• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.575-582
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• 1991

To increase the production of glutathione by the expression of recombinant gsh plasmids, two genes responsible for the biosynthesis of glutathione were isolated and cloned. To clone a gshI gene, the GS903 mutant strain, which is deficient in $\gamma$-glutamylcysteine synthetase activity, has been raised. A gshI gene was cloned using pBR322 plasmid as a 3.6 Kb PstI DNA fragment isolated from E. coli K-12 chromosomal DNA. Also a gshIl gene was cloned using pUC13 plasmid as a 2.2 Kb PstI-BamHI DNA fragment. To study the effects of plasmid copy number and passenger DNA size on the expression levels of the gsh genes, various recombinant plasmids containing different sets of genes were constructed. The expression levels of the gsh genes were increased approximately twice higher in pUC series plasmids than that in pBR322 plasmid. But the sizes of the passenger DNA containing the gsh genes in the vector plasmid did not affect on the expression levels of the gsh genes.

• Restorative Dentistry and Endodontics
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• v.20 no.2
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• pp.577-609
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• 1995

Bacteria have been regarded as one of the most important factors in pulpal and periapical diseases. Streptococci are frequently isolated facultative anaerobes in infected root canals. Recently molecular biological techniques have been rapidly progressed. This study was designed to apply the molecular biological tools to the identification and classification of streptococci in the endodontic microbiology. Streptococci isolated from infected root canals were identified with both Vitek Systems and API 20 STREP. Identification results were somewhat different in several strains of streptococci. Eighteen streptococci and enterococcal was difficult so to digest plasmid DNA using Hind III and EcoRI to differentiate strains by restriction enzyme analysis of plasmid DNA. 16S rDNA of chromosome was amplified by polymerase chain reaction(PCR) and then restricition fragment length polymorphism(RFLP) using several restriction enzymes was observed. The molecular mass of 16S rDNA of chromosomal DNA was approximately 1.4kb. There were three to five RFLP patterns using eight restriction enzymes. RFLP patterns digested with CfoI which recognizes four base sequences were identical in all stains. Hind III which recognizes six base sequences could not digest the 16S rDNA. Restriction enzymes which recognize five base sequences were suitable for RFLP pattern analysis. At least three different restriction enzymes were needed to compare each strains. 16S rDNA PCR-RFLP was simple and rapid to differentiate and classify strains and could be used in the epidemiological study of root canal infections.

• Microbiology and Biotechnology Letters
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• v.31 no.4
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• pp.322-328
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• 2003

The Transformation-Associated Recombination (TAR) cloning technique allows selective isolation of chromosomal regions and genes from complex genomes. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosomal region of interest. This technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector that has 5'and 3' gene targeting sequences. In this study, we examined the minimum size of specific hooks required for a single-copy gene isolation and compared the utility of different TAR vectors, radial and unique vectors, by cloning the same single-copy gene. The efficiency of TAR cloning of the hHPRT gene was same using hooks varying from 750 to 63 bp. The number of transformants decreased approximately 20-fold when the TAR vector contained two unique hooks versus using a radial vector, but the percentage of positive recombinants increased over 2-fold when a unique TAR vector was used. Therefore, we suggest that the two-unique TAR vector is suitable for general TAR cloning given its high selectivity, and the radial TAR vector is more suitable when genomic DNA is in limited quantity, for example, DNA isolated from pathological specimens. Moreover, we confirm the minimal length of a unique sequence in a TAR vector is approximately 60 bp for a single-copy gene isolation.

• Korean Journal of Breeding Science
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• v.42 no.2
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• pp.163-168
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• 2010

This study was carried out to determine the chromosomal localization of the 5S and 18S-26S ribosomal DNA(rDNA) genes by means of fluorescence in situ hybridization(FISH) techniques, and the constitutive heterochromatin detected by means of Gimsa C-banding technique in rye(Secale cereale L.). The somatic chromosomes number was 2n=14. The karyotype consists of four pairs of metacentrics(chromosomes 1, 2, 3, and 7) and three pairs of submetacentrics(chromosomes 4, 5, and 6). Secondary constrictions appeared in the short arm of chromosome 1. The 5S rDNA genes have been located on two pairs of chromosomes 1 and 5, and 18S-26S rDNAs genes have been located on one pair of chromosome 1. 5S rDNA genes were detected on the distal region of the secondary constrictions in nucleolus organizer regions(NOR) in chromosome 1, and other detected on the intercalary region in the short arm of chromosome 5.

