• Korean Journal of Microbiology
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• v.23 no.1
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• pp.13-19
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• 1985

Acc I endonuclease has been isolated from 300g (wet weight) cells of Acinetobacter calcoaceticus. The cells were broken by using French press at 20, 000p.s.i. After ammonium sulfate fractionation, the enzyme was further purified by heparin agarose, DEAE-sephades, Affi.-gel Blue, phosphocellulose, and hydroxylapatite column chromatography. The purified Acc I endonudlease has a single polypeptide species and its subunit molecular weight was 45,000 ${\pm}$ 1,000 daltons as judged by 10% SDS-polyacrylamide gel electrophoresis. The isolated enzyme was essentially free of contaminating nucleases as judged by homochromatography by using a $^{32}P-labeled$ oligonucleotide. The enzyme showed maximum activity at pH values between 8.0 and 11.0 and in the presence of $MgCl_2$. Acc I endonuclease was maximally active in the absence of NaCl and was completely inhibited at 200 mM NaCl.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2001.05a
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• pp.559-560
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• 2001

The objective of the present study was to analyze genetic variation and characteristics in rainbow trout(Oncorhynchus mykiss) using amplified fragment length polymorphism(AFLP) method as molecular genetic technique, to evaluate the usefulness of AFLP as genetic markers, and to compared the efficiency of agarose and polyacrylamide sequencing gels. The amplified products were performed by agarose and sequencing gel electrophoresis to detect AFLP band patterns, respectively. Using 9 primer combinations, total of 141 AFLP bands were produced, 108 bands(82.4％) of which were polymorphic in agarose gels. In sequencing gels, total of 288 bands were generated, and 220 bands (76.4％) were polymorphic. The level of bandsharing(BS) ranged from 0.18 to 0.32 for the 9 primer combinations tested, with a mean of 0.24. Consequently, AFLP markers of these rainbow trout could be used as genetic information such as species identification, genetic relationship or analysis of genome structure, and selection aids for genetic improvement of economically importment traits in fish species.

• Korean Journal of Microbiology
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• v.25 no.3
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• pp.180-183
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• 1987

Stu I, type II restriction endonuclease, has been purified to homogeneity from Streptomyces tubercidicus (ATCC 25502), and its catalytic properties have been studied. For the purification of Stu I endonuclease free of nonspecific nucleases, DEAE-Sephadex (A-50), QAE-Sephadex (A-50) and Heparin-agarose column chromatography have been performed after ammonium sulfate fractionation of the crude extract. The enzyme was further purified by gel filtration using Sephadex G-100 column to obtain homogeneous form of protein. The single polypeptide species of Stu I endonuclease has a subunit molecular weight of 34,000 $\pm$ 1,000 daltons as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Stu I endonuclease requires $Mg^{2+}$ ion for its activity and is maximally active at neutral pH (7.0-8.0) in the absence of NaCl.

• Journal of the Korean Society of Tobacco Science
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• v.12 no.1
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• pp.9-18
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• 1990

To study the determinants which are involved in the loss of pathogenicity in wilt-inducing Pseudomoms solamcewum, several physlologica I functions were compared in a virulent P. solanacearum strain and an avirulent, spontaneously derived mutant strain. The polyacrylamide gel electrophoresis showed the distinction between two strains in the patterns and the relative intensity of proteins produced intracellularly or extracellularly. Enzyme assays showed that the level of polygalacturonase activity in the culture filtrate of the avirulent mutant was markedly reduced, while carboxymethylcellulase(rondoglucanase) activity in both strains were nearly negligible. These results suggest that the loss of pathogenicity in mutant strain is attributed in part to the reduced production of polygalacturonase. In audit ion, comparative analyses by agarose gel electrophoresis of DNA molecules isolated from both strains show that the pathogenicity genes of p. solanaceerum are not located on plasmid but are on chromosome.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.405-409
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• 2000

We have studied the genome of an obligately commensal thermophile, Symbiobacterium toebii. The chromosome was extracted from pure cultures of S. toebii recently established. Total DNA of S. toebii was resolved by pulsed-field gel electrophoresis (PFGE) into discrete numbers of fragments by digenstion with the endonuclease SspI, SpeI, XbaI, and HpaI. Estimated sizes of fragments produced by the four enzymes and their sum consistently yielded a total genome size of 2.8 Mb. Because restriction endonucleases NotI and SwaI, recognizing 8 bp, released too many fragments, these enzymes could not be used for the estimation of the genome size. Considering no mobility of undigested genome under PFGE, the genome of S. toebii appears to be circular. The presence of extrachromosomal DNA in S. toebii was excluded by the results of the conventional 1% agarose gel electrophoresis and the field inversion gel electrophoresis of undigested S. toebii DNA.

