• BMB Reports
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• v.31 no.6
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• pp.578-584
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• 1998

In a previous paper (Kim et al., 1996a), the immediate 5' -flanking region and coding region of the human UDP-N -acetylglucosamine:-D-mannoside-1,4-Nacetylglucosaminyltransferase III (N-acetylglucosaminyitransferase- III; GnT-III) gene was reported, isolated and analyzed. Herein, we report on amplification of a new 5' -noncoding region of the GnT-III mRNA by single-strand ligation to single-stranded cDNA-PCR (5' -RACE PCR) using poly(A)+ RNA isolated from human fetal liver cells. A cDNA clone was obtained with 5' sequences (96 bp) that diverged seven nucleotides upstream from the ATG (+1) start codon. A concensus splice junction sequence, TCTCCCGCAG, was found immediately 5' to the position where the sequences of the cDNA diverged. The result suggested the presence of an intron in the 5' -noncoding region and that the cDNA was an incompletely reversetranscribed cDNA product derived from an mRNA containing a new noncoding exon. When mRNA expression of GnT-III in various human tissues and cancer cell lines was examined, Northern blot analysis indicated high expression levels of GnT-III in human fetal kidney and brain tissues, as well as for a number of leukemia and lymphoma cancer cell lines. Promoter activities of the 5' -flanking regions of exon 1 and the new noncoding region were measured in a human hepatoma cell line, HepG2, by luciferase assays. The 5'-flanking region of exon 1 was the most active, whilst that of exon 2 was inactive.

• Microbiology and Biotechnology Letters
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• v.17 no.3
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• pp.263-268
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• 1989

To maximize the performance of recombinant cell fermentation process through optimizing environmental conditions, the production of invertase from recombinant Saccharomyces cerebisiae Containing SUC2 gene was studied as a model. The recombinant cells showed biphasic growth on glucose. Since the promoter of the SUC2 is regulated by the concentration of glucose in the medium, expression of invertase by recombinant yeast began when the glucose concentration decreased in a range of 0.25-0.4 g/L during the batch culture. Plasmid segregation occured frequently during glucose fermentation, and infrequently during ethanol oxidation. A rapid appearance of invertase activity with glucose was observed under nonaerated condition, and the maximum specific invertase activity was about 1.5 times as high as under aerobic condition, In fed batch culture, when n low level of glucose was continuously supplied to the tormentor after the time of glucose depletion during growth phase, specific and total invertase activity increased about 1.7 and 2.9 fold, respectively, in a batch culture.

• Journal of Life Science
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• v.28 no.12
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• pp.1432-1437
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• 2018

Clara cell secretory protein (CCSP) plays an important role in protecting the lungs from inflammation. This research focuses on identifying the cis-element for binding the repressor of CCSP gene expression. A DNase I footprinting experiment revealed three protected regions between -812 and -768 bp (45 bp) of the mCCSP promoter. One motif (D3: GCCTGGGAA) was 100% conserved across rat, hamster, and human. The addition of excess amounts of the D3 motif exhibited high competition within that 45 bp range in an electrophoretic mobility shift assay. However, when mutated D3 ($G{\underline{AA}}TG{\underline{TT}}AA$) was used, the competition was significantly reduced. This demonstrates that the D3 motif within that 45 bp region of the mCCSP promoter is an important site for the protein-DNA interaction. Transient transfection assays with -756 Luc resulted in highly decreased expression of CCSP than those with -812 Luc, suggesting that the 45 bp could function as a binding site for the repressor. Co-transfection of KLF4 exhibited significant repression of the -812 Luc but not the -768 Luc which clearly shows that KLF4 might function as a repressor for the CCSP gene and also suggests that the D3 motif is strongly involved in the binding of KLF4. In addition, when anti-KLF4 antibody was added, super-shifted bands were observed. This result demonstrates that KLF4 could function as a repressor by binding to this 45 bp region of the CCSP promoter and that the D3 motif might be involved in the specific binding of KLF4.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.101-108
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• 2002

Human Prourokinase (proUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase that is associated with clinical benefits in patients with acute peripheral arterial occlusion. For production of transgenic cow as human proUK bioreacotor, we conducted this study to establish efficient production system for bovine transgenic embryos by somatic cell nuclear transfer (NT) using human prourokinase gene transfected donor cell. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human prourokinase target gene into a pcDNA3 plasmid. Cumulus cells were used as donor cell and transfected with the expression plasmid using the Fugene 6 as a carrier. To increase the efficiency for the production of transgenic NT, development rates were compared between non-transfected and transfected cell in experiment 1, and in experiment 2, development rates were compared according to level of GFP expression in donor cells. In experiment 1, development rates of non-transgenic NT embryos were significantly higher than transgenic NT embryos (43.3 vs. 28.4％). In experiment 2, there were no significant differences in fusion rates (85.4 vs. 78.9％) and cleavage rates (78.7 vs. 84.4％) between low and high expressed cells. However, development rates to blastocyst were higher in low expressed cells (17.0 vs. 33.3％), and GFP expression rates in blastocyst were higher in high expressed cells (75.0 vs. 43.3％), significantly.

