http://chineseinput.net/에서 pinyin(병음)방식으로 중국어를 변환할 수 있습니다.
변환된 중국어를 복사하여 사용하시면 됩니다.
Reconstitution of Artificially Designed inaz Gene and Its Expression in vivo
BAHK, JEONG DONG,CHO, MOO JE,LEE, DAE SIL 경상대학교 유전공학연구소 1992 遺傳工學硏究所報 Vol.11 No.-
In order to express the artifically designed inaz gene, which was deduced from the InaZ protein of Pseudomonas syringae, in Escherichia coli, a fused protein was planned. One unit of the fused protein consists of N-term. 50 amino acids of LacZ protein of E. coli and one unit of the repeated 48 amino acids from the core domain of InaZ protein of Ps. syringae. Each of them consists of eight synthetic DNA fragments. All of them were phosphorylated, block-ligated and reconstituted. Theresfter, the reconstituted DNA stretah was inserted into the plasmid vector pASI containing a P_L promoter of bacteriophage lambda. The recombinant plasmid pASLF was transformed to an expression host, E. coli N4839-1. After performing expression under a permissible or impermissible temperature, the cultures were applied to the buffer gradient gel and ckecked. At the near of 12-13 kD region a clear thick band was observed to be the fused INA protein.
Baek, Dong-won,Nam, Jae-sung,Koo, Yoon-Duck,Kim, Doh-Hoon,Lee, Ji-young,Jeong, Jae-Cheol,Kwak, Sang-Soo,Chung, Woo-Sik,Lim, Chae-Oh,Bahk, Jeong-Dong,Hong, Jong-Chan,Lee, Sang-Yeol,Maki Kawai-Yamada,Hi Plant molecular biology and biotechnology research 2004 Plant molecular biology and biotechnology research Vol.2004 No.-
An Arabidopsis protoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsis mesophyll protoplasts expressing murine Bax cDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivo targeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N-acetyl-L-cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of Arabidopsis Bax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together. our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.
Moon, Hae-Jeong,Lee, Bo-Young,Choi, Giltsu,Shin, Dong-Jin,D. Theertha Prasad,Lee, Ok-Sun,Kwak, Sang-Soo,Kim, Doh-Hoon,Nam, Jae-Sung,Bahk, Jeong-Dong,Hong, Jong-Chan,Lee, Sang-Yeol,Cho, Moo-Je,Lim, Cha Plant molecular biology and biotechnology research 2003 Plant molecular biology and biotechnology research Vol.2003 No.-
NDP kinases (NDPKs) are multifunctional proteins that regulate a variety of eukaryotic cellular activities, including cell proliferation, development, and differentiation. However, much less is known about the functional significance of NDPKs in plants. We show here that NDPK is associated with H_(2)O_(2)-mediated mitogen-activated protein kinase signaling in plants. H_(2)O_(2)stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Proteins from transgenic plants overexpressing AtNDPK2 showed high levels of autophosphorylation and NDPK activity, and they have lower levels of reactive oxygen species (ROS) than wild-type plants. Mutants lacking AtNDPK2 had higher levels of ROS than wild type. H_(2)O_(2) treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H_(2)O_(2)-activated A. thaliana mitogen-activated protein kinses. In the absence of H_(2)O_(2) treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the myelin basic protein phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a previously uncharacterized regulatory role in H_(2)O_(2)-mediated MAPK signaling in plants.
