• Journal of fish pathology
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• v.8 no.1
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• pp.37-46
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• 1995

The amino acid analysis of polyhedrin protein and nucleotide sequence of polyhedrin gene in H. cunea nuclear polyhedrosis virus (HcNPV) genome have been studied. Polyhedrin had three polypeptide bands in SDS - polyactylamide gel electrophoresis. The major polypeptide had a molecular weight of 25 kd. The polyhedrin was composed of 17 different amino acids. HcNPV DNA was digested with EcoRI restriction enzyme and hybridized with ($\alpha^{32}P$) -labelled AcNPV polyhedrin gene cDNA. The polyhedrin gene was located on the fragment of EcoRI-H. The EcoRI - H fragment containing polyhedrin gene was cloned into the EcoRI site of pUC8 vector which was confirmed with southern blotting, and the recombinant plasmid containg polyhedrin gene was designated as hPE-H. The promoter region of polyhedrin genomic DNA was sequenced. The sequences identified as the TATA box was found at the 5' flanking region of the polyhedrin genomic DNA approximately -79 bp upstream from the transcriptional start site. But CAAT-like box was not shown near the TATA-like box in the polyhedrin gene. Four tandem repeats with the sequence 5' -CTAATAT-3' and 5'-TAAATAA-3' were found between -141 and -108 or -83 upstream and -52 bp downstream from the translation start site. About -141 bp region upstream from the translational start site was highly AT (78%) rich. The coding region for the polyhedrin starts and ends with ATG and TAA, respectively.

• Current Research on Agriculture and Life Sciences
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• v.4
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• pp.36-41
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• 1986

The transformation of Saccharomyces cerevisiae with YIp26, YRp7 and YEp13 was investigated. Transformation frequences of YIp5, YIp26, YRp7 and YEp13 in Escherichia coli HB101 was $5.1{\times}10^{-4}$, $1.5 {\times}10^{-3}$, $1.3{\times}10^{-3}$, $3{\times}10^{-3}$, respectively. When plasmids were used in covalently closed circular form, transformation frequency of YEp13 was $1.2{\times}10^{-4}$ in S. cerevisiae DBY747 and $3.3{\times}10^{-4}$ in S. cerevisiae MC16 and that of YRp7, YIp26 was $3{\times}10^{-6}$, below $6{\times}10^{-8}$ respectively in S. cerevisiae DBY747 by the method of Ito. Cotransformation of YIp26 and YEp13 in linear form increased the frequency of transformation with efficiences 270-fold higher than transformation of YIp26 only in S. cerevisiae DBY747. In cotransformation of YIp5+YEp13 and YIp26+YRp7 with S. cerevisiae DBY747 by Beggs' method. Expression frequency of YIp5+YEp13 and YIp26+YRp7 was $4{\times}10^{-6}$, $1.5{\times}10^{-6}$, respectively. The recombinant plasmid of cotransformant was thought that YIp26 and YEp13, YIp5 and YEp13, and YIp26 and YRp 7 were ligated in vivo in S. cerevisiae DBY747.

• Microbiology and Biotechnology Letters
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• v.47 no.3
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• pp.343-349
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• 2019

Gul jeotgals (GJs) were prepared using solar salt aged for 3 years. One sample was fermented using starters, such as Bacillus subtilis JS2 and Tetragenococcus halophilus BS2-36 (each $10^6CFU/g$), and another sample was fermented without starters for 49 days at $10^{\circ}C$. Initial counts of bacilli and lactic acid bacteria (LAB) in non-starter GJ were found to be $3.20{\times}10^2$ and $7.67{\times}10^1CFU/g$ on day 0, and increased to $1.37{\times}10^3$ and $1.64{\times}10^6CFU/g$ on day 49. Those of starter GJ were found to be $2.10{\times}10^5$ and $3.30{\times}10^7CFU/g$ on day 49, indicating the growth of starters. The pH values of GJ were $5.93{\pm}0.01$ (non-starter) and $5.92{\pm}0.01$ (starter) on day 0 and decreased to $5.78{\pm}0.01$ (non-starter) and $5.75{\pm}0.01$ (starter) on day 49. Amino-type nitrogen (ANN) production increased continuously during fermentation, and $407.19{\pm}15.85$ (non-starter) and $398.04{\pm}13.73$ (starter) mg% on day 49. Clone libraries of 16S rRNA genes were constructed from total DNA extracted from non-starter GJ on days 7, 21, and 42. Nucleotide sequences of Escherichia coli transformants harboring recombinant pGEM-T easy plasmid containing 16S rRNA gene inserts from different bacterial species were analyzed using BLAST. Uncultured bacterium was the most dominant group and Gram - bacteria such as Acidovorax sp., Afipia sp., and Variovorax sp. were the second dominant group. Bacillus amyloliquefaciens (day 7), Bacillus velezensis (day 21 and 42), and Bacillus subtilis (day 42) were observed, but no lactic acid bacteria were detected. Acidovorax and Variovorax species might play some role in GJ fermentation. Further studies on these bacteria are necessary.

