• 한국미생물·생명공학회지
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• 제24권1호
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• pp.72-76
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• 1996

To investigate the expression efficiency of new transfer vector of Bombyx mori nuclear polyhedrosis virus (BmNPV), Escherichia coli lacZ gene was inserted into new transfer vector pBmKSK1, under the control of polyhedrin promoter and expressed in BmN-4 cells and larvae of silkworm, Bombyx mori. The recombinant virus containing lacZ gene was isolated from BmN-4 cells coinfected with transfer vectro pBmKSK1-LacZ and wild type BmNPV genome, and analysed by Southern blotting. The expression of ${\beta}$-galactosidase was characterized by SDS-PAGE, Western blotting and ${\beta}$-galactosidase activity assay. The results showed that the level of expression in silkworm larvae was higher than that of BmN-4 cells.

• Journal of Microbiology
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• 제43권1호
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• pp.44-48
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• 2005

In our previous study, the first structural gene (GGTI) encoding ${\gamma}-glutamyl$ transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of ${beta}-galactosidase$ from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.

• Applied Biological Chemistry
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• 제38권1호
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• pp.7-12
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• 1995

본 연구는 lacZ' 유전자의 promoter 개발을 위하여 착수하였다. lacZ' 유전자의 heterologous promoter I과 II를 효모 염색체의 Bam HI DNA 단편에서 분리하였다. Promoter I의 크기는 2.5 Kb 정도이고 ${\beta}-galactosidase$ 활성은 124.6 U/mg protein이었으며 promoter II의 크기와 효소활성은 4.0 Kb와 168.8 U/mg이었다. 형질 전환체에서의 YEp plasmid 안정성은 52.7%에서 67.4% 정도였다. YEp plasmid로부터 YIp plasmid를 재조합하였으며 이 YIp plasmid는 대장균에서나 효모에서도 발현되었다. 효모로부터 분리한 promoter I과 II는 재조합된 YEp와 YIp plasmid의 promoter로서 이용 가능하였다.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.976-983
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• 2003

Seven Escherichia coli cells defective with aerobic growth were isolated by the insertion of ${\lambda}placMu53$, a hybrid bacteriophage of ${\lambda}$ and Mu, which created a transcriptional fusion to lacZY. These insertion mutant cells were tested on an XG ($5-bromo-4-chloro-3-indolyl-{\beta}-D-galactopyranoside$) medium for anaerobic expression of lacZ by fusion to a promoter. The chromosomal DNA from these strains were digested by EcoRI, and the EcoRI fragments that contained the fused gene and lacZ sequence were identified by Southern hybridization, using lacZ containing plasmid as a probe. The EcoRI fragment from each strain was cloned and sequenced. The sequence data were compared with the GenBank database. The mutated gene of three strains, CYT4, CYT5, and OS11, was found to be identical, and it was nrdAB that encoded ribonucleoside diphosphate reductase. The gene nrdAB was at min 50.5 on the Escherichia coli linkage map and 2,348,084 on the physical map, and is involved in hemAe-related reduction-oxidation reaction. OS6 and OS14 mutant strains had insertion at min 8.3 and the mutated gene was hemB. The hemB encodes 5-aminolevulinate dehydratase or porphobilinogen synthase. The OS3 mutant had insertion in cydB at min 16.6. The cydAB encodes cytochrome d oxidase. In the case of OS1, the fusion was made with sucA, the E1 component of ${\alpha}-ketoglutarate$ dehydrogenase.

• Biotechnology and Bioprocess Engineering:BBE
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• 제1권1호
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• pp.9-12
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• 1996

In order to produce the single chain precursor of a novel human insulin analogue, (B30-Homoserine) insulin, the fermentative behaviors of Escherichia coli JM103 were studied, which harbors pKBA plasmid carrying a hybrid gene in which the gene for a single chain precursor was fused with lacZ gene under tac promoter. The maximal induction of gene expression was achieved when more than 0.05 mM of isopropyl-$\beta$-D-thiogalactopyranoside(IPTG) was supplemented to fermentation medium after 4 h cultivation of E. coli, and followed by longer than 2-h fermentation. The hybrid protein of the single chain insulin precursor was isolated from cytoplasmic inclusion bodies by dissolving in 8M urea solution, and purified through DEAE-Sephacel and Sephadex G-200 column chromatographies with a recovery of 35%. The finally purified hybrid protein showed a single band on sodium dodecyl sulfate-polyacrylamide gel.

