• The Korean Journal of Food And Nutrition
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• v.9 no.2
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• pp.123-136
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• 1996

IPSKCDNA gene(1.8 kbp) encoding rat brain IP3K enzyme contained Not I restric site in open reading frame. The Not I sequence, GCGGCCGC, was converted to GCAGCCGC by site-directed mutagenesis. The mutated IP3KcDNA was digested with EcoR I and ligated with EcoR I-restricted psp72·Not2 vector. The resulting psp72 · Not2-IP3KCDNA was digested with the Not I restriction enzyme and then subcloned into the Not I -digested PZIP · NeoSV(X) mammalian expression vector. The PZIP · NeoSV(X) -IPSKCDNA was transfected into CCL39 hamster lung fibroblast cells. The efficiency of the expressed IPSKCDNA gene was significantly higher than expected generally, not only a mean 5-fold increase in the amount of enzyme, but also 16-fold increase in enzyme activity from tractsfected CCL39 cells by the method of Western blot using anti-lP3K antibodies. Both distribution of IPSK in various rat tissues and biochemical properties were discussed.

• Journal of the Korean Magnetic Resonance Society
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• v.5 no.2
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• pp.130-137
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• 2001

Backbone dynamics of the catalytic residues in free and steroid-bound $\Delta$$^{5}$ -3- ketosteroid isomerase from Pseudomonas testosteroni has been examined by $^{15}$ N relaxation measurements. The relaxation data were analyzed using the model-free formalism to extract the model-free parameters (S$^2$, $\tau$$_{e}$, and R$_{ex}$). Tyr-34 and Asp-99 exhibit enhanced high-frequency (pico- to nanosecond) internal motions in the free enzyme, which are restricted upon ligand binding, while Asp-38 experiences severe restriction of the internal motions in the fee enzyme, suggesting that Tyr-14 and Asp-99 are more actively involved in the ligand binding than Asp-38. The results also indicate that the H-bond network in the catalytic cavity might be slightly strengthened upon ligand binding, which may have some implications on the enzyme mechanism.he enzyme mechanism.m.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1170-1177
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• 2005

Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to monitor inoculated oil-degrading microorganisms during bioremedial treatability tests. A pair of universal primers, fluorescently labeled 521F and 1392R, was employed to amplify small subunit rDNA in order to simultaneously detect two bacterial strains, Corynebacterium sp. IC10 and Sphingomonas sp. KH3-2, and a yeast strain, Yarrowia lipolytica 180. Digestion of the 5'-end fluorescence/labeled PCR products with HhaI produced specific terminal-restriction fragments (T-RFs) of 185 and 442 bases, corresponding to Corynebacterium sp. IC10 and Y. lipolytica 180, respectively. The enzyme NruI produced a specific T-RF of 338 bases for Sphingomonas sp. KH3-2. The detection limit for oildegrading microorganisms that were inoculated into natural environments was determined to be $0.01\%$ of the total microbial count, regardless of the background environment. When three oil-degrading microorganisms were released into oil-contaminated sand microenvironments, strains IC10 and 180 survived for 35 days after inoculation, whereas strain KH3-2 was detected at 8 days, but not at 35 days. This result implies that T-RFLP could be a useful tool for monitoring the survival and relative abundance of specific microbial strains inoculated into contaminated environments.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.334-339
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• 1999

More innovative molecular markers were investigated for rapid and consistent differentiation of Metarhizium anisopliae var. majus from M. anisopliae var. anisopliae. A total of 28 isolates were obtained from various countries and hosts: 13 isolates of M. anisopliae var. anisopliae, 12 isolates of M. anisopliae var. majus, and 3 isolates of M. anisopliae collected in Korea. This study involved restriction enzyme digestions of a PCR product amplified from nuclear internally transcribed spacer (ITS) and a portion of the 28S rDNA regions. Among 11 different restriction enzymes used in this study, MboⅠ digestion particularly produced a restriction pattern that had characteristics of M. anisopliae var. majus. This restriction pattern was consistent in all isolates of M. anisopliae var. majus regardless of their geographic origins and insect hosts. Mapping experiments revealed that MboⅠ sites of M. anisopliae var. majus are identical to those of M. anisopliae var. anisopliae with an exception for the presence of an additional site in the PCR product. Results from this study provide an additional method for identification and differentiation of isolates of these two varieties of M. anisopliae for use in the field and laboratory experiments.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.104-110
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• 1999

