• Korean Journal of Microbiology
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• v.37 no.4
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• pp.259-264
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• 2001

It has been well known that the expression of S. typhimurium cadBA operon requires at least two extracellular signals: low pH and high concentration of lysine. To better understand the nature of pH-dependent and lysine dependent signal transduction, mutants were isolated in JF2238(cadA-lacZ) by Tn10 insertion, spontaneous mutagenesis, and EMS treatment. Two mutants were isolated from JF2238, expressed as a cadA-lacZ operon fusion in various growth conditions, and analyzed to have mutations in cadC, a gene encoding a function necessary for transcriptional activation of cadBA. One isolate (cadC6) conferred pH-independent and lysine-independent cadBA expression and the other(cadC4) showed pH-independent and lysine-dependent cadBA expression. cadR::Tn10 and cadR4 mutants were expressed in the absence of exogenously added lysine. They were also resistant to thiosine and complemented by lysP clone from E. coli. Thus, in the absence of exogenous lysine, cadR is a negative regulator of cadBA expression. Cadaverine, the product of lysine decarboxylation, was shown to inhibit expression of cadA-lacZ fusion in cad $C^+$ cell.

• Microbiology and Biotechnology Letters
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• v.25 no.2
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• pp.144-150
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• 1997

After DNA damage, umuDC is the only SOS operon that must be induced to promote SOS mutagenesis in Escherichia coli. The recombinant plasmid pBC401 and pBC402 were constructed to fuse the lac structural genes with promoter region of umuDC operon to induce the expression of lacZ gene by DNA damage. We transformed the plasmid pBC401 and pBC402 into E. coli MC1061, lacZ deleted strain and determined the activity of $\beta$-galactosidase for various mutagen; UV, mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroqunoline-1-oxide (NQO), ethyl methanesulfonate (EMS). The $\beta$-galactosidase activities of PBC401 and pBC402 for UV, MMC, and NQO were increased in proportion to expression time until 3 hours thereafter, the activities were constant or slightly decreased. The activities for MNNG and EMS were not so high as for UV, MMC, and NQO. When MNNG and EMS were treated, $\beta$-galactosidase activity of pBC402 was slightly lower than pBC401 but when UV, MMC, and NQO were treated in pBC402, $\beta$-galactosidase activity was slightly higher than in pBC401. Therefore, the pBC402 was better than the pBC401 in terms of sensitivity for frameshift mutagen. We suggest that the plasmid pBC401 and pBC402 are easy to detect mutagens which cause frameshift mutation rather than point mutation.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.259-268
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• 2003

Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinoma cells and is known to prevent symptomatic rotavirus infections. In this study, we tried to transfer the human lactadherin gene to the Chinese Hamster Ovary (CHO) cells using retrovirus vector system and tested inducible expression of the gene under the tetracycline-controllable promoter. At first, tetracycline-mediated inducibility was tested using E.coli LacZ marker gene. NIH3T3 cells co-infected with RevTet-On and RevTRE-LacZ retrovirus vectors showed that the cells responded to doxycycline (a derivative of tetracycline) in a dose-dependent manner, and prominent induction of the lacZ gene expression was observed from 1 $\mu\textrm{g}$/ml of doxycycline concentration. Based on the results of the pilot experiment, inductional expression of the human lactadherin gene was conducted using RevTet-On and RevTRE-Ltd retrovirus vectors. Analysis with the RT-PCR demonstrated successful inductional expression of the lactadherin gene in the Chinese Hamster Ovary (CHO) cells. Considering that constitutive overexpression of the exogenous genes in the target cells causes serious physiological imbalance, the results obtained in this study will be very useful especially in the studies of gene therapy and transgenic animal production.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1357-1363
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• 2008

The $P_{entC}$ promoter of the entCERA operon encoding enzymes for enterobactin biosynthesis in Escherichia coli is tightly regulated by the availability of iron in the culture medium. In iron-rich conditions, the $P_{entC}$ promoter activity is strongly repressed by the global transcription regulator Fur (ferric uptake regulator), which complexes with ferrous ions and binds to the Fur box 19-bp inverted repeat. In this study, we have constructed the expression vector pOS2 containing the $P_{entC}$ promoter and characterized its repression, induction, and modulation by quantifying the expression of the lacZ reporter gene encoding $\beta$-galactosidase. $\beta$-Galactosidase activities of E. coli transformants harboring pOS2-lacZ were highly induced in the presence of divalent metal ion chelators such as 2,2'-dipyridyl and EDTA, and were strongly repressed in the presence of excess iron. It was also shown that the basal level $\beta$-galactosidase expression by the $P_{entC}$ promoter was drastically decreased by incorporating the fur gene into the expression vector. Since the newly developed iron chelator-inducible expression system is efficient and cost-effective, it has wide applications in recombinant protein production.