Moon, Hae-Jeong,Baek, Dong-Won,Lee, Bo-Young,D. Theertha Prasad,Lee, Sang-Yeol,Cho, Moo-Je,Lim, Chae Oh,Choi, Myung-Suk,Bahk, Jeong-Dong,Kim, Myeong-Ok,Hong, Jong-Chan,Yun, Dae-Jin Plant molecular biology and biotechnology research 2002 Plant molecular biology and biotechnology research Vol.2002 No.-
Bax, a mammalian proapoptotic member of the Bcl-2 family, can induce cell death when expressed in yeast or plant cells. To identify plant Bax inhibitors, we cotransformed a soybean cDNA library and the Bax gene into yeast cells and screened for expressed genes that prevented Bax-induced apoptosis. From the Bax-inhibiting genes isolated, ascorbate peroxidase (aAPX) was selected for characterization. The transcription of aAPX in plants was specifically induced by oxidative stress. Moreover, overexpression of aAPX partially suppressed the H_(2)D_(2)-sensitive phenotype of yeast cytosolic catalase T (Δctt)- and thermosensitive phenotype of cytochrome c peroxidase (Δccp)-deleted mutant cells. Examination of reactive oxygen species (ROS) production using the fluorescence method of dihydrorhodamine 123 oxidation revealed that expression of Bax in yeast cells generated ROS, which was greatly reduced by coexpression with sAPX. Our results collectively suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Bax-induced cell death in yeast.
Jeong, Jin Yong,Cho, Moo Je,Bahk, Jeong Dong 慶尙大學校 기초과학연구소 1992 基礎科學硏究所報 Vol.8 No.-
대장균 플라스미드 p15A 유도체인 플라스미드 pACYC184의 복제 개시점 하류에 위치 한 159-nt 영역에서 하나의 단일 가닥 복제 개시(ssi)signal이 발견 되었다. 이 ssi signal은 돌연변이 M13파아지(M13Δlac182)를 이용한 plaque morphology assay를 이용하여 선별하였다. M13Δlac182는 상보 가닥 DNA를 합성하는 복제 개시 부위(ori_c)의 많은 부분이 결실 되었기 때문에 작고 탁한 plaque을 형성한다. 159-nt의 ssi 단편을 가지고 있는 재조합된 phage(M13Δlac182/pACYC184ssi)는 야생형 M13mp18 phage와 거의 같은 효율로 생육 하였다. 그러므로 159-nt 단편은 M13Δlac182의 결실된 복제 개시점으로 부터 18-nt 아래에 위치한다. 플라스미드pACY184에서 발견된 ssi signal은 다른 여러가지 플라스미드에서 발견된 ssi signal들의 DNA 염기배열과 높은 homology를 보여 주었다. 159-nt 단편 중에서 가능한 2차 구조를 그려볼 수 있었으며 이 2차 구조에서 몇몇 conserved sequences(n'단백질 인식 부위; dnaB, dnaC, 그리고 dnaG 의존형 복재 개시 signal ; primer RNA 개시 부위)가 발견되었다. A single-strand initiation(ssi) signal for phage DNA synthesis was identified in the 159-bp region of plasmid pACYC184, a derivative of plasmid p15A of Escherichia coli. The ssi signal was identified by using a plaque morphology assay with a mutant M13 phage(M13Δlac182) which forms small turbid plaques because it lacks the greater part of the origin of complementary DNA strand synthesis(ori_c). The recombinant phage (M13Δlac182/pACYC184ssi) carrying the 159-bp insertion grew as efficiently as wild-type M13mp 18 phage and thus this 159-bp DNA segment can recover the defect in phage replication of M13Δlac182. This region is located 18-nt downstream from p15A origin of DNA replication. The direction of chain elongation in DNA synthesis is opposite to that of the leading strand. The ssi signal of plasmid pACYC184 shows sequence homology to the ssi signals of some other plasmids. In this region, we found a potential stem and loop structure. Its stem region contains the consensus sequence, 5'-CGCTCGCCGCAT-3', known as dnaB, dnaC and dnaG protein-dependent initiation signal and 5'-GAAGCGG-3', known as an n' protein recognition site. Furthermore, trinucleotide sequence, 5'-CTG-3', known as a primer RNA initiation site, was also maintained.
