• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.509-516
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• 1998

A plasmid vector, pXGPRMATG-lac-Tgx, was developed for overproduction of $\beta$-galactosidase in Escherichia coli using the genetic components of groEx, a heat-shock gene cloned from symbiotic X-bacteria in Amoeba proteus. The vector is composed of intragenic promoters P3 and P4 of groEx, the structural gene of lac operon, transcription tenninator signals of lac and groEx, and ColEl and amp'of pBluescript SKII. The optimized host, E. coli DH5$\alpha$, transfonned with the vector constitutively produced 117,310-171,961 Miller units of $\beta$-galactosidase per mg protein in crude extract. The amount of enzyme in crude extract was 53% of total water-soluble proteins. About 43% of the enzyme could be purified to a specific activity of 322,249 Miller units/mg protein after two-fold purification, using two cycles of precipitation with ammonium sulfate and one step of gel filtration. Thus, the expression system developed in this study presents a low-cost and simple method for purifying overproduced $\beta$-galactosidase in E. coli.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.7
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• pp.1015-1022
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• 2007

The spermatogonial stem cell (SSCs) is unique in that it is the only cell in the adult male that can contribute genes to a subsequent generation. Permanent modification of the germ cell line may be realized if stem cells could be cultured, transfected with unique genes, and then transplanted into recipient testes. We developed a culture system that supported long-term viability of SSCs. We used a retrovirus vector (pMSCV including ${\beta}$-galactosidase) to stably transfect spermatogonia following long-term culture using the system developed. Expression of the reporter gene ${\beta}$-galactosidase controlled by the retroviral vector was stable in long-term cultured SSCs. We confirmed the retroviral-mediated ${\beta}$-galactsidase gene could be expressed in germ cells in recipient mice following SSCs transplantation.

• 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).

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.5
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• pp.449-454
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• 1995

The present study was conducted to assess gene expression of bacterial lacZ driven by the SV40 promoter at early developmental stages of bovine embryos. The lacZ gene was linearized with BamHI digestion and introduced into the pronucleus by microinjection at 20 hrs after the commencement of in vitro fertilization. Intact bovine blastocysts were not stained with X-Gal, suggesting that there is no endogenous beta-galactosidase activity in these blastocysts. In contrast, the bovine blastocyst cells microinjected with the lacZ gene exerted a characteristic greenish-blue color originating from the bacterial beta-galactosidase activity, albeit at a low rate, i.e. 2.1% of the total fertilized oocytes injected. It was concluded, therefore, that the lacZ gene driven by the SV40 promoter could be used for an indirect screening method in which the presence of transgene is evaluated from the product of transgene expression.

• Molecules and Cells
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• v.20 no.1
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• pp.43-50
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• 2005

Glutaredoxin (Grx) is a small, heat-stable redox protein acting as a multi-functional glutathione (GSH)-dependent disulfide oxidoreductase. We have cloned the monothiol Grx5 gene from the genomic DNA of the fission yeast Schizosaccharomyces pombe. It has 1,904 bp, with one intron, and encodes a putative protein of 146 amino acids with a molecular mass of 16.5 kDa. Recombinant Grx5 produced functional Grx in S. pombe cells. NO-generating sodium nitroprusside (SNP, 1.0 and 2.0 mM) and potassium chloride (KCl, 0.2 and 0.5 M) increased the synthesis of ${\beta}$-galactosidase from a Grx5-lacZ fusion gene, and transcription of Grx5 was also enhanced by SNP and KCl. Synthesis of ${\beta}$-galactosidase from the Grx5-lacZ fusion was lower in Pap1-negative TP108-3C cells than in wild type KP1 cells, and when Pap1 was overproduced in KP1 cells, the level of ${\beta}$-galactosidase increased. We also found that Pap1 is involved in the induction of Grx5 by SNP and KCl. S. pombe Grx5 may play a crucial role in responses to nitrosative and osmotic stresses.

• 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.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.12-19
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• 1994

KEM1 is known to control the spindle pole body or microtubule function, probably in response to the cellular nutritional conditions in Saccharomyces cerevisiae. Transposon insertions were performed in the cloned KEM1 gene using mini-Tn10-LUK element carrying E. coli ${\beta}$-galactosidase structural gene. A collection of ranfom Tn10-LUK insertions defined an approximately 3.5 kb region required for the KEM1 function. From this collection functional KEM1::lacZ protein fusions were identified. Indirect immunofluorescence using anti-${\beta}$-galacatosidase antibodies localized the KEM1::lacZ fusion protein to the periphery of the nucleus.

• 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.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.758-761
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• 2001

In this study, the nar promoter of E. coli was characterized to see whether the nar promoter cloned onto pBBR122 can be used as an expression promoter of gram negative microbes. For this purpose, a plasmid with lacZ gene expressing ${\beta}-galactosidase$ instead of the structural genes of nar operon in a gram negative host strain(Agrobacterium.tumefaciens) was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate concentration, maximally inducing the nar promoter, the amount of expressed ${\beta}-galactosidase$ and induction ratio(specific ${\beta}-galactosidase$ activity after maximal induction/specific ${\beta}-galactosidase$ activity before induction). The following results were obtained from the experiments: the growth of Agrobacterium with E.coli nar promoter was not much affected by nitrate concentration in the shake-flask; induction of nar promoter was optimal when Agrobacterium was grown in the presence of 1% nitrate ion at the beginning of culture and when overnight culture was completely grown in the shake-flask before being transferred to other shake-flask; the amount of ${\beta}-galactosidase$ per cell and per medium volume was maximal when Agrobacterium was grown under aerobic condition to $OD_{600}$ of 1.7; then the nar promoter was induced under microaerobic and anaerobic condition made by lowering dissolved oxygen level(DO). After 2-3h of induction in the YEP medium selected as a main culture medium, the specific ${\beta}-galactosidase$ activity became about 17,000 Miller units in the fermentor cluture.