• Journal of Life Science
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• v.24 no.10
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• pp.1046-1054
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• 2014

Beta-1,3-glucanase is widely used in various biotechnological and industrial processes, with over-production required to enable versatile utilization. We examined the overexpression of ${\beta}$-1,3-glucanase (EXGA) from Aspergillus oryzae using ${\delta}$-sequence-mediated integration. We constructed $pRS{\delta}$-exgA and $pRS{\delta}K$-exgA plasmids for integration of the EXGA gene into various chromosomes of Saccharomyces cerevisiae. These plasmids contain the ADH1 promoter for constitutive expression, a signal sequence (exoinulinase signal sequence [INU1 s.s]) for secretory production, and a ${\delta}$-sequence for integration of ${\beta}$-1,3-glucanase. The $pRS{\delta}$-exgA plasmid was transformed into the S. cerevisiae $BY4742{\Delta}exg1$ strain, and ${\beta}$-1.3-glucanase was stably overexpressed and secreted. Another plasmid, $pRS{\delta}K$-exgA, was introduced into the S. cerevisiae $BY4742{\Delta}exg1$ (YKY082) strain, and overexpression of ${\beta}$-1,3-glucanase was examined by inducible integration under geneticin selection. The activity of ${\beta}$-1,3-glucanase increased in accordance with a rise in the geneticin concentration, with 0.8 mg/ml of geneticin suitable for overexpression of ${\beta}$-1,3-glucanase. Subsequently, $pRS{\delta}K$-exgA was repeatedly transformed for sequential ${\delta}$-integration. The activity of ${\beta}$-1,3-glucanase reached about 0.063 unit/ml/$OD_{600}$, 0.095 unit/ml/$OD_{600}$, 0.131 unit/ml/$OD_{600}$ and 0.165 unit/ml/$OD_{600}$ by the first, second, third, and fourth round of integration, respectively. According to the increase in the activity of ${\beta}$-1,3-glucanase by sequential ${\delta}$-integration, the copy number (integration rate) of the EXGA gene also increased in various chromosomes. These results suggest that recombinant ${\beta}$-1,3-glucanase activity can be sequentially increased by repeated ${\delta}$-sequence integration.

• Microbiology and Biotechnology Letters
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• v.37 no.4
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• pp.389-398
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• 2009

Development of expression vectors is important for the basic and applied researches on kimchi LAB (lactic acid bacteria). An expression vector, pSJE6c was constructed by inserting P6C promoter sequence from Lactococcus lactis into pSJE, a shuttle vector for E. coli and Leuconostoc species. To test the efficiency of pSJE6c, aga ($\alpha$-galactosidase) and lacZ ($\beta$-galactosidase) genes were expressed in Lactobacillus brevis 2.14. Compared to the pSJE, expression levels of both genes were increased, indicating P6C promoter was better than indigenous promoters. Enzyme activities of L. brevis cells harboring pSJE6caga (pSJE6c with aga) or pSJE6Z (pSJE6c with lacZ) were 1.5-2 fold higher than those with pSJEaga (pSJE with aga) or pSJEZ (pSJE with lacZ). More RNA transcripts were detected in cells harboring pSJE6c based recombinant plasmid. The results indicated that heterologous gene expressions in kimchi LAB could be improved significantly by use of efficient expression vectors.

• Applied Biological Chemistry
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• v.39 no.6
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• pp.443-448
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• 1996

In order to investigate the function of xylA promoter(Pxyl) as regulatory region Pxyl-lacZ fusion gene was constructed by the insertion of xylA promoter to the multiple cloning site of upstream of lacZ gene in a multicopy numbered plasmid pMC1403 containing promoterless lac operon, which was designated pMCX191, and Pxyl-lacZ fragment from pMCX191 was inserted to low copy numbered plasmid pLG339, designated pLGX191. The expressions of ${\beta}-galactosidase$ in these recombinant plasmids containing Pxyl-lacZ fusion gene were induced strongly by the addition of xylose, repressed by the addition of 0.2% glucose in the presence of xylose. The catabolite repressions were derepressed by the addition of 1 mM cAMP as same as native xylA gene. The fragment of xylA promoter was partially deleted from the upstream of xylA promoter by exonuclease III to investigate the regulation site of xylA promoter and the degrees of deletion derivatives of xylA promoter were analyzed by the DNA base sequencing. By the investigations of the induction by xylose, repression by glucose and derepression by cAMP on xylose isomerase production, the regulation site of xylA promoter may be located in segment between -165 and -59 bp upstream from the initiation site of xylA translation.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.497-506
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• 1998

