• International Journal of Industrial Entomology and Biomaterials
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• v.1 no.2
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• pp.115-122
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• 2000

A Bacillus thuringiensis designated NT0423, belonging to B. thuringiensis subsp. aizawai (H 7), was isolated from samples of dust and soil of sericultural farms. B. thuringiensis NT0423 having dualspecificity against Lepidoptera and Diptera produced bipyramidal inclusions consisting of two major polypeptides of approximately 130- and 70-kDa. Proteolytic processing by trypsin and gut juice of Bombyx mori yielded predominant proteins with molecular masses of about 66-kDa. The whole crystal protein of B. thuringiensis NT0423 immunologically was related to that of B. thuringiensis subsp. aizawai. PCR analysis showed that B. thuringiensis NT0423 has at least five crystal protein genes including cryIA(a), cryIA(b), cryIC, cryID and cryIIA, and southern blot was determined the location of each gene on intact and enzyme-digested plasmid DNA fragments. Except for cryIA(a) gene on the high molecular weight plasmid of 165-kb, all of four genes were located on the plasmid of 66-kb. The production of $\beta$-exotoxin from B. thuringiensis NT0423 was identified by the HPLC analysis. In addition, the $\beta$-exotoxin showed its ability to prevent pupation of treated larvae of house flies (Musca domestica) from developing into normal adults.

• International Journal of Industrial Entomology and Biomaterials
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• v.3 no.1
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• pp.19-23
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• 2001

Two B. thuringiensis strains, which possess mosquitocidal activities, were isolated from Korean soil samples and named K-1205-1 and K-1381-1. Serological studies indicated that K-1205-1 and K-1381-1 belonged to B. thuringiensis subsp. kurstaki (H3a3b3c) and subsp. aizawai (H7), respectively. K-1205-1 produced typical bipyramidal parasporal inclusions, but K-1381-1 produced irregular bipyramidal shape. Total plasmid DNA patterns analysis shewed that K-1205-1 and K- 1381-1 were different from their reference strains, subsp. kurstaki and subsp. aizawai, respectively, in high molecules, whereas their crystal protein patterns showed no difference. The cry gene contents of K-1205-1 and K-1381-1 were identical with those of the reference strains. Mosquitocidal activities of crystal proteins produced by K-1205-1 and K-1381-1 were significantly high by about 40-50 folds at $LC_50$ when compared to those of subsp. kurstaki and subsp. aizawai. Finally, in southern blot analysis using cry1A-type specific probe, K-1205-1 and K-1381-1 had different bands from subsp. kurstaki and subsp. aizawai, respectively. In conclusion, our results suggest that K-1205-1 and K-1381-1 appear to be new moquitocidal B. thuringiensis strains isolated from Korean soil.

• International Journal of Industrial Entomology and Biomaterials
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• v.3 no.1
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• pp.37-41
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• 2001

The ecdysteroid UDP-glucosyltransferase (egt) gene isolated from Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain was compared to its homologue from Autographa californica NPV (AcNPV) and Bm NPV T3. The egt gene of BmNPV-K1 encoded 506 amino acid open reading frame, and was 99.6% identical at the amino acid level and 99.2% identical at the nucleotide level to BmNPV T3. The BmNPV-K1 egt gene showed highly identity to AcNPV and BmNPV T3 strain. The BmNPV-K1 egt gene was different from amino acid sequence at 2 positions, 19 and 72, in BmNPV T3. The genomic location of egt gene in the BmNPV-K1 was confirmed by Southern blot analysis and its expression patterns at the transcriptional level in the infected cells were confirmed by Northern hybridization analysis. Transcripts of the egt of Bm NPV-K1 peaked around 12 hrs postinfection (p.i.) and reduced at 24 hrs p.i.

