Increase of Yeast Survival under Oxidative Stress by the Expression of the Laccase Gene from Coprinellus congregatus

Dongsik Kim,Eunju Kwak,최형태 한국미생물학회 2006 The journal of microbiology Vol.44 No.6

Coprinellus congregatus secreted a laccase isozyme when the culture was transferred to an acidic liquid medium (pH 4.1). The laccase cDNA gene (clac2) was used as a probe for cloning of the genomic laccase gene (lac2) including the promoter (Plac2). The open reading frame (ORF) of lac2 had 526 deduced amino acids and four conserved copper binding domains as other fungal laccases. Recombinant plasmid (pRSlac2p-cDNA) of lac2 cDNA with its own promoter was transformed in Saccharomyces cerevisiae. Expression of the transformed lac2 gene was induced by oxidative stress (H2O2) in yeast and the survival rate of the transformed yeast strain was greatly increased when compared with that of the control strain transformed with pRS316 yeast vector.

Increase of Yeast Survival under Oxidative Stress by the Expression of the Laccase Gene from Coprinellus congregatus

Kim, Dong-Sik,Kwak, Eun-Jung,Choi, Hyoung-T. The Microbiological Society of Korea 2006 The journal of microbiology Vol.44 No.6

Coprinellus congregatus secreted a laccase isozyme when the culture was transferred to an acidic liquid medium (pH 4.1). The laccase cDNA gene (clac2) was used as a probe for cloning of the genomic laccase gene (lac2) including the promoter (Plac2). The open reading frame (ORF) of lac2 had 526 deduced amino acids and four conserved copper binding domains as other fungal laccases. Recombinant plasmid (pRSlac2p-cDNA) of lac2 cDNA with its own promoter was transformed in Saccharomyces cerevisiae. Expression of the transformed lac2 gene was induced by oxidative stress ($H_2O_2$) in yeast and the survival rate of the transformed yeast strain was greatly increased when compared with that of the control strain transformed with pRS316 yeast vector.

Expression of lac and gal operons in Zymomonas mobilis

CHO, DONG-WUK,ROGERS, PETER L.,DELANEY, STEPHEN F. 한국미생물 · 생명공학회 1994 Journal of microbiology and biotechnology Vol.4 No.2

Two Zymomonas mobilis strains (ZM63 and ZM6307), containing both lactose and galactose operons, were constructed. β-Galactosidase and galactokinase assays indicated that both operons were expressed in both strains. The transport systems available for lactose uptake by Zymomonas mobilis were investigated using ^14C-labelled lactose. After the outer membrane, which was considered to be a possible barrier to lactose uptake, was disrupted by treatment with EDTA and Ca^2+ ions, some increase in lactose uptake was observed in ZM6306 (lac^+) and ZM6307 (lac^+ gal^+), but not in the parent, ZM6. This suggested that the outer membrane of Zymomonas mobilis acts as a barrier to lactose uptake to some degree, and also that the lactose permease is operational in Zymomonas mobilis.

환경 오염물질의 진보된 독성 평가 기법

류재천,최윤정,김연정,김형태,방형애,송윤선 한국환경독성학회 1999 환경독성보건학회지 Vol.14 No.1

Recently, several new methods for the detection of genetic damages in vitro and in vivo based on molecular biological techniques were introduced according to the rapid progress in toxicology combined with cellular and molecular biology. Among these methods, mouse lymphoma thymidine kanase (tk) gene forward mutation assay, single cell gel electrophoresis (comet assay) and transgenic animal and cell line model as a target gene of lac I (Big Blue) and lac Z (Muta Mouse) gene mutation are newly introduced based on molecular toxicological approaches. The mouse lymphoma tk^(+/-) gene assay (MOLY) using L5178Y tk+i- mouse lymphoma cell line is one of the mammalian forward mutation assays, and has many advantages and more sensitive than hprt assay. The target gene of MOLY is a heterozygous tk^(+/-) gene located in 11 chromosome, so it is able to detect the wide range of genetic changes like point mutation, deletion, rearrangement, and mitotic recombination within tk gene or deletion of entire chromosome 11. The comet assay is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakages in mammalian cells. Also, transgenic animal and cell line models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease process, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Also in vivo acridine orange supravital staining micronucleus assay by using mouse peripheral reticulocytes was introduced as an alternative of bone marrow micronucleus assay. In this respect, there was an International workshop on genotoxicity procedure (IWGTP) supported by OECD and EMS (Environmental Mutagen Society) at Washington D, C, in March 25-26, 1999. The objective of IWGTP is to harmonize the testing procedures internationally, and to extend to finalization of OECD guideline, and to the agreement of new guidelines under the International Conference of Harmonization (ICH) for these methods mentioned above. Therefore, we introduce and review the principle, detailed procedure, and application of MOLY, comet assay, transgenic mutagenesis assay and supravital staining micronucleus assay.

Molecular Cloning and Expression of Genes Related to Antifungal Activities from Enterobacter sp.B54 Antagonistic to Phytophthora capsici

Yoon, Sang Hong 한국미생물 · 생명공학회 1999 Journal of microbiology and biotechnology Vol.9 No.3

Enterobacter sp. B54 inhibited growth of the fungus Phytophthora capsici on potato dextrose agar (PDA). Three mutants with antifungal activities (denoted M54-47, M54-113, and M54-329) which were lost or increased, through P1::Tn5 lac mutagenesis, were used to isolate genes responsible for fungal inhibition on PDA. Two clones were selected from the partially EcoR1-digested genomic library of the wild-type strain by probing with genomic flanking sequences of each mutant. We have isolated a 20-kb EcoR1 genomic DNA fragment from this strain that contains genes involved in hyphal growth inhibition of P capsici on PDA. Subcloning and expression analysis of the above DNA fragment identified a 8-kb region which was necessary for antifungal activities. A 8-kb HindⅢ DNA fragment covers three genomic loci inserted by Tn5 lac in each mutant. This suggested that all genes which are related to antifungal activities might be clustered in simple forms of at least 5-8kb sizes.

