• Biomedical Science Letters
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• v.2 no.2
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• pp.231-240
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• 1996

Human blood clotting (coagulation) factor 9 cDNA which codes for 461 amino acid has been cloned by screening human fetal liver cDNA library using PCR. This 1.4 kb cDNA spanning from the ATG initiation codon to the TAA termination codon was cloned into bacterial .expression vector pGEX-2T, generating pGEX-F9 plasmid. The plasmid pGEX-F9 expresses about 73 kDa GST (Glutathione S-transferase)-Factor 9 fusion protein when introduced into E. coli. Western blot analysis using polyclonal antibody raised against human factor 9 confirmed this fusion protein contains factor 9 protein. The level of GST-factor 9 expression was about 20% of total protein and the purification of fusion protein was efficiently achieved by using GST agarose bead based on one step purification protocol.

• Archives of Pharmacal Research
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• v.21 no.2
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• pp.128-134
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• 1998

Nitric oxide synthase, NOS (EC.1.14.13.39), was purified from bovine pancreas over 5,500-fold with a 7.6% yield using 30% ammonium sulfate precipitation, and $2^1$,$5^1$-ADP-agarose and calmodulin-agarose affinity chromatography. The purified bovine pancreatic NOS (bpNOS) showed a single band on SDS-PAGE corresponding to an apparent molecular mass of 160 kDa, whereas it was 320 kDa on non-denaturating gel-filtration. This indicated a homodimeric nature of the enzyme. The specific activity of the purified bpNOS was 31.67 nmol L-citrulline fored/mtn/mg protein and an apparent $K\textrm{m}$ for L-arginine was 15.72 $\mu\textrm{M}$, The enzyme activity was dependent on $Ca^{2+}$ and calmodulin, and to a lesser extent on NADPH, FAD and FMN. $H_4B$ was not required as a cofactor for the activity. In an inhibition experiment with L-arginine analogues, $N^G$-nitro-L-arginine (NNA) had the most potent inhibitory effect on bpNOS, and $N^{G}$, $N^{G1}$-dimethyl-L-arginine (symmetric; sDMA) did not have any inhibitory effect. Immunohistochemical analysis of the bovine pancreas using brain type NOS antibody (anti-bNOS antibody) revealed that acinar cells showed strong immunoreactivity against the antibody.

• Korean Journal of Plant Tissue Culture
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• v.28 no.3
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• pp.153-157
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• 2001

Protoplasts were isolated from hypocotyls, cotyledons, and young leaves of Chinese cabbage grown under in vitro environmental condition. An enzyme mixture of 1% Cellulysin and 0.5% Macerozyme in combination with 0.4 M mannitol was most effective condition for protoplast isolation. The highest yield of protoplasts, 7.6$\times$10$^{5}$ protoplast/g of fresh weight, was obtained from the treatment of leaves for 12~16 hours at 27~28$^{\circ}C$ with shaking at 30 rpm. The most suitable medium for an initial cell division was K8p basal medium supplemented with 5 mg/L 2,4-D and 2 mg/L kinetin. Within 7~10 days, protoplasts derived from hypocotyl and cotyledon tissues formed cell colonies. When the cells were grown at the size of 8~10 cells, they were embedded into semi-solid medium containing 0.2% agarose. Calli derived from protoplast culture were transferred to the 100 different types of plant regeneration media, but no completely regenerated plants from inbred lines of Chinese cabbage used for this study wore obtained, though frequent rooting took place in several media tested.

• Journal of Sericultural and Entomological Science
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• v.35 no.2
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• pp.120-128
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• 1993

The objective of this study is to identify plasmids of Bacillus thuringiensis var. kurstaki HD-1(B. t k HD-1) toxic to lepidopteran larvae. The results from agarose gel electrophoresis indicated that the bacterium contained 9 plasmids with approximate sizes of 1.4, 4.9, 5.4, 9.3, 10, 29, 44, 52, and 150 megadaltons(Md). By treating the wild type of B. t k HD-1 with either SDS or EtBr as curing agent, 26 cured mutants of the bacterium were obtained, 9 of them were crystallifereous(cry+) and the others acrystallifereous(cry-). Plasmids from B. t k HD-1 were transferred to B. cereus 569 strR cry- recipients(Bc569 M1). Among 13 isolates of Bc569 M1 transcipient, 11 of them were capable of producing the crystal toxic proteins. The plasmid patterns of Bc569 M1 transcipients and partially curved mutants of B. t k HD-1 on agarose gel electrophoresis suggested that the 29 and 44Md plasmids should be involved in the production of crystalline toxic proteins.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.270-274
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• 1988

Samples used for the enrichment culture were collected from the sea water of suspected chronic petroleum contamination in the vicinity of Pusan, Chungmu and Ulsan ports, Korea. Alkaline Iysis and agarose gel electrophoresis techniques were employed to screen these isolates for the presence of plasmid DNA. There were n little differences in the percentage of isolates containing plasmids between sampling sites of unpolluted sen water (22％) and polluted son water (25％). Bacterial isolates taken from the Bunker-C oil enriched culture showed significantly more plasmid incidence (29％). About two thirds of strains grown on a variety of hydrocarbons were Gram negative strains of which 33％ contained one or more plasmids. Multiple plasmids were observed in 23％ of the plasmid-carrying strains. Forty one percent of the plasmids detected were estimated to have a mass of 20 kb or more.