The expression in Escherichia coli of a cloned insecticidal protein (ICP) gene from Bacillus thuringiensis var. kurstaki HD1 in pHLN1-80 (+) and pHLN2-80(-) plasmids was investigated through deletions in promoters, transcription start point, and termination region. Six recombinant plasmids were constructed in an attempt to analyze the overexpression of the ICP in relations to its gene structure. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone was not overexpressed which having only -80 bp (contained BtI promoter) part of the ICP gene promoter (without Plac promoter), the right-oriented ICP gene and the termination region. Removal of 350 bp from upstream region of the Plac of the clone pHLN2-80 (-) resulted in overexpression of the ICP. One clone was not overexpressed in which the clone consisted of -72 bp part of the ICP promoter without the transcription start point and the transcriptional termination region, and having the right-oriented ICP gene sequence. One clone consisting of the inverted ICP gene sequence, the -72 bp ICP gene promoter, and without the termination region caused overexpression. One clone which consisted of the inverted ICP gene, the -72 bp ICP gene promoter and the termination sequence was overexpressed. These results indicated that the Plac promoter, transcription termination region, the inverted ICP gene insertion, and the -80 bp or -72 bp part of the ICP gene promoters were concerned in the overexpression of the ICP gene in the recombinant plasmid, and also the overexpression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.7
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• pp.922-929
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• 2019

Objective: Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. Methods: Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3'-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3'-UTR and pGL3 Control LDLR mutant-3'-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine premiR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3'-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. Results: Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR3'-UTR-driven (p<0.01), but not mutant LDLR-3'-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = -0.656, p<0.05). Conclusion: LDLR is a potential target of miR-20a, which might directly bind the LDLR 3'-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.112-118
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• 2002

Sporulation in the fission yeast Schizosaccharomyces pombe has been regarded as an important model of cellular development and differentiation. S. pombe cells proliferate by mitosis and binary fission on growth medium. Deprivation of nutrients especially nitrogen sources, causes the cessation of mitosis and initiates sexual reproduction by matting between two sexually compatible cell types. Meiosis is then followed in a diploid cell in the absence of nitrogen source. DNA fragment complemented with the mutations of sporulation gene was isolated from the S. pombe gene library constructed in the vector, pDB 248' and designated as pDB(spo5)1. We futher analyzed six recombinant plasmids, pDB(spo5)2, pDB(spo5)3, pDB(spo5)4, pDB(spo5)5, pDB (spo5)6, pDB(spo5)7 and found each of these plasmids is able to rescue the spo5-2, spo5-3, spo5-4, spo5-5, spo5-6, spo5-7 mutations, respectively. Mapping of the integrated plasmid into the homologous site of the S. pombe chromosomes demonstrated that pDB(spo5)1, and pDB(spu5)Rl contained the spo5 gene. Transcripts of spo5 gene were analyzed by Northern hybridization. Two transcripts of 3.2 kb and 2.5kb were detected with 5kb Hind Ⅲ fragment containing a part of the spo5 gene as a probe. The small mRNA(2.5kb) appeared only when a wild-type strain was cultured in the absence of nitrogen source in which condition the large mRNA (3.2kb) was produced constitutively. Appearance of a 2.5kb spo5-mRNA depends upon the function of the meil, mei2 and mei3 genes.

• The Korean Journal of Nuclear Medicine
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• v.38 no.4
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• pp.294-299
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• 2004

Purpose: Both human NIS and mutant $D_2R$ transgenes are proposed as reporting system in transplanted cell tracking. Using hepatoma cell lines, we constructed a dual reporter system containing human sodium-iodide symporter (hNIS) and dopamine 2 receptor ($D_2R$) and compared its characteristics. Materials and Methods: The recombinant plasmid ($pIRES-hNIS/D_2R$) was constructed with IRES (internal ribosome entry site) under control of the CMV promoter $pIRES-hNIS/D_2R$ was transfected to human hepatoma SK-Hep1 cell line with lipofectamine. HEP-ND ($SK-Hep1-hNIS/D_2R$) cells stably expressing hNIS and $D_2R$ was established by selection with G418 for two weeks. RT-PCR was performed to investigate the expression of both hNIS and $D_2R$ genes. The expressions of hNIS and $D_2R$ were measured by $^{125}I$ uptake assays and receptor binding assays. Specific binding of $D_2R$ to $[^3H]spiperone$ was verified by Scatchard plot with (+) butaclamol as a specific inhibitor. $K_d\;and\;B_{max}$ values were estimated. The correlation between hNIS and $D_2R$ expression was compared by using each clone. Results: Similar quantities of hNIS and $D_2R$ genes were expressed on HEP-ND as RT-PCR assays. HEP-ND cells showed 30 to 40 fold higher radioiodine uptakes than those of parental SK-Hep1 cells. $^{125}I$ uptake in HEP-ND cells was completely inhibited by $KClO_4$, a NIS inhibitor Specific binding to HEP-ND cells was saturable and the $K_d\;and\;B_{max}$ values for HEP-ND cells were 2.92 nM, 745.25 fmol/mg protein and 2.91nM, 1323 fmole/mg protein in two clones, respectively. The radioiodine uptake by hNIS activity and $D_2R$ binding was highly correlated. Conclusion: We developed a dual positron and gamma imaging reporter system of hNIS and $D_2R$ in a stably transfected cell line. We expect that $D_2R$ and hNIS genes can complement mutually as a nuclear reporting system or that $D_2R$ can be used as reporter gene when hNIS gene were used as a treatment gene.