Incorportation of Foreign Gene with Ti Plasmid Vector System: (I) Introduction of E. coli Thioredoxin Gene into A. tumefaciens.

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Soon-Joo,Kim, Seong-Wan,Lim, Chang-Jin,kim, Young-Myeong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

외래 유전자를 도입함으로써 식물체에 새로운 유전정보를 부여하려는 노력이 다각적으로 수행되어 왔으며 최근에는 넓은 숙주범위를 갖는 Ti plasmid를 vector로 이용한 외래 유전자의 도입이 활발히 진행되고 있다. 본 연구에서는 Ti plasmid vector의 일종인 pGA658을 이용하여 광합성 조절 등 여러가지 기능을 갖고 있는 것으로 알려져 있는 thioredoxin 유전자를 식물체내로 도입하여 식물체내에서의 그 역할을 조사할 목적으로 DNA 재조합을 시도하였다. E. coli thioredoxin 유전자를 함유하는 pCJF 4의 Hind III-BamHI DNA fragment를 pGA658의 Hind III-Bgl II site에 삽입시키고 E. coli에 transformation 한 후 형질전환체의 확인은 항생물질 marker에 대한 저항성으로, 재조합 DNA의 유전자 지도는 여러가지 제한효소를 이용한 절편의 크기 확인으로, 그리고 도입 유전자의 발현은 효소 활성 측정에 의 하여 확인하였다. 이렇게 확인된 재조합된 plasmid pKDB3를 담배세포로 도입하기 위한 전 단계로서 freeze-thaw 방법으로 Agrobacterium에 transformation 한 후 항생물질 저항성과 제한효소를 이용한 절편의 크기 확인 및 효소 활성 측정에 의하여 pKDB3가 도입되어 안정하게 유지됨을 확인할 수 있었다. In this part of study on the incorporation of foreign gene into plant cells, a derivative of Ti plasmid vector (pGA658), containing E. coli thioredoxin gene, was prepared and introduced into Agrobacterium tumefaciens. A recombinant plasmid, pKDB3, was constructed by transferring HindlII-BamHI DNA fragment of pCJF4, including E. coli thioredoxin gene, into HindIII-BglII restriction sites of plasmid pGA658. By doing this, E. coli thioredoxin gene is expected to express from nos promoter of pGA658 after the incorporation into plant cells. The structure of DNAs isolated from kanamycin-resistant E. coli transformants was convinced by restriction mapping. As a preceding step before incorporation into plant cells, the recombinant plasmid pKDB3 was transformed into A. tumefaciens by freeze-thaw procedure. In Agrobacterium transformants, the expression of E. coli thioredoxin gene was positively observed, and this suggested the stable existence of the E. coli gene.

Incorporation of Foreign Gene with Ti Plasmid Vector System: (II) Expression of E. coli Thioredoxin Gene in Cultured Tobacco Cells.

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Soon-Joo,Kim, Seong-Wan,Lim, Chang-Jin,Kim, Young-Myeong 생화학분자생물학회 1988 한국생화학회지 Vol.21 No.4

본 연구에서는 E. coli thioredoxin 유전자를 함유하고 있는 재조합된 plasmid pKDB3의 담배세포내로의 도입을 시도하고, 도입된 thioredoxin 유전자의 발현을 조사하였다. 담배(Nicotina tabacum cv Xanthi) 세포로의 재조합 plasmid pKDB3의 도입은 담배잎 절편과 재조합 DNA 및 helper Ti plasmid pTiBo 542를 함유하고 있는 Agrobacterium tumefaciens strain A281과의 cocultivation 방법을 이용하여 행하여졌으며, 형질전환된 담배세포는 항생물질 저항성 배지에서의 callus 형성 여부로 선별되었고, 선별된 형질전환된 calli는 shoot와 root 형성을 위해 적절한 MS agar 배지에서 계속 키워졌다. 이와 같이 형질전환된 담배세포에서 완전한 식물체로 재생된 담배잎에서의 E. coli thioredoxin 유전자의 발현을 조사한 결과 thioredoxin 활성이 형질전환된 담배세포가 정상세포에 비해 9배 정도 더 큰 것으로 나타났다. 이러한 일련의 결과들은 E. coli thioredoxin 유전자가 성공적으로 담배세포에 들어가서 높은 수준으로 발현됨을 보여주고 있다. This study was performed to observe the expression of E. coli thioredoxin gene incorporated in tobacco cells. The recombinant DNA used, pKDB3, had been constructed from a Ti plasmid vector pGA658 and a bacterial plasmid pCJF4 harboring E. coli thioredoxin gene, as described in the preceding paper (Lee et al., 1988). The leaf discs of plant (N. tabacum cv Xanthi) were transformed to kanamycin resistance by the cocultivation with Agrobacterium A281 containing plasmid pKDB3. Transformed leaf discs were cultured in MS agar medium with kanamycin for callus induction. Kanamycin-resistant tobacco calli were continuously grown in MS agar medium for shoot induction, and then in MS agar medium for root induction. Expression of E. coli thioredoxin gene in the plant tissue regenerated from transformed tobacco cells was confirmed by DTNB assay. The thioredoxin activity of transformed tobacco cells was much higher (about 9 times) than that of normal tobacco cells. Our results suggest that E. coli thioredoxin gene was successfully incorporated into tobacco cells, and the incorporated bacterial gene could be expressed at a high level.

