인 DFR 유전자를 분리하였다. 염기서열분석을 수행하여 분리유전자의 크기가 1279 bp이며 이 중 coding region은 1063 bp임을 확인하였다. 이미 밝혀진 다른 식물체의 DFR 유전자와 서로 염기서열의 일치성을 비교해 본 결과 Cheddar pink, 카네이션, 양배추, 개나리, 페튜니아, cup flower, 장미, 과꽃 및 거베라에서 각각 62% 이상을 나타내었다. 분리유전자의 발현을 확인하기 위하여 Northern blot 분석 및 인위적으로 기내에서의 transcription과 translation을 수행하였고, 분리한 유전자의 효소활성을 측정해 본 결과 leucopelargonidin의 작은 peak를 확인하였다. Southern blot 분석 결과 안개초의 DFR 유전자는 다른 대부분의 식물체와 유사하게 한 개가 존재함을 확인하였다.

• Journal of Photoscience
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• 제9권2호
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• pp.269-271
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• 2002

To investigate the visual and extra-ocular photoreception, we cloned the opsin genes in ayu (Plecoglossus allivelis). Amplified fragments encoding exon-4 (-5) of opsin cDNAs were cloned from the retina and brains of ayu, and sequenced. One clone was identified as rod (AYU-Rh), two as green cone (AYU-GI, -G2), one as red cone (A YU-R), two as ultraviolet cone (AYU-UVl, UV2), one as VA (AYU-VA), and one as extra-ocular rod (AYU-ExoRh) opsins. 335 amino acids sequence deduced from the full-length cDNA of AYU-Rh showed high identity with that of other fish. Southern blotting analysis indicated that ayu possess two 'rhodopsin' genes, one is visual rhodopsin and the other is non-visual extra-ocular rhodopsin. In situ hybridization showed that the mRNA of AYU-Rh was localized only in rod cells in the retina. On the other hands, AYU-ExoRh was expressed only in the pineal. We cloned two isoforms (AYU-VAM and -VAL) of VA opsin from ayu. The deduced amino acid sequences of these variants were identical to each other within the first 342 residues, but they showed divergence in the C-terminal sequence. AYU- VAL corresponded to the long isoform found in other fish, and AYU-VAM was identified as a new type of VA opsin variant. Pal-VAM is a new probably functional non-visual photoreceptive molecule in fish.

• Journal of Microbiology and Biotechnology
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• 제7권5호
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• pp.293-298
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• 1997

A 4.6-kb HindIII fragment encompassing the complete 140-kDa ${\alpha}$-amylase gene of Lactobacillus amylovorus B 4540 was cloned into pBR322 by the shot gun method. Southern blotting and restriction mapping for the insert were performed. The recombinant 9.0-kb plasmid, pFML1, conferred ${\alpha}$-amylase activity to E. coli and Lactococcus lactis hosts when introduced by electroporation. SDS-PAGE and zymography confirmed the production of 140-kDa ${\alpha}$-amylase and its proteolytic degradation products with enzyme activity in transformants. Total ${\alpha}$-amylase activity of E. coli $DH5{\alpha}$ cells harboring pFML1 was 1.8 units and most activity was detected from cell pellets. Total enzyme activity of L. lactis subsp. lactis MG1363 transformant was five to ten-fold lower than that of E. coli cell but more than half of the activity was detected in the culture supernatant.

• Journal of Microbiology and Biotechnology
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• 제20권2호
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• pp.265-270
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• 2010

Two major endoglucanase genes (cel7B and cel5A) were cloned from Penicillium decumbens 114-2 using the method of modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The result of Southern blotting suggested that P. decumbens has a single copy of the cel5A gene and a single copy of the cel7B gene in its chromosomal DNA. The expression levels of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that the two genes were coordinately expressed, and repressed by glucose and induced by cellulose. Both endoglucanase genes were expressed in Saccharomyces cerevisiae and the recombinant proteins were purified. The recombinant Cel7B and Cel5A were both optimally active at $60^{\circ}C$ and pH 4.0. The recombinant Cel7B showed more than 8-fold, 30-fold, and 5-fold higher enzyme activities toward carboxymethyl cellulose, barley $\beta$-glucan, and PASC, respectively, in comparison with that of Cel5A. However, their activities toward pNPC and Avicel showed minor differences. The results suggested that Cel7B is a strict endoglucanase, whereas Cel5A showed processivity because of its relative higher ability to hydrolyze the crystal cellulose.

• Parasites, Hosts and Diseases
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• 제43권3호
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• pp.101-110
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• 2005

In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited $42-57\%$ homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GOSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cock-roach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.

• Journal of Plant Biotechnology
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• 제46권4호
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• pp.323-330
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• 2019

The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicum-specific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.

• 원예과학기술지
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• 제29권4호
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• pp.345-351
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• 2011

원예작물의 내병성 육종에 있어 $F_2$와 그 이후 분리 세대에서 유용하게 사용할 수 있는 선발방법의 모델 시스템을 개발하고자 하였다. 상용 토마토 품종인 '모모따로 요크'에 제초제 저항성 유전자를 도입한 후 획득된 형질전환체의 도입 유전자 수와 후대 분리비를 분석하고, 제초제 저항성 유전자와 기존 병 저항성 유전자가 연관된 개체를 선발하였다. 총 60개의 형질전환체 중에서 42개체에 Southern blot을 실시하여 도입 유전자 수를 확인하고, 제초제 저항성 유전자에 대한 분리비를 비교 분석하여 Southern 결과와 도입된 유전자의 표현형의 분리비가 일치하지 않으며 여러 개의 copy가 삽입된 대부분의 경우, 실제 도입된 copy 수보다 더 적은 copy 수를 가지는 것처럼 분리비를 나타내는 것을 알 수 있었다. 삽입된 제초제 저항성 유전자와 기존 병 저항성 유전자가 가깝게 연관된 개체를 찾기 위해 $T_1(F_2)$세대 개체에 대하여 two-stepwise screening 방법을 실시하여 위조병 저항성 I2 유전자(11번 염색체)와 제초제 저항성 patII 유전자가 대략 12-13cM으로 가깝게 연관된 개체(T-20)를 선발하였다.

