• Journal of Microbiology and Biotechnology
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• 제17권2호
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• pp.234-243
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• 2007

Vibrio vulnificus is an opportunistic pathogen that causes septicemia in humans. To identify the genes associated with its pathogenicity, in vivo expression technology (IVET) was used to select genes specifically expressed in a host, yet not significantly in vitro. Random lacZ-fusions in the genome of V vulnificus strain MO6-24/O were constructed using an IVET vector, pSG3, which is a suicide vector containing promoterless-aph and -lacZ as reporter genes. A total of ${\sim}18,000$ resulting library clones were then intraperitoneally injected into BALB/c mice using a colony forming unit (CFU) of $1.6{\times}10^6$. Two hours after infection, kanamycin was administered at $200{mu}g$ per gram of mouse weight. After two selection cycles, 11 genes were eventually isolated, which were expressed only in the host. Among these genes, VV20781 and VV21007 exhibiting a homology to a hemagglutinin gene and tolC, respectively, were selected based on having the highest frequency. When compared to wild-type cells, mutants with lesions in these genes showed no difference in the rate of growth rate, yet a significant decrease in cytotoxicity and the capability to form a biofilm.

• Journal of Microbiology
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• 제33권2호
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• pp.120-125
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• 1995

.betha.-galactosidase activity of Escherichia coli cells containing operon fusion nhaA'-'lacZ was monitored to study the regulation of expression of nhaA gene under various conditions. The expression of the fusion was enhanced only by chemicals containing Na$^{+}$ or Li$^{+}$. This Na$^{+}$ or Li$^{+}$. This Na$^{+}$(Li$^{+}$)-specific enhancement of .betha.-galactosidase activity represented the increase in the rate of synthesis of .betha.-galactosidase rather than the decrease in the breakdown rate. The induction pattern was influenced by copy numbers of the gene. Induction by Na$^{+}$ or Li$^{+}$ was concentration and time dependent, reaching maximum 5-6 fold induction after 2 hours at 0.4-0.5 M for Na$^{+}$ or at 0.25-0.35 M for Li$^{+}$, Although the expression was induced at much lower concentration of Na$^{+}$ at alkaline pH values than at neutral pH in the presence of Na$^{+}$, alkaline pH itself did ot induced the expression of the fusion in the absence of Na$^{+}$. Temperature shift and growth phase of culture did not affect the level of induction.he level of induction.

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.678-682
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• 2010

The deletion of sti from the Streptomyces plasmid pIJ101 made its derivative pHZ1358 an efficient vector for gene disruption and replacement. Here, pHZ1358 was further optimized by the construction of a derivative plasmid pJTU1278, in which a cassette carrying multiple cloning sites and a lacZ selection marker were introduced for convenient plasmid construction in E. coli. In addition, the oriT region of pJTU1278 was also deleted, generating a vector (pJTU1289) that can be used specifically for PCR-targeting. The efficient usage of these vectors was demonstrated by the deletion of the gene involved in avermectin biosynthesis in S. avermitilis.

• 대한의생명과학회지
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• 제9권2호
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• pp.67-74
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• 2003

Viral and non-viral vectors have been used in the delivery of genetic materials into animal cells and tissues, with each approach having pros and cons. Non-viral vectors have many useful merits such as easy preparation, low immunity and size tolerance of a transgene when compared to those of viral vectors. Delivery specificity may be achieved by complex formation between receptor ligands and a non-viral vector. In the present study, non-viral vector systems are investigated in an effort to find a practical delivery means for gene therapy, Receptor-ligand interaction between transferrin-receptor and transferrin was utilized for efficient gene transfer into cancer cells. A plasmid vector, pcDNA3 (LacZ) was ligated with a small duplexed oligo fragment in which a Biotin- VN$^{TM}$ phosphoramidite was placed in the middle of the oligo. The plasmid vector labeled by biotin was then conjugated with biotin-labeled transferrin via streptavidin. This trimeric conjugates were delivered to a hepatoma cell line, HepG2. The delivery efficiency of the trimeric conjugate was 2-fold higher than that of cationic liposomes used for transfection of a plasmid vector. These results demonstrate that a plasmid vector can be efficiently transferred into cells by forming a trimeric complex of plasmid vector-linker-ligand.

