• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.872-880
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• 2003

Three vectors, pSG1, 2, and 3, which facilitate in vivo expression technology (IVET) in Gram-negative bacteria, were developed. Vectors pSG1and 2 are derivatives of ColE1, and pSG3 is a derivative of an R6K replicon. These vectors contain oriT sites that allow mobilization when the RK2 Tra functions are provided in trans. These vectors contain promoterless lacZ (pl-lacZ) and promoterless aph (pl-aph) transcriptionally fused together, which allow qualitative and quantitative measurements of the expression of genes in the genome of bacterial cells. pSG1 and 3 contain gentamicin-resistance genes, and pSG2 carries a streptomycin-/spectinomycin-resistance gene, allowing for selection of recombinants generated by a single crossover between a library fragment cloned into a pSG vector and the identical region in the genome of a bacterial species from which the library fragment originated. These vectors were successfully applied to the generation of random fusions at high rates in the genomes of four representative Gram-negative bacteria. In addition, the expression level of ${\beta}-galactosidase$ and the degree of resistance to kanamycin in cells with fusions generated by these vectors were found to be linearly correlated, proving that these vectors can be used for IVET.

• Environmental Engineering Research
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• v.14 no.1
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• pp.63-67
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• 2009

Escherichia coli K-12 (E. coli K-12) is a representative indicator globally used for distinguishing and monitoring dynamic fates of pathogenic microorganisms in the environment. This study investigated how to most critically quantify lacZ ($\beta$-galactosidase) gene in E. coli K-12 by two different real-time polymerase chain reaction (real-time PCR) in association with three different DNA extraction practices. Three DNA extractions, i.e., sodium dodecyl sulfate (SDS)/proteinase K, magnetic beads and guanidium thiocyanate (GTC)/silica matrix were each compared for extracting total genomic DNA from E. coli K-12. Among them, GTC/silica matrix and magnetic beads beating similarly worked out to have the highest (22-23 ng/${\mu}L$) concentration of DNA extracted, but employing SDS/proteinase K had the lowest (10 ng/${\mu}L$) concentration of DNA retrieved. There were no significant differences in the quantification of the copy numbers of lacZ gene between SYBR Green I qPCR and QProbe-qPCR. However, SYBR Green I qPCR obtained somewhat higher copy number as $1{\times}10^8$ copies. It was decided that GTC/silica matrix extraction or magnetic beads beating in combination with SYBR Green I qPCR can be preferably applied for more effectively quantifying specific gene from a pure culture of microorganism.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.63-68
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• 2000

GroEL is well known as a molecular chaperone. In order to determine the dynamic stress response of the Escherichia coli groE promoter, a groE-lacZ operon fusion in the chromosome was constructed. Stress leading to ${\sigma}^{32}$ synthesis induces transcription from E. coli groE promoter, since the promoter is ${\sigma}^{32}-regulated$. When the strain was stressed with ethanol, phenol, and sodium chloride, clear inductions of ${\beta}-galactosidase$ were observed. Two types of simultaneous stresses of sodium chloride and phenol induced the enze much more than either of the two alone, suggesting that stress was an additive. The combined stress resulted in the highest induction of the enzyme in this system. The groE-lacZ fusion strain developed in this study can conveniently be used to detect other harmful pollutants in the environment. Stress treatment of cells containing recombinant proteins, which need GroEl, by ethanol, phenol, or sodium chloride, might have a tendency to increase their biological activities.

• Genomics & Informatics
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• v.1 no.2
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• pp.113-118
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• 2003

Among a number of antigens characterized in M leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M leprae. We have previously determined a sequence specific element in the 18 kDa gene of M leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M bovis BCG or M smegmatis in front of LacZ gene resulted in normal $\beta$­galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, $\beta$-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the $\beta$-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.

