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Cloning and Expression of Escherichia coli Ornithine Transcarbamylase Gene, argI
류기중,유장걸,고영환,김찬식,송성준,오영선,이선주,Riu, Key-Zung,U, Zang-Kual,Ko, Young-Hwan,Kim, Chan-Shik,Song, Sung-Jun,Oh, Young-Seon,Lee, Sun-Joo 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.2
Escherichia coli의 오르니틴 트란스카바밀라제는 오르니틴과 카바밀인산으로부터 시트룰린의 합성을 촉진시키는 효소이다. 이 효소의 기능과 구조와의 상관관계, 반응메카니즘 등 생화학적 연구를 하기 위하여 대량의 효소를 추출할 필요가 있다. 본 연구는 오르니틴 트란스카바밀라제의 대량생산 시스템을 확립하기 위하여 E. coli argI 유전자를 E. coli $DH5{\alpha}$ 세포의 염색체 DNA를 추출한 후에 PCR 방법으로 증폭시켜 얻었다. 증폭된 argI 유전자를 단핵생물 단백질 발현벡터인 pKK223-3에 접합시킨 후, 오르니틴 트란스카바밀라제가 존재하지 않은 E. coli TB2 세포에 클로닝 시켰다. 이 세포로부터 생산된 오르니틴 트란스카바밀라제는 암모늄염에 의한 분할, 열변성, 크로마토그래피 등을 사용하여 순수하게 분리하였다. SDS 단백질 전기영동 결과 약 38 kDa 크기의 효소가 순수하게 얻어졌다. 반응속도론적 실험결과 $K_{cat}$은 $1{\times}10^5m^{-1}$, $K_M$은 오르니틴에 대하여는 0.35 mM, 카바밀인산에 관하여는 0.06 mM이 각각 얻어졌다. 이 결과는 야생형 오르니틴 트란스카바밀라제의 반응속도 인자들과 비슷한 값이다. 본 연구는 이들 결과로부터 오르니틴 트란스카바밀라제의 기능을 하는 E. coli argI 유전자가 클로닝 되었음을 확인하였다. Escherichia Coli ornithine transcarbamylase is the enzyme which catalyzes the L-citrulline biosynthesis from L-ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many biochemical studies such as structure and function relationships and catalytic mechanisms, the cloning and expression of E. coli argI gene for ornithine transcarbamylase was conducted. argI was amplified from genomic DNA of E. coli strain of $DH5{\alpha}$, by polymerization chain reaction (PCR) method. The amplified argI gene was ligated to the prokaryotic expression vector pKK223-3 and used for transformation of E. coli TB2 which was deficient of ornithine transcarbamylase. The over-produced enzyme by the tnansformant was purified by ammonium sulfate fractionation, heat denaturation and affinity chromatography. The result of SDS denaturation gel electrophoresis for the purified enzyme showed a single band of about 38 kDa of ornithine transcarbamylase. Kinetic data for the expressed enzyme gave almost the s?????? values as those of the wild type enzyme. The $k_{cat}$, of the enzyme was $1.0{\times}10^5min^{-1}$, and $K_ms$ for ornithine and carbamyl phosphate were 0.35 mM and 0.06 mM, respectively.
Agrobacterium rhizogenes 를 이용한 Populus tremuloides 의 형질전환
류기중(Key Zung Riu),소인섭(In Sup So),유장걸(Zang Kual U),고영환(Yong Hwan Ko),이선주(Sun Joo Lee),(Wesley P . Hackett) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.2
Several factors affecting on transformation efficiency were studied to establish a Agrobacterium rhizogenes mediated system for the transformation of Populus species, and We could obtaine tansgenic plantlets expressing the introduced gene. Leaf sections were more sensitive than stem sections to kanamycin and thought to be better material for transformant screening. The bacterial density did not affect on the efficiency of transformation over the range of 4×10^5∼7×10^9 cfu. The optimum period for co-cultivation was one day or shorter. Both of the optimum concentrations of cefotaxime and ampicillin in the medium were 250 ㎍/㎖ for elimination of bacteria from the inoculated leaf sections. The addition of acetosyringone in the bacterial culture medium increased transformation rate, and the highest rate was obtained at 50 μM of acetosyringone. The transformed galls could be selectively induced and gown on the growth regulator-free medium or on the medium containing 100 ㎍/㎖ or higher contrition of kanamycin. The roots were induced from the galls incited by A rhizogenes within 3 weeks on the growth regulator-free medium as well as on the medium containing growth regulators. The plantlets were regenerated from the galls cultured for 6 weeks on the medium containing 0.05 ㎎/㎖ of NAA and 0.5 ㎎/㎖ of BA. The expressions of the introduced opine gene in the transformed galls and plantlets were confirmed by the analysis of agropine and mannopine.