Ti Plamid Vector System 을 이용한 외래 유전자의 도입 : ( 2 ) E . coli thioredoxin 유전자의 배양된 담배세포내 발현

이희봉,주충노,홍순주,김성완,임창진,김영명 ( Hee Bong Lee,Chung No Joo,Soon Joo Hong,Seong Wan Kim,Chang Jin Lim,Young Myeong Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

This study was performed to observe the expression of E. coli thioredoxin gene incorporated in tobacco cells. The recombinant DNA used, pKDB3, had been constructed from a Ti plasmid vector pGA658 and a bacterial plasmid pCJF4 harboring E. coli thioredoxin gene, as described in the preceding paper (Lee et al., 1988). The leaf discs of plant (N. tabacum cv Xanthi) were transformed to kanamycin resistance by the cocultivation with Agrobacterium A281 containing plasmid pKDB3. Transformed leaf discs were cultured in MS agar medium with kanamycin for callus induction. Kanamycin-resistant tobacco calli were continuously grown in MS agar medium for shoot induction, and then in MS agar medium for root induction. Expression of E. coli thioredoxin gene in the plant tissue regenerated from transformed tobacco cells was confirmed by DTNB assay. The thioredoxin activity of transformed tobacco cells was much higher (about 9 times) than that of normal tobacco cells. Our results suggest that E. coli thioredoxin gene was successfully incorporated into tobacco cells, and the incorporated bacterial gene could be expressed at a high level.

Ti plasmid vector 를 이용한 진핵세포 유전자의 도입에 관한 연구

이희봉,주충노,홍순주,김성완,임창진,김영명 ( Hee Bong Lee,Chung No Joo,Sun Joo Hong,Seong Wan Kim,Chang Jin Lim,Young Myeong Kim ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.1

DNA recombination using plasmid pGA658, a Ti plasmid vector, was tried to study the role of yeast homoserine dehydrogenase after introducing its gene into plant cells. Two recombinant plasmids with opposite orientation of the gene DNA under nos promoter of vector pGA658, pKDB1 and pKDB2, were constructed by the aid of two intermediate vectors, pUC7 and pUC119. Transformed E. coli and Agrobacterium tumefaciens were confirmed by the resistance to appropriate antibiotics and by sizing DNA restriction fragments after plasmid isolation. Assay of homoserine dehydrogenase activity showed that a little increase in its activity was detected in transformed E. coli. but no increase in transformed Agrobacterium tumefaciens, compared with its untransformed strain.

The Incorporation of Eucaryotic Gene Using Ti Plasmid Vector: I. The Introduction of Yeast Homoserine Dehydrogenase Gene into Agrobacterium tumefaciens

이희봉,주충노,홍순주,김성완,임창진,김영명,Lee, Hee-Bong,Joo, Chung-No,Hong, Sun-Joo,Kim, Seong-Wan,Lim, Chang-Jin,Kim, Young-Myeong 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.1