• Journal of Microbiology
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• v.42 no.4
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• pp.319-327
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• 2004

An additional amylase, besides the typical $\alpha-amylase,$ was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a malto­genic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the $\beta-galactosidase$ activity produced from the bbmA promoter fused to the amino terminus of the lacZ struc­tural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The pro­moter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing $\beta--cyclodextrin\;(\beta-CD),$ maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the $\beta-CD$ hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regu­latory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the $\beta-CD$ hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing $\beta-CD$ in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and $\beta-CD$ utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.

• BMB Reports
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• v.40 no.5
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• pp.791-800
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• 2007

Ethylene performs an important function in plant growth and development. 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), the key enzyme involved in ethylene biosynthesis, has been the focus of most ethylene studies. Here, a cotton ACS gene referred to as Gossypium hirsutum ACS1 (GhACS1), was isolated. The full-length cDNA of GhACS1 encodes for a 476-amino acid protein which harbors seven conserved regions, 11 invariant amino acid residues, and the PLP binding active site, all of which characterize ACC synthases. Alignment analysis showed that GhACS1 shared a high degree of identity with other known ACC synthases from different species. Two introns were detected in the genomic DNA sequence, and the results of Southern blot analysis suggested that there might be a multi-gene family encoding for ACC synthase in cotton. From the phylogenetic tree constructed with 24 different kinds of ACC synthases, we determined that GhACS1 falls into group II, and was closely associated with the wound-inducible ACS of citrus. The analysis of the 5' flanking region of GhACS1 revealed a group of putative cis-acting elements. The results of expression analysis showed that GhACS1 displayed its transient expression nature after wounding, abscisic acid (ABA), and $CuCl_2$ treatments. These results indicate that GhACS1, which was transiently expressed in response to certain stimuli, may be involved in the production of ethylene for the transmission of stress signals.

• The Journal of Korean Society of Virology
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• v.26 no.2
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• pp.151-162
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• 1996

Pseudorabies is caused by Pseudorabies virus (PRV: Aujeszky's disease virus) of Herpesviridae that is characterized by 100 to 150nm in size with a linear double-stranded DNA molecule with of approximately $90{\times}10^6Da$. This disease affects most of domestic animals such as swine, cattle, dog, sheep, cat, chicken, etc. causing high mortality and economic losses. In swine, young piglets show high mortality and pregnant sows, reproductive failures. However the adult swine reveals no clinical signs in general. But they become a carrier state and play an important role for propagation of the disease. In this study, the nucleotide sequence of major casid protein gene of PRV, Yangsan strain isolated from the diseased swine in Korea was analyzed, and the recombinant MCP was produced by expression of the MCP gene in Sf-9 cell using baculovirus transfer vector system. As result, in BamHI digestion, MCP gene locus of PRV YS strain showed different from that of Indiana S strain. The patterns of enzyme mapping were also found to be unidentical each other. The sequence of the MCP gene partially analyzed showed 98.09% identity to Indiana S strain. The expression of MCP in Sf-9 cell cotransfected by pVLMCP-44 baculovirus expression vector was characterized by Southern blot hybridization, immunofluoresent and immunocytochemical tests, SDS-PAGE and Western blotting. The rMCP with M.W. 142kDa was most effectively expressed in Sf-9 cells at the 3-4th days post inoculation of the recombinant baculovirus by 2 moi.

• Plant Biotechnology Reports
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• v.5 no.2
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• pp.147-156
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• 2011

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with ${\beta}$-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with $1mg\;l^{-1}$ indole-3-butyric acid and $15mg\;l^{-1}$ hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for ${\beta}$-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of $3.9{\pm}0.39$ transgenic plantlets per explant was achieved in the present transformation system. It took only 2-3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1329-1333
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• 2004