• 생명과학회지
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• 제9권2호
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• pp.169-175
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• 1999

Salmonella typhimurum은 생활사 동안에 다양한 환경과 접하게 된다. S. typhimurium은 오염된 웅덩이에서 숙주의 phgolysosome에 이르기까지 생태계에 널리 분포하면서 산성 스트레스를 경험하며 또한 생존할 수 있다. Salmonella 이와 같은 산저항능은 Acid Tolerance Response (ATR)에 의해 획득된다. 약산의 pH에 적응된 S. typhimurium은 낮은 pH 영역에서 생존할 수 있는 일종의 대항능을 갖게되며, 이런 생존 능력은 복잡한 유전적 유도현상 결과로 해석된다. Salmonella 있어서 ATR은 RpoS 의존적 그리고 RpoS 비의존적 현상으로 구분되며, 특히 혐기적 조건(5% $CO_2$, 5% H$_2$, 90% $N_2$)에서 rpoS$\Omega$Ap는 UK1과 동일한 ATR을 나타내어, 혐기적 조건의 ATR은 RpoS 비의존적인 것으로 판명되었다. P22와 MudJ (Km, lacZ)를 이용한 gene fusion기법과 sodium acetate (pH4.5)를 이용한 돌연변이체 분리방법을 병행하여 산민감성의 형질을 나타내는 LE487 aatA::MudJ를 얻었다. antA 는 Salmonella의 유전자 지도상에서 12min에 위치하는 것으로 조사되었다. antA는 혐기적 조건(5% $CO_2$, 5% H$_2$, 90% $N_2$)하의 pH4.3 조건에서 야생형 Salmonella 비해서 매우 높은 산민감성을 보여주었다. 그러므로 antA는 혐기적 ATR에 있어서 산적응 기전에 관여하는 중요한 유전자로 확인되었다.

• 한국미생물·생명공학회지
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• 제39권4호
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• pp.337-344
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• 2011

이전 연구에서, bacteriophage ${\Phi}FC1$이 Enterococcus faecalis KBL703에서 UV induction을 통해 분리 동정되었으며, ${\Phi}FC1$은 phage attachment site인 attP와 bacterial attachment site인 attB 사이에서 site-specific integration을 촉매하는 integrase를 가지고 있다는 것을 밝혀냈으며 이를 MJ1이라 명명하였다. 이 연구에서는 이를 바탕으로 MJ1에 의한 site-specific integration의 효율을 Escherichia coli와 NIH3T3 cell에서 확인 하기 위해 attP, attB, MJ1을 각각의 벡터에 삽입하였다. MJ1 인테그라제에 의한 재조합을 수행하기 위해서 기질 벡터 pABLP를 $DH5{\alpha}$에 형질전환시킨 후, LB 배지에서 $37^{\circ}C$ 1시간 배양한 후 암피실린(ampicillin)과 테트라싸이클린(tetracycline) 항생제 플레이트로 pGMJ1과 pABLP 같이 가지고 있는 colony 들을 선별하여, LacZ 유전자가 불활성화 된 흰색 콜로니 개수를 세고 통계를 낸 결과 integration의 frequency가 99% 이상인 것으로 나타났다. 또한, 실제로 재조합이 일어났는 지를 확인하기 위해서 콜로니 PCR을 수행하여 재조합의 산물인 attL 150 bp을 확인하였다. PCR 산물은 염기서열분석을 통해 정확한 site-specific integration이 일어났음을 확인하였다. MJ1에 의한 integration을 보이기 위해 attP와 attB를 가지고 있는 vector를 MJ1 expression vector와 함께 NIH3T3 cell에 cotransfection 했으며 GFP를 reporter로 사용해 그 activity를 관찰하였다. NIH3T3 cell에서 GFP의 발현을 형광 현미경을 통해 알아본 결과, MJ1에 의한 sitespecific integration이 다른 accessory protein의 도움 없이 일어난다는 것을 볼 수 있었다. 마찬가지 방법으로, attR과 attL 간의 excision을 GFP로 알아본 결과, GFP는 발현하지 않았으며, 이는 MJ1에 의한 excision이 일어나지 않았음을 보여주었다. 이와 같은 결과로 볼 때, MJ1의 host만이 아니라 넓은 범위안에서도 integration을 수행할 수 있다는 것을 보여주었다. 따라서 MJ1을 이용한 site-specific integration system의 개발은 gene therapy를 위한 gene delivery system의 구축에 있어서 좋은 시작이 될 수 있다.