• KSBB Journal
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• v.19 no.4
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• pp.327-330
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• 2004

An inducible expression system of poly[(R)-3-hydroxybutyrate] (PHB) depolymerization was established in metabolically engineered Escherichia coli with the PHB biosynthesis genes. The Ralstonia eutropha PHB depolymerase gene was cloned in a vector system containing the PHB biosynthesis genes and expressed under inducible promoter. Recombinant E. coli harboring the PHB biosynthesis genes and depolymerase gene was first cultured for the accumulation of PHB, and then the depolymerase was expressed resulting in the degradation of accumulated PHB into (R)-3-hydroxybutyric acid (R3HB). R3HB could be produced with the concentration of 7.6 g/L in flask culture. Two different PHB biosynthesis genes from Alcaligenes latus and R. eutropha were compared for the production of R3HB. This strategy can be used for the production of enantiomerically pure (R)-hydroxycarboxylic acids with high concentration.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.507-513
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• 1992

Serratia marecescens RH1 isolated from soil samples produced large amount of extracellular proteases. One of the genes encoding an extracellular protease form S. marcescens RH1 was cloned in Escherichia coli by shot gun cloning method. The cloned protease, SSP, was stably expressed by its own promoter and excreted into the extracellular medium from E. coli host (ORF) of 3.135 nucleotides corresponding to 1.045 amino acids (112 kDa). The nucleotide and deduced amino acid sequence of SSP showed high overall homology (88%) to one of the S. marcescens protease (27), but low homology to other serine protease families. The optimal pH and temperature of the enzyme were pH 9.0 and 45.deg.C respectively. The activity of protease was inhibited by phenylmethylsulfonyl fluoride (PMSF), which suggests that the enzyme is a serine protease.

• BMB Reports
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• v.34 no.5
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• pp.428-433
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• 2001

A defect in uvsC of Aspergillus nidulans caused high methyl methansulfonate (MMS)-sensitivity, hyporecombination, and a lack of UV induced mutation. The uvsC gene of Aspergillus nidulans shares a sequence similarity with the RAD51 gene of Saccharomyces cerevisiae. In this study, in vitro and in vivo tests were conducted in order to determine whether or not the UVSC protein had functional similarities to RAD51, the recombination enzyme in yeast. The purified recombinant UVSC protein, following expression in Escherichia coli, showed binding activity to single-stranded DNA (ssDNA), when both ATP and magnesium are present. In addition, ATPase activity was also demonstrated and its activity was stimulated in the presence of ssDNA. The UVSC protein that was expressed under the ADH promoter in S. cerevisiae suppressed in part the sensitivity to MMS of the rad51 null mutant. Similarly, when the uvsC cDNA was expressed from the nmt promoter, the MMS sensitivity of the rhp51 null mutant of Schizosaccharomyces pombe was partially complemented. These results indicate that the A. nidulans UVSC protein is a functional homologue of the RAD51 protein.

• Molecules and Cells
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• v.43 no.11
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• pp.909-920
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• 2020

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.540-545
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• 2006

T-DNA-mediated transformation is a common method for generating transgenic plants with insertional mutagenesis. In order to identify important genes involved in shoot development, a system of promoter trap insertional mutagenesis was employed in Arabidopsis thaliana. For this system, an efficient promoter trap vector, pFGL561 was developed. The pFGL561 includes a basta-resistant gene, an intron with multiple splicing donor and acceptor sites, and a promoter-less GFP reporter gene. Using floral-dipping method, we made total 300 $T_1$ promoter-trap lines which were screened for GFP expression. GFP signals in the $T_1$ plants were detected with high frequency, 26.7%, and the signals were reconfirmed in $T_2$ plants. To isolate the genes that are involved in shoot development, phenotypes were analyzed in $T_2$ plants of the 19 $T_1$ lines that had GFP signals in shoot apex, and 6 $T_1$ lines were selected that had abnormal shoot development. These lines will be very useful for the investigation of shoot development.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.109-113
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• 1998

In this study we tried to transform an antimicrobial peptide gene (Shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) into tobacco and potato plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified ie nucleotide sequence to increase antimicrobial activity. Transgenic tobacco plants were regenerated and their seeds were tested on the media containing kanamycin (500 mg/L). The results of PCR amplification and genomic Southern blot hybridization confirmed the integration of construct (tPAL5 promoter-Shiva-NOS-GUS-NOS) into chromosome. We observed that one of the transgenic tobacco plants showed chromosome rearrangement when integrated. In case of potato transformation, the efficiency of regeneration was maximized at the medium containing Zeatin 2mg/L, NAA 0.01mg/L, GA$_3$ 0.1mg/L. We also observed the high expression of GUS (${\beta}$-glucuronidase) enzyme which was located next to the terminator sequence of nopaline synthase gene (NOS) in the vascular tissue of stem, leaves of transgenic potatoes. This result suggested that a short sequence of Shiva gene (120 bp) and NOS terminator sequence might be served as a leader sequence of transcript when translated.