• Korean Journal of Microbiology
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• v.45 no.2
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• pp.215-221
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• 2009

Site-specific incorporation of unnatural amino acids into proteins in vivo can be achieved by co-expression of an orthogonal pair of suppressor tRNA and engineered aminoacyl-tRNA synthetase (ARS) that specifically ligates an unnatural amino acid to the suppressor tRNA. As a step to establish this technique, here we generated an Escherichia coli reporter strain DH10B(Tn:lacZam) by integrating amber mutated lacZ gene into the chromosome of E. coli DH10B strain. In vivo expression of E. coli amber suppressor $tRNA^{Gln}$ produced blue colonies in culture plates containing X-Gal as well as dramatically increased $\beta$-galactosidase activity. In addition, expression of an orthogonal pair of Saccharomyces cerevisiae suppressor $tRNA^{Tyr}$ and tyrosyl-tRNA synthetase also produced blue colonies as well as moderate increase of $\beta$-galactosidase activity. These data demonstrate that our reporter strain will provide an efficient method to assess amber suppression in both qualitative and quantitative manners.

• KSBB Journal
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• v.11 no.4
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• pp.431-437
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• 1996

The nar promoter of Escherichta coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an expression promoter. The modified nar promoter, in which several bases in the -10 region was mutated to the consensus sequence by site-directed mutagenesis, was characterized in E. coli, on which chromosome the fnr gene affecting expression of the nar promoter according to dissolved oxygen level was mutated. The E. coli lacZ gene was used as a reporter gene. The following effects were investigated to find optimal conditions to induce the modified nar promoter: induction methods, optimal nitrate concentrations, the amount of ${\beta}$-galactosidase expressed at the different growth conditions, and induction characteristics. The following results were obtained from the experiments : expression of ${\beta}$-galactosidase from the modified nar promoter was not affected much by nitrate concentrations. The maximal specific ${\beta}$-galactosidase activity was obtained when E. coli was grown under aerobic conditions, and then the modified nar promoter was induced at OD600=2.2 under microaerobic conditions (DO=1∼2%), under which conditions the maximal specific ${\beta}$-galactosidase activity was 13,000 Miller units. However, the specific ${\beta}$-galactosidase activity was approximately 6,000 Miller units even before the modified nar promoter was induced. Therefore, the modified nar promoter seemed to be useful when the cloned gene wants to be expressed in E. coli constitutively.

• The Korean Journal of Zoology
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• v.38 no.2
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• pp.196-203
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• 1995

The c-myc proto-oncogene, one of the immediately earlY genes, is expressed in various mammalian cell types and heavily involved in the regulation of cell proliferation and differentiation. To determine endogeneous expression pattern of c-myc gene in preimpBantation mouse embwos, we employed a reverse transcription coupled to polvrnerase chain reaction (RT-PCR). Transcript of c-myc was detected at fertilized embryos as a maternal transcript. At the early two-cell stave, transcript of c-myc gene was hardly detected, bu, appeared at late two-cell embryos as a zygotic transcript. The level of c-myc expresion was increased at later stases and peaked at blastocvst stage. To examine the functional role of promoter region for c-myc gene transcription, we fused the 5'upstream region (1.8 kb) including econ 1 of c-myc genomic DNA with E. coli lacE gene fnamed as pcMYC-laczl. pcMYC-lacZ was microiniected into the pronscleus of mouse one-cell embryovs, and p·salactosidase activity was determined tv histochemical staining with X-gal at different stases. f-galactosidase activity was detected only at blastocyst, but not at the earlier stage embryos. This result indicates that c-myc gene is transcriptionallv active during mouse preimplantation development.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• Microbiology and Biotechnology Letters
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• v.15 no.6
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• pp.396-401
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• 1987

Protein productivities of a cloned gene ($\beta$-galactosidase) and the cultural performances of two recombinant Escherichia coli strains, which use different host-vector systems, were studied. E. coli JM109/pTBG10 strain which carries Tac promoter had higher protein productivity than E. coli MH3000 (pRKc1857)/pASI(lacZ) strain which carries pL promoter. Induction of protein syn-thesis was optimum at the initial-and mid-logarithmic growth phases for both strains. Oxygen demand was observed to be very high during the cloned gene expression, and could be alleviated to some extent through pH control. The ratio of specific growth rates of plasmid-harboring to plasmidfree cell, $\mu$+ /$\mu$-, of the high productivity strain was observed to be lower than that of the low productivity one. Plasmid stability was analyzed for 20-30 generations, and it was found that the traction of plasmid-harboring cells dropped to l0% level in about 25 generations for both strains when the cloned gene expression was induced.

• Journal of Life Science
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• v.14 no.2
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• pp.325-330
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• 2004

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA-RNA helicase activities. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, the RAD3 homolog gene was isolated by screening of genomic DNA library. The isolated gene was designated as HRD3 (Homologue of RAD3 gene). The over-expressed HRD3 protein was estimated to be a 75 kDa in size which is in good agreement with the estimated by the nucleotide sequence of the cloned gene. Two-dimensional gel electrophoresis showed that a number of other protein spots dramatically disappeared when the HRD3 protein was overexpressed. The overexpressed RAD3 protein showed a toxicity in E. coli host, suggesting that this protein may be involved in the inhibition of protein synthesis and/or degradation of host protein. To determine which part of HRD3 gene contributes to the toxicity in E. coli, various fusion plasmids containing a partial sequence of HRD3 and lac'Z gene were constructed. These results suggest that the C-terminal domain of HRD3 protein may be important for both toxic effect in E. coli and for its role in DNA repair in S. pombe.