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Pseudomonas sp. Strain DJ77에 존재하는 Glutathione S-Transferase 아미노 말단잔기의 Site-directed Mutagenesis
우희종,박용춘,김성재,정용제,정안식,김영창 한국산업미생물학회 1997 한국미생물·생명공학회지 Vol.25 No.4
Pseudomonas sp. DJ77로부터 분리된 glutathione S-transferase(GST)의 N-말단 아미노산 서열은 MKLFISPGACSL로 전체 아미노산 서열의 상동성은 E. coli와는 45%, Haemophilus influenzae와는 20%로 알려져 있다. N-말단의 tyrosine 잔기는 박테리아 뿐 아니라 알려진 모든 세포성 GSTs에 존재하며 포유동물의 α, μ, π유형의 GSTs에서는 기능적인 활성자리라고 알려져 있다. 이런 점 때문에 Pseudomonas sp. DJ77의 GST에서 N-말단에 tyrosine 대신에 Phe-4와 Ile-5가 존재하는 것은 매우 특이한 일이다. Pseudomonas sp. DJ77 GST의 반응기작을 이해하기 위한 한 방법으로 site-directed mutagenesis를 이용하여 이 두 아미노산들을 각각 tyrosine으로 바꾸었다. 이 두 돌연변이주의 CDNB에 대한 효소 활성은 야생형와 비교해 거의 비슷한 활성을 나타내었고 in vivo에서의 CDNB에 의한 성장 저해 분석을 통해서도 같은 결과를 얻을 수 있었다. 이러한 결과들은 Pseudomonas sp. DJ77 GST가 적어도 포유류의 GST와는 다른 반응기작을 가진다는 것을 의미하는 것이다. Glutathione S-transferase (GST) was purified from Pseudomonas sp. DJ77, and its N-terminal sequence was determined to be MKLFISPGACSL. A specific tyrosyl residue in the vicinity of the N terminus is conserved in all the known cytosolic GSTs and has been shown to function as a catalytic residue in α, μ, π class GSTs from mammals. However, Pseudomonas sp. DJ77 GST has the Phe-4 and Ile-5 instead of Tyr in N-terminus. Its replacement with tyrosine did not significantly affect the enzyme activity. Results from in vitro biochemical analyses were confirmed by the in vivo activity-based CDNB growth inhibition analyses. Our results clearly indicate that GST of Psedomonas sp. DJ77 has a novel reaction mechanism different from that of mammalian GSTs.
우희종,신공식,이기종,권순종,조용구,서석철 한국식물생명공학회 2010 JOURNAL OF PLANT BIOTECHNOLOGY Vol.37 No.2
Selectable marker gene systems are vital for the development of transgenic plants, but the presence of selectable marker genes encoding antibiotic or herbicide resistance in genetically modified plants poses a number of problems. A lot of research results and various techniques have been developed to produce marker-free GM plants. The aim of this review is to describe the principal methods used for eliminating selectable marker genes to generate markerfree GM plants, concentrating on the three significant methods (co-transformation, site-specific recombinase-mediated excision,non-selected transformation) in several marker-free techniques.
우희종,신공식,이기종,권순종,조용구,서석철,Woo, Hee-Jong,Shin, Kong-Sik,Lee, Ki-Jong,Kweon, Soon-Jong,Cho, Yong-Gu,Suh, Seok-Cheol 한국식물생명공학회 2010 식물생명공학회지 Vol.37 No.2
Selectable marker gene systems are vital for the development of transgenic plants, but the presence of selectable marker genes encoding antibiotic or herbicide resistance in genetically modified plants poses a number of problems. A lot of research results and various techniques have been developed to produce marker-free GM plants. The aim of this review is to describe the principal methods used for eliminating selectable marker genes to generate marker-free GM plants, concentrating on the three significant methods(co-transformation, site-specific recombinase-mediated excision, non-selected transformation) in several marker-free techniques.
우희종,이승범,Yang Qin,임명호,이진형,신공식,조현석,박순기 한국식물생명공학회 2015 Plant biotechnology reports Vol.9 No.6
Public concern about the safety of genetically modified (GM) crops and potential pleiotropic effects of selectable marker gene insertion and expression can be avoided by developing a selectable marker-free transformation system. Various techniques have been developed to produce marker-free transgenic plants; however, an approach that does not require the use of selectable marker genes would not require post-transformation processes. We report such a method for generating of GM rice plants. To develop transgenic rice resistant to lepidopteran insects, a marker-free binary vector harboring the Bacillus thuringiensis mcyr1Ac gene was constructed and used for Agrobacterium tumefaciens-mediated transformation of rice scutellar calli. Initial screening of putative T0 transgenic rice plants was performed using polymerase chain reaction (PCR) on pools of DNA from 15 to 30 regenerated shoots, followed by genomic DNA PCR and Cry1Ac ImmunoStrip assays of individual plant extracts. Although the overall rice transformation efficiency of the non-selection approach is lower than that of traditional rice transformation by using marker genes, we derived six independent homozygous T3 events from Cry+ T0 shoots. Transgenes were detected and inheritance confirmed using DNA PCR, RT-PCR, and Southern blot analysis, and insertion sites were characterized by the sequencing of DNA flanking the transgenes. The transgene expression of cry1Ac was also stable and effective against rice leaf folder at levels comparable to those of a transgenic plant that had been derived by selection. This method could be applied to other plant species with similar transformation efficiencies to create selectable-marker-free transgenic plants for research and commercial uses.
우희종,정찬미,신공식,지현소,이기종,서석철,권순종,조용구 한국식물생명공학회 2009 식물생명공학회지 Vol.36 No.2
Genetic components introduced into approved GM crops are a key subject for safety assessment and provide a basis for the development of detection methods for GM crops. In order to understand the genetic components in approved GM crops comprehensively, we screened the genetic vector maps of GM crops that had been approved for commercialization around the world. A total of 64 varieties from 5 major GM crop species (maize, canola, cotton, soybean, and tomato) were subjected to analysis. The genetic components included genes, promoters, terminators, and selection marker. This survey may be useful for researchers who develop GM crops and methods for detecting GM crops.
우희종,이승범,임명호,권순종,이진형,신공식,조현석 한국식물생명공학회 2013 식물생명공학회지 Vol.40 No.3
A less simple approach developed for generation of marker-free transgenic plants is to select transformants without the use of selective marker genes. Some results about development of marker-free transgenic plants were obtained using a non-selective approach in several crops such as rice, potato and tobacco. However, the study did not provide evidence on detailed characterization of introduced gene on genome, a critical step for confirming the stable integration and transmission of a foreign gene. In this study, we evaluated structure and integration sites of transgene (mCry1Ac) in the transgenic Bt rice plants which were made via conventional Agrobacterium-mediated transformation by non-selective method. Structure and integration sites of transgene in these transgenic plants had similar fashion as those recovered under selection.