Glyphosate Toxicity: III. Detection of QB Protein in Thylakoid Membrane of Tomato Apical Meristem Using an Antibody Raised from Hybrid Protein of psb A and lac Z Gene

김태완,니콜라스 암라인,Kim, Tae-Wan,Amrhein, Nikolaus 한국잡초학회 1995 Weed&Turfgrass Science Vol.15 No.3

Glyphosate를 토마토의 동화산물 공급부위에 처리하였을 때, 제초제결합 단백질인 QB 단백질을 Escherichia coli 내에서 ${\alpha}$-galactosidase가 발현되기 위해 lac Z 유전자의 3' 말단에 cloning된 시금치 psb A 유전자에 의해 발현되는 hybrid 단백질에 대한 항체를 형성시킨 후 이것을 이용하여 immunoblotting을 실시하였다. G1yphosate는 thylakoid 막의 Photosystem II내에 있는 D1 단백질의 붕괴에 영향을 주었다. LHC II 복합체내의 D1 단백질의 기능 이상은 glyphosate 의 다면발현적 효과였다. Glyphosate(N-[phosphonomethyl]glycine) applied to the assimilate-exporting leaves(i.e. third old leaf) of tomato(Lycopersicon esculentum Mil var. Moneymaker). Herbicide binding protein, QB protein(D1), has been immunoblotted using the antibodies raised against the hybrid-protein expressed by a part of spinach psb A gene cloned in frame with the 3'end of lac Z gene to allow expression of the ${\beta}$-galactosidase(EC 3.21.23) in Escherichia coli. Glyphosate has an effect on a turnover of D1 within photosystem II of thylakoid membrane. The dysfunction of D1 protein within light harvesting complex(LHC-II) seems to be a pleiotropic effect of glyphosate.

Effects of Gene Expression of Photobacterium leiognathi CuZn Superoside Dismutase (PSOD) by lacZ Promotor Control under Oxidative Stress

Kim, Young-Gon The Microbiological Society of Korea 1992 미생물학회지 Vol.30 No.6

The effect of PSOD expression on lacZ-sodP fusion (pYK4) was explored in Escherichia coli sodA sodB mutants (QC774) under oxidative stress. In this system, although .betha.-galactosidase activity was not fully induced by isopropyl-1-thio-.betha.-galactosidase (IPTG) and was inhibited by glucose, functional PSOD was under lacZ promotor control and was induced by IPTC, lactose, PQ and copper isons, finally, the results show that higher PSOD expression leel was consistently importnat in defending against superoxide radicals.

土壤病害 拮抗性 Pseudomonas maltophilia B-14의 拮抗遺傳子探索

구본성,서영우,김용환,윤상홍,오상수,박경수,류진창,은무영 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.6

서로 다른 성접합형 A와 a를 가지는 Rhodosporidium toruloides가 생산하는 invertase를 조사한 결과, 접합형 A는 세포내, 세포벽 결합, 세포의 분비성 invertase의 3가지를 생산하였으나, 접합형 a는 세포외 invertase를 생산하지 않는 접합형 특이성이 있는 것을 알았다. 양 접합형 세포로부터 세포내 invertase를 부분정제하여 이들의 성질을 비교한 결과, 접합형 A세포 유래의 효소는 Zn^2+에 의하여 11%의 활성 상승효과와 Mn^2+에 의한 42.2%의 감소를 보이는 반면, a세포 유래의 효소는 Mn^2+에 의한 15%의 활성 감소만을 보였다. 효소 저해제의 효과에서 a세포의 invertase는 2-mercaptoethanol, sodium dodecyl sulfate, phenol에만 약간 저해를 받을 뿐이었다. 이들 양 효소의 열 안정성은 모두 80℃ 이상에서 급격히 실활되는 양상을 보였으며 최적 온도가 60℃로 둘 다 비슷하였다. a세포 유래의 invertase는 pH 3에서 pH 10까지 넓은 pH의 범위에서 안정하며 최적 pH가 4.0인 반면, A세포의 invertase는 pH 3에서 pH 6까지 산성의 조건에서만 안정하였으며 최적 pH는 5.0으로 나타났다. 그리고, A세포와 a세포 유래 invertase의 K_m값은 각각 2.5×10^-3 M과 3.O×10^-3 M이였다. This work was carried out to study physiological characteristics of Rhodosporidium toruloides cells having two different mating types. The mating type A produces internal, cell wall-bound, and external invertases while type a produces only two invertases except external invertase. Comparing their characteristics after partial purification of internal invertases from both mating type cells, invertase from type a has decreased 15% of invertase activity only by Mn^2+ while invertase from type A has been increased 11% of invertase activity by Zn^2+ and decreased 15% of invertase activity by Mn^2+. On the effect of enzyme inhibitor, invertase of type a was inhibited from 12% to 57% by 2-mercaptoethanol, sodium dodecyl sulfate, phenol, but invertase of type A was slightly inhibited only by phenol. The thermal stability of both invertases has showed steep inactivation at above 80℃ and their optimal temperatures were similar at 60℃ Invertase from type A showed stability only on condition of acid from pH 3 to 6 and its opimal pH was 5.0, while invertase from type a showed stability at the wide range of pH 3∼10 and its optimal pH was 4.0. And the K_m values of invertases from type A and type a were 2.5×10^-3 M and 3.4×10^-3 M, respectively.