Field infectious bursal disease viruses (IBDVs) were isolated from IBDV-suspected commercial chickens. The variable region in VP2 gene of six Korean IBDV isolates (K-IBDVs) and IBD vaccines was examined using the reverse transcriptase / polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay. With all K-IBDVs and vaccine IBDVs, a 474-bp fragment of the VP2 gene was amplified and tested with various restriction enzymes. Restriction enzymes BstNI and StyI differentiated K-IBDV isolates and IBD vaccines into four groups. Restriction enzyme profiles of K-IBDV isolates were different from them of IBD vaccines. K-IBDV isolates except for 310 isolate had specific SspI and TaqI recognition sites, which were recognized in highly virulent IBDVs, but IBD vaccines had no those sites. This study showed that RT/PCR-RFLP assay was thought to be valuable tool for differentiation of IBDVs and identification of highly virulent IBDV.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.217.2-217
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• 2005

• Mycobiology
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• v.31 no.1
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• pp.32-35
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• 2003

Restriction enzyme-mediated integration(REMI) was used to transform uracil auxotrophs of Pleurotus ostreatus to prototrophy. When protoplasts of Pleurotus ostreatus were treated by the reaction mixture containing 10 units of BamHI, the frequency of REMI was about 64 transformants per 1 ${\mu}g$ of DNA. This efficiency was increased by 14.2 times compared with that of the conventional PEG transformation. The optimal condition for REMI of P. ostreatus was achieved when 1 ${\mu}g$ of linearized pTRura3-2 DNA was added into $1{\times}10^7$ protoplasts along with 10 units BamHI. Southern blot analysis revealed that about 50% of transformants examined were caused by REMI event and 30% carried single copy insertion at the genome. This suggested that the REMI method might be a useful tool for efficient transformation and tagging mutagenesis of P. ostreatus.

• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.536-543
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• 1995

Multicopy integration vector is a very useful vector system in that they can be integrated into chromosomal DNA in several copies and stably maintained under non-selective conditions. To develop a multicopy integration vector system in the yeast Yarrowia lipolytica, P-type ribosomal DNA was cloned from Y lipolytica. A HindIII-BglII fragment of the cloned rDNA and a promoterless URA3 gene were inserted into pGEM1, generating multicopy integration vectors, pMIYL-1 and pMIYL-2. The rDNA fragment is for targeted homologous recombination between the vector and the chromosomal DNA of Y. lipolytica, and the promoterless URA3 gene is a defective selection marker for inducing multicopy integration. pMIYL-1 and pMIYL-2 have an unique restriction enzyme site, KpnI, and two unique restriction enzyme sites, KpnI and EcoRI, repectively, which can be used for targeting of the vectors into the rDNA of Y. lipolytica chromosomal DNA. After transformation of the vectors into Y. lipolytica, copy number and stability were analyzed by Southern hybridization. The vectors were found to be present in less than 5 copies per cell and were stably maintained during growth in non-selective media.

• Journal of Environmental Science International
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• v.5 no.5
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• pp.605-610
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• 1996

The bacteriophage from the fresh oyster, Crassostrea Virginica which is specific to the marine bacterium was isolated and characterized. Among the foci different vibrio species and the five different serotypes of Vibrio parahaemolyticus host strains tested, only two strains of the parahaemolyticus possessing K17, K52 antigens were highly sensitive to the phage. The size of the isolated plaque was 0.4mm and the electron microscopic head size of the isolated phage was about 67 nm long and 83 nm wide. PFU/ml was 1.25$\times$ $10^{11}$. The phase was sensitive to chloroform but resistant to acetone or methanol. The assay of the isolated phase nucleic acid was deoxyribonucleic acid. The restriction enzyme pattern showed 14 fragment from Hind III and 4 fragments from Eco R I. Two different antigenic groups showed-similar restriction enzyme patterns.

• The Journal of the Korean Society for Microbiology
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• v.35 no.1
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• pp.49-60
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• 2000

ITSI-5.8S-ITSII rDNA region was amplified from the reference strains and clinical isolates with ITS1 and ITS4 primers. These primers amplified DNA fragments of 550 bp in Microsporum audouinii and Trichophyton violaceum, 700 bp in Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, and Trichophyton tonsurans, and 750 bp in Microsporum ferreugineum and Microsporum canis. The restriction enzyme patterns of PCR products digested with 13 restriction enzyme including PstI were distint among the genera, whereas identical in the same species. Examination of the ITS (Internal Transcribed Spacers)1 nucleotide sequence revealed that there was the genetic difference in each genera and species. Phylogenetic relationship among each species showed that the Trichophyton mentagrophytes was more closely related Trichophyton tonsurans than Trichophyton rubrum, and Microsporum gypseum was less related than Microsporum spp..