Escherichia coli 오르니틴 트란스카바밀라제의 유전자 argⅠ 의 클로닝 및 발현
류기중(Key Zung Riu),유장걸(Zang Kual U),고영환(Young Hwan Ko),김찬식(Chan Shik Kim),송성준(Sung Jun Song),오영선(Young Seon Oh),이선주(Sun Joo Lee) 한국응용생명화학회 1995 Applied Biological Chemistry (Appl Biol Chem) Vol.38 No.2
Escherichia Coli ornithine transcarbamylase is the enzyme which catalyzes the L-citrulline biosynthesis from L-ornithine and carbamyl phosphate. To facilitate the purification of enzyme which will be used for many biochemical studies such as structure and function relationships and catalytic mechanisms, the cloning and expression of E. coli argI gene for ornithine transcarbamylase was conducted. argI was amplified from genomic DNA of E. coli strain of DH5α, by polymerization chain reaction (PCR) method. The amplified argI gene was ligated to the prokaryotic expression vector pKK223-3 and used for transformation of E. coli TB2 which was deficient of ornithine transcarbamylase. The over-produced enzyme by the tnansformant was purified by ammonium sulfate fractionation, heat denaturation and affinity chromatography. The result of SDS denaturation gel electrophoresis for the purified enzyme showed a single band of about 38 kDa of ornithine transcarbamylase. Kinetic data for the expressed enzyme gave almost the scone values as those of the wild type enzyme. The k_(cat), of the enzyme was 1.0×10^5 min^(-1) and K_ms for ornithine and carbamyl phosphate were 0.35 mM and 0.06 mM, respectively.
아피오스(Apios americana M.) 도입 생산을 위한 기초 연구
강시용(Si-Yong Kang),류기중(key Zung Riu),강영길(Young Kil Kang),강봉균(Bong Kyoon Kang),김동섭(Dong Sub Kim),박인숙(In Sook Park),송희섭(Hi Sup Song) 한국자원식물학회 2005 한국자원식물학회지 Vol.18 No.3
Newly introduced two lines of apios (Apios americana Medikus, red-vine and green-vine) were grown in Jeju island, to clarify their growth and production characteristics as well as to develope as a new edible crops in Korea. Both lines bloomed but did not develop to pod and seed. The red-viny line showed the habit of more early growth and maturity compared with green-viny line. Fresh tuber yields per 10a harvested in late November ranged from about 500kg to 800kg as according to the lines and cultural condition. Fresh tuber yield of red-viny line was relatively greater than that of green-viny line, mainly due to their higher tuber number per plant. Among the planting dates(April 1, April 16 and May 1) of seed tubes, highest tuber yield was obtained on May 16 planting. And the stacking cultivation culture was better than non-stacking cultivation in respect of tuber yield and disease avoidance. These results indicate that apios can produce in Jeju island, and in order to extend its cultivation to farmers it will be needed to develope some cultivars with high yields as well as labor-saving cultivation methods.
제주산 식물을 이용한 Tyrosinase 억제 활성, Hyaluronidase 억제 활성, 라디칼 소거 활성 검색
이선주,정덕상,부희정,양홍철,류기중,이남호,Lee, Sun-Joo,Jung, Deok-Sang,Bu, Hee-Jeong,Yang, Hong-Chul,Riu, Key-Zung,Lee, Nam-Ho 한국생약학회 2001 생약학회지 Vol.32 No.3
Solvent extracts of 17 plants collected in Cheju Island were investigated for their biological properties related to cosmeceuticals such as tyrosinase and hyaluronidase inhibition and also radical scavenging effects. The chloroform fraction of Phytolacca esculenta root exhibited strong inhibition against tyrosinase activity. No fraction showed significant hyaluronidase inhibition. Some solvent extracts of plants such as Achyranthes japonica and Artemisia princeps showed considerable radical scavenging activities.