효모에 있어서 threonine과 methionine 생합성에 관여하는 효소들 중 하나인 homoserine dehydrogenase가 식물체내로 도입된 후, 그 역할을 조사하고자 Ti plasmid에서 유도된 pGA658 vector와 homoserine dehydrogenase 유전자 함유 DNA의 재조합을 시도하였다. 그 결과 pGA658 vector의 nos promoter 다음에 효모의 homoserine dehydrogenase 유전자를 함유한 DNA 절편이 방향이 반대로 삽입된 재조합 plasmids가 성공적으로 만들어졌다. 형질전환된 E. coli와 Agrovbacterium tumefaciens는 적절한 항생물질 함유배지에서 저항성을 나타내었으며, 그들의 plasmid를 분리하여 여러가지 제한효소를 이용한 크기를 조사해 보니, 예상되는 크기와 일치하는 것을 관찰할 수 있었다. 또한 형질전환된 E. coli와 A. tumefaciens내에서의 yeast homoserine dehydrogenase 유전자 발현을 조사하기 위해 그 효소의 활성을 조사해 본 결과, 형질전환된 E. coli에서는 대조군에 비해 약간의 증가를 나타내었으나, 형질전환된 A. tumefaciens에서는 변화가 없었다. DNA recombination using plasmid pGA658, a Ti plasmid vector, was tried to study the role of yeast homoserine dehydrogenase after introducing its gene into plant cells. Two recombinant plasmids with opposite orientation of the gene DNA under nos promoter of vector pGA658, pKDB1 and pKDB2, were constructed by the aid of two intermediate vectors, pUC7 and pUC119. Transformed E. coli and Agrobacterium tumefaciens were confirmed by the resistance to appropriate antibiotics and by sizing DNA restriction fragments after plasmid isolation. Assay of homoserine dehydrogenase activity showed that a little increase in its activity was detected in transformed E. coli, but no increase in transformed Agrobacterium tumefaciens, compared with its untransformed strain.

Ti Plasmid Vector System 을 이용한 외래 유전자의 도입 : ( 1 ) A . tumefaciens 로의 E . coli Thioredonxin 유전자의 도입

이희봉,주충노,홍순주,김성완,임창진,김영명 ( Hee Bong Lee,Chung No Joo,Soon Joo Hong Seong Wan Kim,Chang Jin Lim,Young Myeong Kim ) 생화학분자생물학회 1988 BMB Reports Vol.21 No.4

In this part of study on the incorporation of foreign gene into plant cells, a derivative of Ti plasmid vector (pGA658), containing E. coli thioredoxin gene, was prepared and introduced into Agrobacterium tumefaciens. A recombinant plasmid, pKDB3, was constructed by transferring HindIII-BamHI DNA fragment of pCJF4, including E. coli thioredoxin gene, into HindIII-BgIII restriction sites of plasmid pGA658. By doing this, E. coli thioredoxin gene is expected to express from nos promoter of pGA658 after the incorporation into plant cells. The structure of DNAs isolated from kanamycin-resistant E. coli transformants was convinced by restriction mapping. As a preceding step before incorporation into plant cells, the recombinant plasmid pKDB3 was transformed into A. tumefaciens by freeze-thaw procedure. In Agrobacterium transformants, the expression of E. coli thioredoxin gene was positively observed, and this suggested the stable existence of the E. coli gene.

Comparative Analysis on the Two Translational Initiation Sites of Escherichia coli Thioredoxin Gene : Enhanced Expression from the Minor Translational Initiation Site

임창진,김영덕,임은주,홍순주,백융기,Lim, Chang-Jin,Kim, Young-Deug,Lim, Eun-Joo,Hong, Sun-Joo,Paik, Young-Ki 생화학분자생물학회 1992 한국생화학회지 Vol.25 No.3

대장균 치오레독신 유전자의 mRNA는 두 개의 강력한 번역시작 부위를 가지고 있는데 그 중 첫번째 것은 잘 알려진 치오레독신보다 19개의 아미노산이 더 긴 단백질을 생성한다. 이전 연구에서 두번째 시작암호를 제거하여 첫번째 시작암호에 의한 번역을 확실하게 증명하였으나 그 발현 정도는 매우 낮았다. 이번 실험에서 우리는 첫번째 시작암호와 그것의 가상적인 Shine-Dalgarno(SD)의 위치 사이에 두 개의 염기를 첨가하여 SD와의 거리를 4개 염기로 만들었다. 첨가된 유전자의 조절부위를 $\beta$-유당분해효소 구조유전자와 결합시킨 뒤 $\beta$-유당분해효소의 활성측정으로서 연장된 경우의 효과를 알아 보았는데 첫번째 시작암호에 의한 번역시작이 현저하게 증가하는 것을 관찰하였다. 또한 연장된 치오레독신의 생성 증가의 효과를 여러 측면에서 조사하여 보았다. An E. coli trxA mRNA contains two potential translational initiations in the nucleotide sequence, the first one of which could initiate synthesis of a protein 19 amino acids longer than the known trxA gene product. Previously, we showed with the removal of the second ATG codon that the translation from the first ATG codon was apparently occurred, but on a very low level. In the present study, we made the insertion of two nucleotides between the first ATG codon and its presumed Shine-Dalgarno (SD) sequence, which resulted in 4 nucleotides on the distance to the SD sequence. When the effect of the extension was measured thru $\beta$-galactosidase activity after translational fusion into $\beta$-galactosidase gene, it significantly enhanced the translation for the first ATG codon. Additionally, the increased production of the extended thioredoxin was in vivo characterized in the various aspects.