• Environmental Mutagens and Carcinogens
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• v.21 no.2
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• pp.89-94
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• 2001

Recently, single cell gel electrophoresis, also known as comet assay, is widely used for the detection and measurement of DNA strand breaks in vitro and in vivo in many toxicological fields such as radiation exposure, human monitoring and toxicity evaluation. As well defined, comet assay is a sensitive, rapid and visual method for the detection of DNA strand breaks in individual cells. Briefly, a small number of damaged cells suspended in a thin agarose gel on a microscope slide were lysed, unwinded, electrophoresed, and stained with a fluorescent DNA binding dye. The electric current pulled the charged DNA from the nucleus such that relaxed and broken DNA fragments migrated further. The resulting images which were subsequently named for their appearance as comets, were measured to determine the extent of DNA damages. However, some variations could be occurred in procedures, laboratories's conditions and kind of cells used. Hence, to overcome and to harmonize these matters in comet assay, International Workshop on Genotoxicity Test Procedure (IWGTP) was held with several topics including comet assay at Washington D.C. on March, 1999. In spite of some consensus in procedures and conditions in IWGTP, there are some problems still remained to be solved. In this respect, we attempted to set the practical optimal conditions in the experimental procedures such as lysis, unwinding, electrophoresis and neutralization conditions and so on. First of all, we determined optimal lysis and unwinding time by using 150 $\mu$M methyl methanesulfonate (MMS) which is usually used concentration. And then, we determined optimal positive control concentrations of benzo(a)pyrene (BaP) and MMS in the presence and absence of S9 metabolic activation system, respectively.

• Journal of Microbiology and Biotechnology
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• v.8 no.2
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• pp.182-184
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• 1998

PCR was used for quantitative counting of Bifidobacterium spp. in a sample mixed with Lactobacillus acidophilus using two primer sets; one set for universal priming and the other set for Bifidobacterium specific priming. DNA products from two independent PCRs with DNA extracted from the mixed sample were found to be easily distinguishable from each other by agarose gel electrophoresis. The concentrations of PCR products correlated with the total number of bacteria and with the number of Bifidobacterium spp. present in the sample.

• The Korean Journal of Mycology
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• v.27 no.2 s.89
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• pp.112-118
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• 1999

Strains of Fusarium oxysporum f. sp. lycopersici isolated from Korea, Japan and U.S.A. were used for electrophoretic karyotype (EK) analysis. Chromosome separations on FastLane agarose gels (FMC BioProducts, Rockland, ME), called pulsed field gel electrophoresis (PFGE), were performed by CHEF-DRII apparatus (Bio-Rad Laboratories, Melville, NY) using TAE as a running buffer. To obtain optimal condition for separation of chromosome sized DNAs, variable running conditions such as field strengths, swithching intervals, and running time were applied in CHEF gel electrophoresis. We were able to resolve 9 to 11 chromosome sized DNAs ranging in size from 0.76 to 6.41 Mb in isolates from Korea and estimate that the total genome size was ranging from 35.29 to 38.92 Mb. Distinct differences in length range and genome size exist among isolates from different countries. Isolates from Japan and U.S.A. were resolved 9 to 11 chromosome sized DNAs ranging in size from 1.24 to 6.85 Mb and estimated that the total genome size was ranging from 35.32 to 43.87 Mb. Isolates from variable provinces in Korea had the same or similar chromosomal polymorphism and showed different chromosomal DNA patterns compared to isolates from the other countries.

• Korean Journal of Microbiology
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• v.32 no.2
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• pp.132-138
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• 1994

L-Ascorbic acid-producing enzyme in Neurospora crassa was found to exist in mitochondria and the activity of this enzyme was increased by the addition of D-fluconno-${\gamma}$-lactone or L-gulono-${\gamma}$-lactone in the media. L-Ascorbic acid-producin enzyme in N. crassa has been purified with ammonium sulfate precipitation. DEAE Sepharose CL-6B ion exchange chromatography. Sephacryl S-200 gel filtration chromatography and Reactive yellow 3-agarose dye affinity column chromatography. The specific activity of this enzyme was increased to 239.6 fold and the yield was 2.1%. The molecular weight of the native enzyme was 150.000 dalton when it was estimated with Sephacryl S-200 gel filtration chromatography. Its molecular weight appeared as 75.000 dalton by SDS-polyacrylamide gel electrophoresis. which suggested that this enzyme was consisted with two identical subunits. The optimal pH for this enzyme was 9.0 and the $K_m$ value for D-galactono-${\gamma}$-lactone was 0.073 mM.

• The korean journal of orthodontics
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• v.9 no.1
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• pp.9-14
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• 1979

The purpose of this study was to pursue the biosynthesis of proteins of human and bovine periodontal ligaments in vitro system. The excised periodontal ligaments from human and bovine were incubated in Krebs-glucose medium containing $^3H$-proline. After incubation the incubated periodontal ligaments were homogenized and the proteins were treated with 0.1%sodium dodecyl sulfate and $\beta$-mercaptoethanol. Separation of the protein fractions was performed with agarose gel column chromatography and SDS acrylamide gel electrophoresis. The results indicated as follow: 1. Only a small percentage of $^3H$-proline incorporated into proteins was hydroxylated to $^3H$-hydroxyproline. 2. The labeled proteins in periodontal ligaments showed a wide distribution of molecular weight. But only small amounts of labeled protein were found that were characteristics of the molecular weight of collagen. 3. In all of the combined fractions of gel filtration, the degree of hydroxylation was small.