• BMB Reports
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• 제39권1호
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• pp.68-75
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• 2006

In higher plants, P450s participate in the biosynthesis of many important secondary metabolites. Here we reported for the first time the isolation of a new cytochrome P450 cDNA that expressed in a stem-specific manner from Camptotheca acuminata (designated as CaSS), a native medicinal plant species in China, using RACE-PCR. The full-length cDNA of CaSS was 1735 bp long containing a 1530 bp open reading frame (ORF) encoding a polypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS contained a heme-binding domain PFGXGRRXCX and showed homology to other plant cytochrome P450 monooxygenases and hydroxylases. Southern blotting analysis revealed that there was only one copy of the CaSS present in the genome of Camptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in a tissue-specific manner, highly in stem and lowly in root, leaf and flower. Our study suggests that CaSS is likely to be involved in the phenylpropanoid pathway.

• Asian Pacific Journal of Cancer Prevention
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• 제13권12호
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• pp.6191-6196
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• 2012

Aims and Background: Traditional chemotherapy strategies for human leukemia commonly use drugs based on cytotoxicity to eradicate cancer cells. One predicament is that substantial damage to normal tissues is likely to occur in the course of standard treatments. Obviously, it is urgent to explore therapies that can effectively eliminate malignant cells without affecting normal cells. Our previous studies indicated that ginsenoside $Rg_1$ ($Rg_1$), a major active pharmacological ingredient of ginseng, could delay normal hematopoietic stem cell senescence. However, whether $Rg_1$ can induce cancer cell senescence is still unclear. Methods: In the current study, human leukemia K562 cells were subjected to $Rg_1$ exposure. The optimal drug concentration and duration with K562 cells was obtained by MTT colorimetric test. Effects of $Rg_1$ on cell cycle were analyzed using flow cytometry and by SA-${\beta}$-Gal staining. Colony-forming ability was measured by colony-assay. Telomere lengths were assessed by Southern blotting and expression of senescence-associated proteins P21, P16 and RB by Western blotting. Ultrastructural morphology changes were observed by transmission electron microscopy. Results: K562 cells demonstrated a maximum proliferation inhibition rate with an $Rg_1$ concentration of $20{\mu}\;mol{\cdot}L^{-1}$ for 48h, the cells exhibiting dramatic morphological alterations including an enlarged and flat cellular morphology, larger mitochondria and increased number of lysosomes. Senescence associated-${\beta}$-galactosidase (SA-${\beta}$-Gal) activity was increased. K562 cells also had decreased ability for colony formation, and shortened telomere length as well as reduction of proliferating potential and arrestin $G_2$/M phase after $Rg_1$ interaction. The senescence associated proteins P21, P16 and RB were significantly up-regulated. Conclusion: Ginsenoside $Rg_1$ can induce a state of senescence in human leukemia K562 cells, which is associated with p21-Rb and p16-Rb pathways.

• 한국가축번식학회지
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• 제27권2호
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• pp.179-185
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• 2003

본 연구는 생체 내 세포 및 조직배양기로서의 면역결핍동물을 개발할 목적으로 proximal Ick promoter와 DT-A유전자를 이용하여 형질전환생쥐를 생산하였고 형질전환생쥐의 면역기관에서 Di-phteria toxin이 발현되어 T-cell이 결핍되는지를 분석하였다. 총 암수 2마리의 형질전환생쥐를 PCR과 South-ern blotting으로 분석하여 얻었으며 이식유전자의 copy수는 약 2∼3 copy가 정착된 것으로 확인되었다. 형질전환생쥐의 thymus, spleen, liver 조직을 분리한 후 total RNA를 추출하여 poly(dT) primer 와 DT 특이적 primer를 이용하여 RT-PCR수행 결과 형질전환생쥐의 thymus, spleen, liver에서 DT gene이 발현되고 있는 것을 확인할 수 있었다. 형질전환생쥐의 이들 조직간에 DT 발현량에는 큰차이는 없는 것으로 확인되었다. 형질전환생쥐의 혈액에서 적혈구, 백혈구 ,혈소판, 헤모글로빈 등이 정상생쥐보다 감소되었고 특히 백혈구수와 혈소판의 수가 크게 감소되어 있는 것으로 나타났다. 또한 형질전환생쥐의 혈액을 CD3 antibody를 이용하여 FACS 분석을 실시하여 형질전환생쥐의 혈액 중 T-cell이 수가 비정상적으로 줄어든 것을 확인할 수 있었다. 본 연구에서는 Ick-DT 형질전환생쥐에서 DT유전자의 발현에 의한 T-cell 결핍을 유도할 수 있는 것으로 나타나 이를 바탕으로 돼지를 이용한 사람의 이종장기 배양용 형질전환동물을 생산하여 응용될 수 있을 것으로 사료된다.