대장균 치오레독신 유전자의 두 번역 시작 부위에 관한 비교분석 : 부수적 번역 시작 부위에서의 발현 증강

임창진,김영덕,임은주,홍순주,백융기 ( Chang Jin Lim,Young Deug Kim,Eun Joo Lim,Sun Joo Hong,Young Ki Paik ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.3

An E. coli trxA mRNA contains two potential translational initiations in the nucleotide sequence, the first one of which could initiate synthesis of a protein 19 amino acids longer than the known trxA gene product. Previously, we showed with the removal of the second ATG codon that the translation from the first ATG codon was apparently occurred, but on a very low level. In the present study, we made the insertion of two nucleotides between the first ATG codon and its presumed Shine-Dalgarno (SD) sequence, which resulted in 4 nucleotides on the distance to the SD sequence. When the effect of the extension was measured thru β-galactosidase activity after translational fusion into β-galactosidase gene, it significantly enhanced the translation for the first ATG codon. Additionally, the increased production of the extended thioredoxin was in vivo characterized in the various aspects.

대장균 변이형 치오레독신의 성질

임창진,최성애,백혜승,이희봉,홍순주 ( Chang Jin Lim,Sung Ae Choi,Hye Seung Back,Hee Bong Lee,Young Ki Paik,Sun Joo Hong 생화학분자생물학회 1993 BMB Reports Vol.26 No.1

The existence of E. coli extended thioredoxin, which contains 19 amino acid residues longer than the well-known thioredoxin of 108 amino acid residues, has previously been confirmed by site-directed mutagenesis. An initiation mutant of thioredoxin gene (trxAE) gives the production of a mutant extended thioredoxin, because the second ATG codon (for methionine) was converted to CTG (for leucine). In order to purify the mutant extended thioredoxin, the protein was labeled with ^35S-methionine using T7 RNA polymerase/promoter system, and traced by radioactivity through purification steps such as ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. The purified mutant extended thioredoxin was characterized by the affinity for E. coli thioredoxin reductase, insulin reduction, and heat stability.

Construction of an E. coli Initiation-mutant Thioredoxin Gene by Site-directed Mutagenesis: An Alternative Translational Initiation for E. coli trxA Gene

임창진,임은주,김영덕,이동권,홍순주,Lim, Chang-Jin,Lim, Eun-Joo,Kim, Young-Deug,Rhee, Dong-Kwon,Hong, Sun-Joo 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.5

대장균 치오레독신 유전자에 대한 mRNA는 두 개의 잠재적인 번역시작 부위를 갖고 있는데, 그 중 하나는 잘 알려진 대장균 치오레독신보다 19개 아미노산이 더 많은 단백질을 합성할 수 있다. 하지만, 이 연장된 치오레독신의 존재가 아직까지는 밝혀진 바 없다. 두 시작 부위로부터의 translation을 분리 확인할 목적으로 두번째 ATG codon을 site-directed mutagenesis에 의하여 CTG로 바꾸었다. 제조된 번역시작 돌연변이체는 단지 연장된 치오레독신만을 생성하여, 대장균 치오레독신 유전자 mRNA의 또다른 번역시작을 입증하였다. 그 결과 존재가 증명된 연장된 치오레독신의 세포내 특성을 여러 측면에서 조사하였다. 이미 알려져 있는 치오레독신과 마찬가지로, 연장된 치오레독신은 T7 DNA polymerase의 한 subunit로 작용하였다. 그러나, methionine sulfoxide reductase의 cofactor로는 사용되지 않았다. The mRNA produced from the E. coli thioredoxin gene (trxA) contains two potential translational initiation sites, one of which could initiate the synthesis of a protein 19 amino acids longer than the E. coli well-known thioredoxin of 108 amino acid residues. However, the expression of extended thioredoxin was not yet confirmed. To dissect translations from two initiation codons, the second ATG codon was converted to CTG by site-directed mutagenesis. The initiation-mutant thioredoxin gene lacking the second ATG codon could produce only the extended thioredoxin, which prove another translational initiation from the first ATG codon of an E. coli trxA mRNA. In vivo characteristics of the extended thioredoxin were examined in the several aspects. Just like the well-known thioredoxin, the extended thioredoxin is able to serve as a subunit of T7 DNA polymerase. In contrast, it doesn't act as a cofactor for methionine sulfoxide reductase.