유장걸(Zang Kual U),류기중(Key Zung Riu),소인섭(In Sup So),홍경애(Kyung Ae Hong) 한국응용생명화학회 1993 Applied Biological Chemistry (Appl Biol Chem) Vol.36 No.6
The neomycin phosphotransferase II gene (nptII) was introduced into geranium (Pelargonium zonale hybrids) protoplast by using PEG or electroporation method. The presence of the introduced DNA in the protoplast and the expressions of the gene in the transformed cells were examined. The presence of the nptII DNA in the protoplasts were detected by polymerase chain reaction. The expressions of nptII gene in the transformed cells were confirmed by the nptII assay.
해녀콩(Canavalia lineata THUNB. DC.) 추출물의 멜라닌 생성 억제 효과
부희정 ( Hee-jung Bu ),류기중 ( Key-zung Riu ),이선주 ( Sunjoo Lee ) 대한화장품학회 2004 대한화장품학회지 Vol.30 No.4
피부에서 melanin은 자외선 차단의 주요한 역할을 한다. Tyrosinase는 멜라닌 생합성과정에서 초기 단계에 관여하는 중요한 효소로서 이것의 조절을 통한 피부 멜라닌화 억제에 관해 많은 연구가 되어져왔다. 본 연구에서는 해녀콩 추출물에서 mushroom tyrosinase 활성억제, B16F10 melanoma 세포를 이용한 dopa oxidase 활성억제 및 멜라닌 합성 억제 효과를 확인하였다. Tyrosinase mRNA 발현에서의 억제 효과를 확인하기 위하여 RT-PCR을 이용하였으며, CHCI<sub>3</sub> 층에서 분리해낸 A 분획에서 tyrosinase mRNA 발현을 억제시킴을 확인하였다. Melanin pigmentation in human skin is a major defensive mechanism against ultraviolet light of the sun. Tyrosinase plays a key role in the biosynthesis of melanin. This is why many researches have been focused on regulations in controlling the epidermal melanization. We found that extract of Canavalia lineata inhibits mushroom tyrosinase activity, dopa oxidase activity, and melanin synthesis in B16F10 melanoma cells. To elucidate mRNA level reverse transcription polymerase chain reaction (RT-PCR) technique was used. It was revealed that A subfraction of CHCI<sub>3</sub> extract of Canavalia lineara reduced the tyrosinase mRNA expression of B16F10 melanoma cells by reverse transcription polymerase chain reaction (RT-PCR) technique.
GMO 격리포장에서의 유전자변형 들잔디로부터 토착미생물로의 수평유전자전달 평가
배태웅,이효연,류기현,이태형,임평옥,윤필용,박신영,류기중,송필순,이용억,Bae, Tae-Wung,Lee, Hyo-Yeon,Ryu, Ki-Hyun,Lee, Tae-Hyeong,Lim, Pyung-Ok,Yoon, Pill-Yong,Park, Sin-Young,Riu, Key-Zung,Song, Pill-Soon,Lee, Yong-Eok 한국식물생명공학회 2007 식물생명공학회지 Vol.34 No.1
The release of genetically modified organisms ($GMO_{s}$) into the environment has the potential risks regarding the possibility of gene transfer from $GMO_{s}$ to natural organisms and this needs to be evaluated. This study was conducted to monitor the possible horizontal gene transfer from herbicide-resistant zoysiagrass (Zoysia japonica Steud.) to indigenous microorganisms. We have first examined the effect of field-released GM zoysiagrass on the microbial flora in the gut of locust (Locusts mlgratoria). The microbial flora was analyzed through determining the 165 rDHA sequences of microorganisms. The comparison of the microbial flora in the gut of locusts that were captured at the field of GM zoysiagrass and of wild-type revealed that there is no noticeable difference between these two groups. This result indicates that the GM zoysiagrass does not have negative impact on microbial flora in the gut of locust. We then investigated whether the horizontal gene transfer occurred from GM zoysiagrass to microbes in soil, rhizosphere and faecal pellets from locusts by utilizing molecular tools such as Southern hybridization and polymerase chain reaction (PCR). When the total DNAs isolated from microbes in GM zoysiagrass and in wild-type zoysiagrass fields were hybridized with probes for bar or hpt gene, no hybridization signal was detected from both field isolates, while the probes were hybridized with DNA from the positive control. Absence of these genes in the FNAs of soil microorganisms as well as microbes in the gut of locust was further confirmed by PCR. Taken together, our data showed that horizontal gene transfer did not occur in this system. These results further indicate that frequencies of transfer of engineered plant DNA to bacteria are likely to be negligible.