To isolate the microsatellites from the chromosomal DNA of the Korean cattle (Hanwoo) and to use those for the genetic selection, four bacteriophage genomic libraries containing the chromosomal DNA of six Hanwoo steers showing the differences in meat quality and quantity were used. Screening of the genomic libraries using $^{32}P-radiolabeled 5'-({CA})_{12}-3$nucleotide as a probe, resulted in isolation of about 3,000 positive candidate bacteriophage clones that contain $(CA)_n$-type dinucleotide microsatellites. After confirming the presence of microsatellite in each positive candidate clone by Southern blot analysis, the DNA fragments that include microsatellite and flanking sequences possessing less than 2 kb in size, were subcloned into plasmid vector. Results from the analysis of microsatellite length polymorphism, using twenty-two PCR primers designed from flanking region of each microsatellite DNA, demonstrated that 208 and 210 alleles of HW-YU-MS#3 were closely related to the economic traits such as marbling score, daily gain, backfat thickness and M. longissimus dorsi area in Hanwoo. Interestingly, HW-YU-MS#3 microsatellite was localized in bovine chromosome 17 on which QTLs related to regulation of the body fat content and muscle ypertrophy locus are previously known to exist. Taken together, the results from the present study suggest the possible use of the two alleles as a DNA marker related to economic trait to select the Hanwoo in the future.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.10
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• pp.1390-1394
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• 2004

We have achieved efficient transformation system for forage-type tall fescue plants by Agrobacterium tumefaciens. Mature seed-derived embryogenic calli were infected and co-cultivated with each of three A. tumefaciens strains, all of which harbored a standard binary vector pIG121Hm encoding the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing $\beta$-glucuronidase (intron-GUS) genes in the T-DNA region. Transformation efficiency was influenced by the A. tumefaciens strain, addition of the phenolic compound acetosyringone and duration of vacuum treatment. Of the three A. tumefaciens strains tested, EHA101/pIG121Hm was found to be most effective followed by GV3101/pIG121Hm and LBA4404/pIG121Hm for transient GUS expression after 3 days co-cultivation. Inclusion of 100 $\mu$M acetosyringone in both the inoculation and co-cultivation media lead to an improvement in transient GUS expression observed in targeted calli. Vacuum treatment during infection of calli with A. tumefaciens strains increased transformation efficiency. The highest stable transformation efficiency of transgenic plants was obtained when mature seed-derived calli infected with A. tumefaciens EHA101/pIG121Hm in the presence of 100 $\mu$M acetosyringone and vacuum treatment for 30 min. Southern blot analysis indicated integration of the transgene into the genome of tall fescue. The transformation system developed in this study would be useful for Agrobacterium-mediated genetic transformation of tall fescue plants with genes of agronomic importance.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.1
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• pp.20-25
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• 2009

Xenotransplantation of pig organs into humans is a potential solution for the shortage of donor organs for transplantation. However, multiple immune barriers preclude its clinical application. In particular, the initial type of rejection in xenotransplantation is an acute cellular rejection by host $CD8^+$ cytotoxic T lymphocyte (CTL) cells that react to donor major histocompatibility complex (MHC) class I. The human cytomegalovirus (HCMV) glycoprotein Unique Short (US) 2 specifically targets MHC class I heavy chains to relocate them from the endoplasmic reticulum (ER) membrane to the cytosol, where they are degraded by the proteasome. In this study we transfected the US2 gene into minipig fetal fibroblasts and established four US2 clonal cell lines. The integration of US2 into transgenic fetal cells was confirmed using PCR and Southern blot assay. The reduction of Swine Leukocyte Antigen (SLA)-I by US2 was also detected using Flow cytometry assay (FACS). The FACS analysis of the US2 clonal cell lines demonstrated a substantial reduction in SLA-I surface expression. The level (44% to 76%) of SLA-I expression in US2 clonal cell lines was decreased relative to the control. In cytotoxicity assay the rate of $CD8^+$ T cell-mediated cytotoxicity was significantly reduced to 23.8${\pm}$15.1% compared to the control (59.8${\pm}$8.4%, p<0.05). In conclusion, US2 can directly protect against $CD8^+$-mediated cell lysis. These results indicate that the expression of US2 in pig cells may provide a new approach to overcome the CTL-mediated immune rejection in xenotransplantation.