• BMB Reports
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• v.34 no.5
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• pp.395-401
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• 2001

Schizosaccharomyces pombe gene encoding redox enzymes, such as thioltransferase (TTase) and thioredoxin (TRX), were previously cloned and induced by oxidative stress. In this investigation, their expressions were examined using $\beta$-galactosidase fusion plasmids. The expression of the two cloned genes appeared to be growth-dependent. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was increased in the medium with the low glucose level, whereas it was significantly decreased in the medium without glucose or with galactose. It was also decreased in the nitrogen-limited medium. The synthesis of galactosidase from the TRX-lacZ fusion was unaffected by galactose or low glucose. However, it was lowered the absence of glucose. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was shown to be enhanced in a higher medium pH. Our findings indicate that S. pombe TTase and TRX genes may be regulated by carbon and nitrogen sources, as well as medium pH.

• Microbiology and Biotechnology Letters
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• v.24 no.1
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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.

• Environmental Mutagens and Carcinogens
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• v.18 no.1
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• pp.15-21
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• 1998

Transgenic animal and cell line models which are recently developed and used in toxicology fields combined with molecular biological technique, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Transgenic 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 processes. The lac I and lac Z gnee most widely used as a mutational target in transgenic systems. The assay is performed by treatment with putative mutagenic agents, isolation of genomic DNA from cells or tissues, exposure the isolated DNA to in vitro packaging extract, plating and sequencing. The results from these processes provide not only mutant frequency as quantitative evaluation but also mutational spectrum as qualitative evaluation of various agents. Therefore we introduce and review the principle, detailed procedure and application of transgenic mutagenesis assay system in toxicology fields especially in mutagenesis and carcinogenesis.

• 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.

• BMB Reports
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• v.38 no.5
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• pp.609-618
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• 2005

$\gamma$-Glutamyl transpeptidase (GGT, EC 2.3.2.2.) catalyzes the transfer of the $\gamma$-glutamyl moiety from $\gamma$-glutamyl containing ompounds, notably glutathione (GSH), to acceptor amino acids and peptides. A second gene (GGTII) encoding GGT was previously isolated and characterized from the fission yeast Schizosaccharomyces pombe. In the present work, the GGTII-lacZ fusion gene was constructed and used to study the transcriptional regulation of the S. pombe GGTII gene. The synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene was significantly enhanced by NO-generating SNP and hydrogen peroxide in the wild type yeast cells. The GGTII mRNA level was increased in the wild-type S. pombe cells treated with SNP. However, the induction by SNP was abolished in the Pap1-negative S. pombe cells, implying that the induction by SNP of GGTII is mediated by Pap1. Fermentable carbon sources, such as glucose (at low concentrations), lactose and sucrose, as a sole carbon source, enhanced the synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene in wild type KP1 cells but not in Pap1-negative cells. Glycerol, a non-fermentable carbon source, was also able to induce the synthesis of $\beta$-galactosidase from the fusion gene, but other non-fermentable carbon sources such as acetate and ethanol were not. Transcriptional induction of the GGTII gene by fermentable carbon sources was also confirmed by increased GGTII mRNA levels in the yeast cells grown with them. Nitrogen starvation was also able to induce the synthesis of $\beta$-galactosidase from the GGTII-lacZ fusion gene in a Pap1-dependent manner. On the basis of the results, it is concluded that the S. pombe GGTII gene is regulated by oxidative and metabolic stress.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.114-119
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• 1990

A yeast chromosomal superkiller gene (SK13) was cloned and expressed in $ski3^{-}$ Saccharomyces cerevisiae strains. The gene was fused to the structural region of E. coli lacZ gene at its C-terminus in a yeast-E. coli shuttle vector, pSR605. The fused gene complemented $ski3^{-}$ strains with SK13 activity and the quantitative level of expression was measured as determined by assaying $\beta$-galactosidase activity. The SDS-polyacrylamide gel electrophoresis and the Western blot analysis of this fused protein showed the immuno-reacted bands with a protein of the estimated molecular size (ca.250Kd).