• Journal of Microbiology
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• 제42권4호
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• pp.319-327
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• 2004

An additional amylase, besides the typical $\alpha-amylase,$ was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a malto­genic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the $\beta-galactosidase$ activity produced from the bbmA promoter fused to the amino terminus of the lacZ struc­tural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The pro­moter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing $\beta--cyclodextrin\;(\beta-CD),$ maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the $\beta-CD$ hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regu­latory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the $\beta-CD$ hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing $\beta-CD$ in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and $\beta-CD$ utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.

• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.541-547
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• 1999

Constructions of a transfer vector and a recombinant baculovirus using the thymidine kinase gene of the Herpes simplex virus type 1 strain F (HSV -1) were carried out. Newly cloned transfer vector, pHcgXIIIB, was constructed by insertion of the glycoprotein gX gene signal peptide sequence of Pseudorabies virus into the baculovirus vector pHcEV-IV. The gX sequence was inserted just downstream from the promoter for the polyhedrin gene of the Hyphantria cunea nuclear polyhedrosis virus (HcNPV). HSV-1 thymidine kinase(tk) gene (1.131 kb) was used as a candidate gene for transferring into the baculovirus expression system. The tk gene was inserted into a BamHI site downstream from the gX sequence-promoter for the polyhedrin gene in the pHcgXIIIB transfer vector and was transferred into the infectious lacZ-HcNPV expression vector. Recombinant virus was isolated and was named gX-TK-HcNPV. The recombinant virus produced a 45 kDa gX-TK fusion protein in Spodoptera frugiperda cells, which was confirmed by Western blot analysis. Microscopic examination of gX-TK-HcNPV-infected cells revealed normal multiplication. Fluorescent antibody staining indicated that the gX-TK fusion protein was present in the cytoplasm. These results indicated that the transfer vector successfully transferred the gX-tk gene into the baculovirus expression system.

• BMB Reports
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• 제43권12호
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• pp.789-794
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• 2010

A novel gene, designated mgt-6, containing four splicing variants, was isolated from a gene trap clone library of C3H/10T1/2 cells transfected with retroviral promoterless gene-trap vector, ROSAFARY. The transcript variants were differentially expressed in murine tissues and cell lines and differentially responded to diverse stimuli including TGF-${\beta}1$ and mitogen-activated protein kinase (MAPK) inhibitors. The mgt-6 gene encoded a protein of 37 or 11 amino acid residuals with cytoplasmic distribution. However, when C3H/10T1/2 cells were treated with 5-azacytidine, the protein translocated into cell nucleus as indicated by fused LacZ or C-terminally tagged EGFP. Our preliminary results suggest that further study on the role of mgt-6 gene in cell transformation and differentiation may be of significance.

• Applied Biological Chemistry
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• 제38권2호
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• pp.111-117
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• 1995

CRP (cAMP receptor protein)은 cAMP와 결합하여 cAMP-CRP 복합체를 형성하여 전사조절의 조절인자로서 작용한다. crp 유전자에 변이를 도입하여 cAMP의 비존재 상태에서 cAMP-CRP와 비슷한 기능을 가진 crp 유전자가 도입된 대장균 MK2001 (crp, cya::km)을 숙주로 사용하여 cAMP 혹은 cGMP의 비존재하에서도 mal 유전자의 발현을 촉진시키는 유전자 sfs (sugar fermentation stimulation) 수 종을 클로닝 하였다. 본 실험에서는 이미 밝혀진 nlp (Ner like protein) 유전자와 같이, sfs의 새로운 유전자를 탐색하여, 그 중 sfs4의 2126 bp 전 염기배열을 결정하고, 잠정적인 sfs4의 promoter 영역에는 CRP 단백질과의 결합 DNA 공통 염기배열(5' AAT TGTGA ACACCA TCACC CGT 3')이 존재함을 확인했다. lacZ 융합 유전자를 작성하여 TP2010R1 MK2001의 균주에서 cAMP를 첨가할 경우 각각 2.3배, 1.8배의 ${\beta}-galactosidase$ 활성이 증가하는 것으로 보아 sfs4는 cAMP-CRP에 의해 발현 조절을 받는 것으로 나타났다.