• Journal of Life Science
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• v.16 no.3 s.76
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• pp.454-458
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• 2006

HSF1 is the heat shock transcription factor in Saccharomyces cerevisiae. S. cerevisiae KNU5377 can ferment at high temperature such as $40^{\b{o}}C$. We have been the subjects of intense study because Hsf1p mediates gene expression not only to heat shock, but to a variety of cellular and environmental stress challenges. Basing these facts, we firstly tried to construct the hsf1 gene-deleted mutant. PCR-method for fast production of gene disruption cassette was introduced in a thermotolerant yeast S. cerevisiae KNU5377, which allowed the addition of short flanking homology region as short as 45 bp suffice to mediate homologous recombination to kanMX module. Such a cassette is composed of linking genomic DNA of target gene to the selectable marker kanMX4 that confers geneticin (G418) resistance in yeast. That module is extensively used for PCR-based gene replacement of target gene in the laboratory strains. We describe here the generation of hsf1 gene disruption construction using PCR product of selectable marker with primers that provide homology to the hsf1 gene following separation of haploid strain in wild type yeast S. cerevisiae KNU5377. Yeast deletion overview containing replace cassette module, deletion mutant construction and strain confirmation in this study used Saccharomyces Genome Deletion Project (http:://www-sequence.standard.edu/group/yeast_deletion_project). This mutant by genetic manipulation of wild type yeast KNU5377 strain will provide a good system for analyzing the research of the molecular biology underlying their physiology and metabolic process under fermentation and improvement of their fermentative properties.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.264-269
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• 2008

To develop an amylolytic industrial yeast strain producing $\beta$-amylase, the BAMY gene encoding Achlya bisexualis $\beta$-amylase was constitutively expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p) in an industrial strain of Saccharomyces cerevisiae. Yeast transformation was carried out by an integration system containing $\delta$-sequences as the recombination site. The integrative cassette devoid of bacterial DNA sequences was constructed that contains the BAMY gene and $\delta$-sequences. Industrial S. cerevisiae transformed with this integrative cassette secreted 45 kDa $\beta$-amylase into the culture medium. The $\beta$-amylase activity of the transformant was approximately 18.5-times higher than that of A. bisexualis. The multi-integrated BAMY genes in the transform ant were stable after 100 generations of growth in nonselective medium. Hydrolysis of soluble starch and various starches with the enzyme released maltose but not glucose or oligosaccharides.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.789-797
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• 2010

The limited information on differential gene expression in the different serotypes of Actinobacillus pleuropneumoniae has significantly hampered the research on the pathogenic mechanisms of this organism and the development of multivalent vaccines against A. pleuropneumoniae infection. To compare the gene expressions in the A. pleuropneumoniae strains CVCC259 (serotype 1) and CVCC261 (serotype 3), we screened the differentially expressed genes in the two strains by performing representational difference analysis (RDA). Northern blot analyses were used to confirm the results of RDA. We identified 22 differentially expressed genes in the CVCC259 strain and 20 differentially expressed genes in the CVCC261 strain, and these genes were classified into 11 groups: (1) genes encoding APX toxins; (2) genes encoding transferrin-binding protein; (3) genes involved in lipopolysaccharide (LPS) biosynthesis; (4) genes encoding autotransporter adhesin; (5) genes involved in metabolism; (6) genes involved in the ATP-binding cassette (ABC) transporter system; (7) genes encoding molecular chaperones; (8) genes involved in bacterial transcription and nucleic acid metabolism; (9) a gene encoding protease; (10) genes encoding lipoprotein/membrane protein; and (11) genes encoding various hypothetical proteins. This is the first report on the systematic application of RDA for the analysis of differential gene expression in A. pleuropneumoniae serotypes 1 and 3. The determination of these differentially expressed genes will serve as an indicator for future research on the pathogenic mechanisms of A. pleuropneumoniae and the development of a multivalent vaccine against A. pleuropneumoniae infection.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.784-788
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• 2007

Growth hormone-releasing hormone (GHRH), a hypothalamic neuropeptide can stimulate the growth hormone secretion from the anterior pituitary. In this study, a porcine GHRH expression plasmid pHC-GHRH was used to enhance growth performance through ectopic expressions in muscle tissues of rats. Rats injected with the plasmid of pHC-GHRH and pCMV-GHRH exhibited cumulative weight gains 6.4% and 1% greater than controls. During a 5-day period, significant weight gain differences were observed as follows compared with that of control: during 5-10 days post-injection (DPI) period, the group pHC-GHRH on average 14.5% heavier than controls, $40.73{\pm}0.88$ g vs. $35.57{\pm}1.23$ g (p = 0.0023); during 10-15 DPI period, the group pHC-GHRH on average 13.6% heavier than controls, $37.49{\pm}2.85$ g vs. $33.00{\pm}1.56$ g (p = 0.0146); during 15-20 DPI period, the group pHC-GHRH on average 17.8% heavier than controls, $25.64{\pm}1.39$ g vs. $21.77{\pm}1.27$ g (p<0.05). In addition, plasmids-treated rats maintained higher serum IGF-I than controls. Significant differences of IGF-I were observed on 13 DPI and on 40 DPI in pHC-GHRH group compared with that of controls. This was accomplished through the use of an improved expression cassette that included the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.5-kilobase portion of porcine ${\alpha}$-skeletal muscle actin promoter.