Jeong, Jin-Yong,Seo, Hak-Soo,Kim, Ho-Yeon,Cho, Moo-Je,Bahk, Jeong-Dong Korean Society for Biochemistry and Molecular Biol 1995 Journal of biochemistry and molecular biology Vol.28 No.4
Using a mutant M13 phage derivative lacking a great part of the complementary strand synthesis origin, we identified six single-strand initiation (ssi) signals for DNA replication in pACYC184, pLG214, pGKV21, and pDPT270 plasmids, and named them $ssiA_{YC}$, $ssiA_{LG}$, $ssiB_{LG}$, $ssiA_{KV}$, $ssiA_{PT}$, and $ssiB_{PT}$, respectively. Two of them were from pDPT270, one from downstream the on of pACYC184, two from pLG214, one from upstream the plus origin of pGKV21. Introduction of these ssi signals into the deleted $ori_c$ site of a mutant filamentous M13 phage ($M13{\Delta}lac182$) resulted in the restoration of growth activity of this phage. These ssi signals were classified into a number of groups on the basis of sequence similarity. $ssiA_{YC}$ and $ssiA_{LG}$ show extensive sequence homology to the n'-site (primosome assembly sites) of ColE1, whereas $ssiB_{PT}$ is homologous to the n'-site of ${\Phi}X174$. $ssiA_{PT}$ belongs to G4-type ssi signals which require only dnaG primase and SSB protein for the priming of replication. In addition, possible biological roles of these ssi signals are discussed.
E . coli cryptic miniplasmid p 15A 에서 유래하는 플라스미드 pACYC 184 의 ssi 시그날에 관한 연구
박정동,Sakai, Hiroshi,Komano, Tohru 생화학분자생물학회 1993 BMB Reports Vol.17 No.3
Single-strand DNA initiation (ssi) signals are DNA requirements for initiation of DNA synthesis on the single-strand DNA templates. For primary screening of ssi signal function, we used plaque morphology method. Plasmid pACYCl84 derived from E. coli minicryptic plasmid p15A had only one ssi signal which consists of 119-nt stretch. This segment played critical roles in ss DNA phage growth activity and performed primase-dependent replication instead of RNA polymerase-dependent one. Three kinds of energetically stable stem and loop structures were expected. Especially, the orientation and location of this 119-nt stretch, when compared with those of the origin of p15A, favor that this ssi signal might be taken part in the lagging strand replication of plasmid pACYCl84.
A proteomic approach in analyzing heat-responsive proteins in rice leaves
Lee, Dong-Gi,Ahsan, Nagib,Lee, Sang-Hoon,Kang, Kyu Young,Bahk, Jeong Dong,Lee, In-Jung,Lee, Byung-Hyun WILEY-VCH 2007 Proteomics Vol. No.
<P>The present study investigated rice leaf proteome in response to heat stress. Rice seedlings were subjected to a temperature of 42°C and samples were collected 12 and 24 h after treatment. Increased relative ion leakage and lipid peroxidation suggested that oxidative stress frequently was generated in rice leaves exposed to high temperature. 2-DE, coupled with MS, was used to investigate and identify heat-responsive proteins in rice leaves. In order to identify the low-abundant proteins in leaves, samples were prefractionated by 15% PEG. The PEG supernatant and the pellet fraction samples were separated by 2-DE, and visualized by silver or CBB staining. Approximately 1000 protein spots were reproducibly detected on each gel, wherein 73 protein spots were differentially expressed at least at one time point. Of these differentially expressed proteins, a total of 34 and 39 protein spots were found in the PEG supernatant and pellet fractions, respectively. Using MALDI-TOF MS, a total of 48 proteins were identified. These proteins were categorized into classes related to heat shock proteins, energy and metabolism, redox homeostasis, and regulatory proteins. The results of the present study show that a group of low molecular small heat shock proteins (sHSPs) were newly induced by heat stress. Among these sHSPs, a low molecular weight mitochondrial (Mt) sHSP was validated further by Western blot analysis. Furthermore, four differentially accumulated proteins that correspond to antioxidant enzymes were analyzed at the mRNA level, which confirmed the differential gene expression levels, and revealed that transcription levels were not completely concomitant with translation. The identification of some novel proteins in the heat stress response provides new insights that can lead to a better understanding of the molecular basis of heat-sensitivity in plants.</P>