• KSBB Journal
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• v.5 no.3
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• pp.247-253
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• 1990

These studies were carried out to obtain the transformant from carrot cells by using binary vector pGA472 with NPT II gene to confer kanamycin resistance in the plant cells. The binary vector pGA472 was mobilized from E. coli MC1000 into A. tumefaciens strains isolated in the Korea, C23-1. K29-1, and disarmed Ti-plasmid PC2760, and A28l using a tri-parental mating method with E. coli HB101/pRK2013. Transconjugants, C23-1/pGA472, K29-1/pGA472, PC2 760/pGA472 and A28l/pGA472 were obtaind on the minimum AB media containing tetracycline and kanamycin, were comfirmed to hold the Ti-plasmid and pGA472 binary vector on the 0.7% agarose gel. Transformed carrot calli were initiated on the MS media supplemented with l00$\mu\textrm{g}$/ml kanamycin and 250$\mu\textrm{g}$/ml carbenicillin after co-cultivation of carrot explant and transconjugant Agrobacteria. Selected callus was grown vigouousley for subculture on the medium containing 100$\mu\textrm{g}$/ml kanamycin, thus indication that the selected callus was transformed with NPT II gene.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.3
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• pp.306-316
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• 1997

Embryogenic calli were induced from mature seed scutella of anther culture-derived rice variety Zhonghua 8. Cell suspension cultures were initiated from friable embryogenic calli and utilized as source material for protoplast isolation. Generally, the older and finer cell suspensions gave higher protoplast yields than younger suspension cultures. Protoplasts exhibited sustained cell division and formed microcalli when cultured in KPR medium supplemented with 0.5 mg $l^{-1}$ 2,4-D, 1.0 mg $l^{-1}$ NAA and 0.5 mg $l^{-1}$ zeatin using the agarose embedding procedure without feeder cells. Protoplast plating efficiencies ranged from 0.20 to 0.54％. Microcalli were transferred to MS medium supplemented with 2.0 mg $l^{-1}$ kinetin and 0.5mg $l^{-1}$ NAA for plant regeneration. The regeneration frequencies were 2 to 12％, depending on the cell suspension lines of Zhonghua 8. The plants were transferred to the glasshouse and were fertile.

• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.219-223
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• 1986

In order to preparr the hybridoma cells which produce a monoclonal antibody to human fibroblast interferon(Hu IFN-$\beta$), spleen cells from BALB/cmice immunized with the purified Hu IFN-$\beta$ were fused with NS-O cells, a myeloma cell line. Forty hybrids with high titer among 1300 hybrids Isolated by an ELISA screening method were subcloned using the soft agarose cloning and limiting dilution methods, and 11 hybrids were selected. As a result of iso-typing the hybrids using the mouse monoclonal typing kit, two hybridomas were found to produce 1gG 2a type of monoclonal an-tibodies. The ascites fluid from nude mice inoculated intraperitoneally with the above hybridomas was removed and purified using a protein A-Sepharose CL-4B. Monoclonal antibody was proven to have only the heavy and light chains on SDS-polyacrylamide gel electrophoresis.

• The Journal of Korean Society of Virology
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• v.30 no.1
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• pp.61-70
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• 2000

Standard plaque assay using agarose-overlay has long been used for titration of many infectious virus particle. Plaque assay for the titration of varicella-zoster virus and its live vaccine requires three intermittent agarose overlay to visualize plaques. Overall procedure of the assay takes at least nine days from virus inoculation and microbe contamination including fungi is frequently accompanied during incubation period. We studied whether an immunofocus assay in conjunction with peroxidase-mediated immunohistochemical reaction may replace the standard plaque assay for the virus titration by comparing the two methods. A linear relationship was observed between number of foci and virus dilution. The number of foci in a given dilution of virus appeared a little higher than counted plaques formed in standard plaque assay. Independent titration results obtained from two assay methods for a given dilution of virus demonstrated a strong correlation ($r^2=0.99$). Foci of virus infected cells as revealed by the enzyme reaction could be counted either 4 days post-infection (p.i.) under low magnification (40X) microscopy, or 6 days p.i. by naked eye observation. Larger size of cell cuture plate, virus adsorption at $35^{\circ}C$, and 10% FBS in diluent appeared to be better conditions for the assay. Immunofocus assay will be an effective and dependable titration method for varicella-zoster virus and its live vaccine in place of the standard plaque assay in respect to accuracy, costs, and experimental convenience.

• Research in Plant Disease
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• v.12 no.1
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• pp.62-64
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• 2006

July in 2005, we collected infected maize plant that showing stripe dwarf disease on maize leaf in Jeonbuk provinces including Gochang-gun and conducted genomic dsRNA extraction and RT-PCR. Genomic dsRNA was extracted directly in infected maize plant and electrophoresis in agarose gel. We confirmed 10 segments of genomic dsRNA. We conducted RT-PCR by genomic dsRNA and specific primer of S7, S8 and S10. As a result, specific band of expected size was confirmed respectively. In the results of dsRNA and RT-PCR analysis, we confirmed Rice black-streaked dwarf fijivirus (RBSDV) from naturally infected maize plant. Occurrence of RBSDV of maize plant was dealt 22 ha's damage in maize field. The occurrence rate was 80% in a lot of places of disease.