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1098-1105
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• 2017

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones ($Mut^s$) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

• Development and Reproduction
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• v.13 no.3
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• pp.191-198
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• 2009

In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat $\beta$-casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to $12.4\;mg/m{\ell}$ for hGH and from 0.04 to $0.13\;mg/m{\ell}$ for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat $\beta$-casein promoter. Therefore goat $\beta$-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.

• Fisheries and Aquatic Sciences
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• v.4 no.2
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• pp.84-87
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• 2001

Multimeric angiotensin-converting-enzyme-inhibitor (ACE}) containing a trypsin cleavable linker peptide between ACEI was constructed. We made synthetic DNA coding for the ACEI peptide with asymmetric and complementary cohesive ends of linker nucleotides. A tandemly repeated DNA cassette for the expression of concatameric short peptide multimers was constructed by ligating the basic units. The resultant multimeric peptide expressed as soluble and trypsin treated peptide was shown at the same retention time with chemically synthetic ACEI by HPLC. The present results showed that the technique developed for the production of the ACEI multimers with trypsin cleavable linker peptides can be generally applicable to the production of short peptide.

• BMB Reports
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• v.36 no.3
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• pp.275-281
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• 2003

An E1B-defective adenovirus, named r2/Ad carrying the neo expression cassette, was constructed by homologous recombination. The construction, selection (using neomycin as a selective marker), and propagation of the recombinant virus was performed in human embryonic kidney 293 cells (HEK 293). An in vitro study demonstrated that this recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as human glioma tumor cells (U251) and human bladder cells (EJ), but not in some cells with functional p53, such as human adenocarcinoma cells (A549) and human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated, under identical conditions, that the U251 cells were more sensitive to r2/Ad replication than the EJ cells. In this paper, we report that r2/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus and has great potential in cancer gene therapy.

• International Journal of Industrial Entomology and Biomaterials
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• v.43 no.2
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• pp.45-51
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• 2021

High blood cholesterol levels and oxidized cholesterol are risk factors for cardiovascular disease, which displays high annual incidence. Although studies on sericulture products, including pupae, silk protein, and blood lymph, as hypocholesterolemic substances have been reported, insufficient research in this field has been focused on silkworm larvae. Six larval extracts (Low temperature distilled water, LW; hot temperature distilled water, HW; and 30-100% ethanol, E30-E100) were prepared, and their effects on cholesterol metabolism were examined. LW most potently reduced the risk of cholesterol-related disorders. Polyphenols were highly represented in LW, corresponding with its increased antioxidant potency. The cholesterol biosynthesis enzyme, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) was strongly inhibited by LW. Hepatocytes over-expressed LDL receptor (LDLR) after LW stimulation, promoting cholesterol elimination from plasma. LW also increased ATP binding cassette transporter 1 (ABCA1) gene expression, upregulating HDL biogenesis. In conclusion, LW exhibited strong antioxidant activity, suppressed cholesterol biosynthesis, improved LDL uptake from plasma, and upregulated HDL biosynthesis. In aggregate, these activities could reduce blood cholesterol levels and prevent cardiovascular disease.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4807-4814
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• 2012

Purpose: The chemoresistance of human hepatocellular carcinoma (HCC) to cytotoxic drugs, especially intrinsic or acquired multidrug resistance (MDR), still remains a major challenge in the management of HCC. In the present study, possible mechanisms involved in MDR of HCC were identified using a 5-fluorouracil (5-FU)-resistant human HCC cell line. Methods: BEL-7402/5-FU cells were established through continuous culturing parental BEL-7402 cells, imitating the pattern of chemotherapy clinically. Growth curves and chemosensitivity to cytotoxic drugs were determined by MTT assay. Doubling times, colony formation and adherence rates were calculated after cell counting. Morphological alteration, karyotype morphology, and untrastructure were assessed under optical and electron microscopes. The distribution in the cell cycle and drug efflux pump activity were measured by flow cytometry. Furthermore, expression of potential genes involved in MDR of BEL-7402/5-FU cells were detected by immunocytochemistry. Results: Compared to its parental cells, BEL-7402/5-FU cells had a prolonged doubling time, a lower mitotic index, colony efficiency and adhesive ability, and a decreased drug efflux pump activity. The resistant cells tended to grow in clusters and apparent changes of ultrastructures occurred. BEL-7402/5-FU cells presented with an increased proportion in S and G2/M phases with a concomitant decrease in G0/G1 phase. The MDR phenotype of BEL-7402/5-FU might be partly attributed to increased drug efflux pump activity via multidrug resistance protein 1 (MRP1), overexpression of thymidylate synthase (TS), resistance to apoptosis by augmentation of the Bcl-xl/Bax ratio, and intracellular adhesion medicated by E-cadherin (E-cad). P-glycoprotein (P-gp) might play a limited role in the MDR of BEL-7402/5-FU. Conclusion: Increased activity or expression of MRP1, Bcl-xl, TS, and E-cad appear to be involved in the MDR mechanism of BEL-7402/5-FU.