• Journal of Microbiology
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• 제34권2호
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• pp.137-143
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• 1996

We isolated a promoter inducible by paraquat, a superoxide-generating agent, from Escherichia coli using a promoter-probing plasmid pRS415. From sequence analysis we found out the promoter is for fpr ENCODING nadph : ferredoxin oxidoreductase. We constructed on operon fusion of lacZ gene with fpr promoter to monitor the expression of the gene in the single-copy state. LacZ expression generators, menadione and plumbagin, also induced the expression of .betha.-galactosidase in the fusion strain. On the other hand, no significant induction was observed by treatment with hydrogen peroxide, ethanol, and heat shock. Induction of .betha.-galactosidase was significantly reduced by introducing a .DELTA. sox 8 :: cat of soxS3 :: Tn10 mutation into the fusion strain, indicating that fpr gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis. Possible roles of fpr induction in superoxide stress were discussed.

• KSBB Journal
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• 제10권1호
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• pp.38-45
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• 1995

Aspergillus oryzae에서 A. nidulans의 glyceral d dehyde-3-phosphate dehydrogenase (gpdA)와 trpC, prmoter의 발현 능력 을 E. coli lacZ gene fusion을 이용하여 비교.분석하였다. A. oryzae 내에서 발현된 E. coli $\beta$galactosidase의 specific activIty를 조사하여 본 결과, gpdA promoter를 가지는 transformant들 에서는 2,000unit/ug of protem 정도의 activity를 보이는 반면, trpC, promater를 가지고 있는 transformant들에서는 10.5~52.3unit/ug of protein 정도의 activity를 보였다. 이 결과로부터 A. oryzae 내에서 A. nidulans의 gpdA promoter가 trpC, promoter에 비해 70 배 정도 더 강한 발현 능력을 보이고 있음을 알 수 있다. Western blot 분석에서도 gpdA promoter를 가지고 있는 transf ormant에서 더 많은 E. coli $\beta$-galactosidase가 발현된 것 을 보여 주고 있다. 또한 southern blot 분석에서는 이러한 강한 발현이 transform된 plasmid의 copy number와 상관 없음을 보여주고 있다.

• 미생물과산업
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• 제14권1호
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• pp.12-16
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• 1988

Allantoin 분해 유전자들중 highly inducible 한 DAL7, DUR1,2및 constitutive한 DAL5 gene의 promoter를 deletion 방법에 의해 발현에 필요한 최소 DNA seqyence 부위를 정한후 이 DNA seqyence를 다시 oligonucleotide 합성방법에 의해 합성하여 Cyc 1-LacZ expression vector에 삽입하여 효모내에서 LacZ의 발현이 삽입한 DNA sequence에 의해 영향을 받는 정도를 측정하여 (.betha.-galactosidase activity) deletion 방법에 의해 결정한 이 DNA dequence들이 직접 발현유도에 관여하는가를 조사하였다.

• 생명과학회지
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• 제8권2호
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• pp.216-225
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• 1998

Metal ion-induced and it’s regulatory genes were screened in virulent salmonella typhimurium UK1 and tested cross-regulation with various stresses. Using the techniqud of P22-MudJ(Km, lacZ)-directed lacZ operon fusion, LF40 cuiA::MudJ and Lf153 cuiD::MudJ which were induced by copper were selected. cuia and cuiD were determined anaerobic coper inducible and copper tolerance response gene, respectively. Also cuiA and cuiD locus were determined at 81 and 8min, respectively, on salmonella Genetic Map. The two regulators were identified as cuaR, and cudR, which controls cuiA and cuiD, respectively. cuaR, and cudR appeared as negative regulators because the expression of cuiA-lac-Z and cuiD-lacZ were increased. Copper adapted UK1 showed high resistance to H$_{2}$O$_{2}$, but cuiD did not. The product of the cudR locus was responsible for decreasing the tolerance to copper and H$_{2}$O$_{2}$. Furthemore cuiA and cuiD locus were found to be part of a regulon under the control of a trans-acting regulators, rpoS, oxyR and relA. Therefore, the results suggest CTR participate with oxidative stress on Salmonella.

• Asian-Australasian Journal of Animal Sciences
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• 제14권2호
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• pp.163-169
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• 2001

Despite the high potency of the retrovirus vector system in gene transfer, one of the main drawbacks of has been difficulty in preparing highly concentrated virus stock. Numerous efforts to boost the virus titer have ended in unsatisfactory results mainly due to fragile property of retrovirus envelope protein. In this study, to overcome this problem, we constructed our own retrovirus vector system producing vector viruses encapsulated with VSV-G (vesicular stomatitis virus G glycoprotein). Concentration process of the virus stock by ultracentrifuge did not sacrifice the virus infectivity, resulting in more than 108 to 109 CFU (colony forming unit) per ml on most of the target cell lines tested. Application of this high-titer retrovirus vector system was tested on chicken embryos. Injection of virus stock beneath the blastoderms of pre-incubated fertilized eggs resulted in chick embryos expressing E. coli LacZ gene with 100% efficiency. Therefore, our results suggest that it is possible to transfer the foreign gene into chicken embryo using our high-titer retrovirus vector.