• International Journal of Oral Biology
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• v.32 no.3
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• pp.113-118
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• 2007

Treatment of oral cancers with chemotherapeutic agents become evaluated as an effective method to reduce cancer cell proliferation. Anti-proliferative and anti-oral cancer activities of momordin I on oral cancer cells were evaluated in this study. Momordin I was originally purified from a natural product, Ampelopsis radix and showed the antiproliferative activity against oral carcinoma, KB cells. Obtained $IC_{50}$ value was approximately $10.4{\mu}g/ml$. Time-and dose-dependent chromosomal DNA fragmentations were observed in momordin I-treated KB cells. Flow cytometry analysis showed time-dependent apoptotic cell appearance after treatment of momordin I. Approximately 18.6% apoptotic cells were observed at 72 hours after $20{\mu}g/ml$ of momordin I treatment. These observation were consistent with the results obtained in DNA fragmentation analysis. These data suggest that momordin I has anti-proliferative effect and induces cell death in KB cells through apoptosis.

• Journal of Genetic Medicine
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• v.4 no.2
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• pp.115-121
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• 2007

Hereditary motor and sensory neuropathy (HMSN; Charcot-Marie-Tooth disease, CMT) was first described by Charcot and Marie in France and, independently, by Tooth in England in 1886. HMSN is the most common form of inherited motor and sensory neuropathy, and is a genetically heterogeneous disorder of the peripheral nervous system. Using positional cloning methods, the chromosomal localization (locus) of more than 40 inherited peripheral neuropathies was found in the last 15 years. However, these genetic analyses also show that many entities do not show linkage to the known loci. This issue deals with a clinical survey of inherited peripheral neuropathies regarding diagnostic approaches based on the molecular findings.

• BMB Reports
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• v.38 no.2
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• pp.191-197
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• 2005

Although the human genome has been nearly completely sequenced, the functions and the roles of the vast majority of the genes, and the influences of single nucleotide polymorphisms (SNPs) in these genes are not entirely known. A modified mutation detection method was developed for large-scale cloning of the possible SNPs between tumor and normal cells for facilitating the identification of genetic factors that associated with cancer formation and progression. The method involves hybridization of restriction enzyme-cut chromosomal DNA, cleavage and modification of the sites of differences by enzymes, and differential cloning of sequence variations with a designed vector. Experimental validations of the presence and location of sequence variations in the isolated clones by PCR and DNA sequencing support the capability of this method in identifying sequence differences between tumor cells and normal cells.

• Biomedical Science Letters
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• v.8 no.2
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• pp.77-82
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• 2002

Ten Listeria monocytogenes were isolated from clinical specimens and mussels, and their physio-biochemical characters were compared with the type strains. Ribotyping was used as a taxonomic tool to determine molecular epidemiological marker. Chromosomal DNA was cleaved with restriction enzymes HindIII and EcoRI. The fragment were subjected to Southern blot hybridization with 165 rDNA from B. subtilis by PCR. EcoRI patterns of Listeria strains showed 6 to 8 bands ranging from 0.75 kb to 11 kb band and they were classified into 6 groups. In comparison, HindIII patterns revealed that 5 to 7 bands ranging from 2.75 kb to 7.75 kb band and they classified into 5 groups. The various patterns of Listeria strains were observed within genus, species and isolated sources. 165 rRNA gene restriction patterns (ribotyping) are useful in epidemiological and taxonomic study.

• Microbiology and Biotechnology Letters
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• v.28 no.1
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• pp.21-25
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• 2000

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in Escherichia. coli TP2139 ( lac, crp). One of the cloned genes, pCKB17, was further analyzed. In order to find whether the increased expression of the gent was under the direction of maltose metabolism, we constructed several recombinant subclones. We have found that the clone, pCKB17AV, codes maltose metabolism stimulation(mms) gene. E. coli transformed with the cloned gene showed increase in the activity of maltose utilzation, The recombinant proteins expressed by multicopy and induction with IPTG, one polypeptide of 29-kDa, was confirmed by SDS-PAGE. The overexpression of maltose-binding proter protein in the presence of mms gene was confirmed by Western blot analysis. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.509.1-509
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• 1986

Recent advances in recombinant DNA techniques have provided a tool for breeding of microorganisms of hyper production. Enzyme production by cloned microorganism has some advantages. They are ⅰ) Enzymes can be produced by a microorganism easily cultured ⅱ) Hyper production. ⅲ) In some cases, such as thermophilic enzyme gene is cloned in a mesophilic bacteria, the enzyme purification procedure can be simplified. One example, production of thermophilic ${\beta}$-galactosidase in B. subtilis will be presented. Bacillus stearothermophilus IAM 11001 produced three ${\beta}$-galactosidases, ${\beta}$-galactosidase I, II and III (${\beta}$-gal-I, II and III). By connecting restriction fragments of the chromosomal DNA to plasmid vector, followed by transformation of Escherichia coli, two ${\beta}$-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were cloned.