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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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The Korean Journal of Microbiology
Journal Basic Information
Journal DOI :
The Microbiological Society of Korea
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Volume & Issues
Volume 41, Issue 4 - Dec 2005
Volume 41, Issue 3 - Sep 2005
Volume 41, Issue 2 - Jun 2005
Volume 41, Issue 1 - Mar 2005
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Effect of Non-homologous Spacing in Target DNA Sequence on the Frequency of Cloning Based Homologous Recombination
Kim Jae-Woo ; Do Eun-Ju ; Yoon Se-Lyun ; Jeong Yun-Hee ; Yoon Young-Ho ; Leem Sun-Hee ; Sunwoo Yangil ; Park In-Ho ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 239~245
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 effect of non-homologous spacing sequence in target hooks on homologous recombination using a plasmid model system. The efficiency of homologous recombination between the modified his3-TRP1-his3 fragments and HlS3 gene on plasmid were analyzed by the characterization of
transformants. The numbers of
transformant showed same level when seven different modified his3-TRP1-his3 fragments were used. But the percentage of positive recombinants.
, dramatically decreased when used the modified his3-TRP1-his3 fragments contained incorrect spacing of nonhomologous region. As a result, we suggest that incorrect spacing inhibits the homologous recombination between target hook and substrate DNA. Therefore, we should consider the correct spacing in target hook when the target hook are used for cloning of orthologue gene.
Characterization of the Neurospora crassa rcm-1 Mutants
Kim Sang-Rae ; Lee Bheong-Uk ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 246~254
Analysis of the complete genome of Neurospora crassa reveals that at least 19 proteins contain tetratricopeptide repeat (TPR) motifs. One of them shows over
homology to Ssn6 of Saccharomyces cerevisiae, a universal repressor that mediates repression of genes involved in various cellular processes. Mutant strains generated by RIP (repeat-induced point mutation) process showed four distinctive vegetative growth patterns and slow growth in various rates. Firstly, a mutant showed denser mycelial growth, yellow, csp, and looked like ropy mutant. Secondly, slower growth, dense mycelial, and conidial phenotype. Thirdly, extremely slower growth and aconidial. And finally, flat, tittle aerial hyphae, acon, and similar with a rco-1 RIP mutant. They are all male-fertile, yet female-sterile and produced little or no perithecium. It seems that various phenotypes were occurred depending upon mostly likely, the degree of RIP. These results indicate that this gene may be involved in several cellular possess during vegetative growth, and asexual and sexual development. Therefore it is pleiotropic. Sequence analysis of cDNA shows that it encodes a putative 102 kDa protein composed of 917 amino acids, and has six introns. It is designated rcm-1 (regulation of conidiation and morphology).
Molecular Cloning and Analysis of the Genes in the Vicinity of Streptomyces griseus Trypsin (SGT) Gene from Streptomyces griseus ATCC10137
Chi Won-Jae ; Kim Mi-Soon ; Kim Jong-Hee ; Kang Dae-Kyung ; Hong Soon-Kwang ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 255~261
A 6.7kb DNA fragment containing the sprT gene encoding Streptomyces griseus trypsin (SGT) was cloned from Streptomyces griseus ATCC 10137, and the complete nucleotide sequence was determined. Nucleotide sequence and deduced amino acid or the EcoRI-HindIII fragment revealed the presence or the six complete ORFs containing the sprT gene and one incomplete ORF, which were named ORF1, SGT, ORF2, ORF3, ORF4, ORF5, and ORF6, respectively. ORF1 has homology with the oxidoreductases from several organisms. ORF2 and ORF3 show similarity with unknown proteins and transcription regulator that belongs to the ArsR family, respectively. ORF4 and ORF5 show homology with the peptidoglycan bound protein with LPXTG motif from Listeria monocytogenes and the membrane protein with transmembrane helix from several organisms, respectively. The last ORF, ORF6, shows homology with the lipoprotein from Streptomyces avermitilis.
Proteome Profiling of Murine Macrophages Treated with the Anthrax Lethal Toxin
Jung Kyoung-Hwa ; Seo Giw-Moon ; Kim Sung-Joo ; Kim Ji-Chon ; Oh Seon-Mi ; Oh Kwang-Geun ; Chai Young-Gyu ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 262~268
Intoxication of murine macrophages (RAW 264.7) with the anthrax lethal toxin (LeTx 100 ng/ml) results in profound alterations in the host cell gene expression. The role of LeTx in mediating these effects is unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, we have used two-dimensional polyacrylamide gel electrophoresis to analyze the protein profile of murine macrophages treated with the LeTx, and have coupled this to protein identification using MALDI-TOF mass spectrometry. Interpretation of the peptide mass fingerprint data has relied primarily on the ProFound database. Among the differentially expressed spots, cleaved mitogen-activated protein kinase kinase (Mek1) and glucose-6-phosphate dehydrogenase were increased in the LeTx treated macrophages. Mek1 acts as a negative element in the signal transduction pathway, and G6PD plays the role for the protection of the cells from the hyper-production of active oxygen. Our results suggest that this proteomic approach is a useful tool to study protein expression in intoxicated macrophages and will contribute to the identification of a putative substrate for LeTx.
Application of a PCR Method for the Detection of Mycoplasma in Veterinary Live Viral Vaccines
Jeon Woo-Jin ; Kim Byoung-Han ; Jung Byeong-Yeal ; An Dong-Jun ; Yi Chul-Hyun ; Jang Hwan ; Chung Gab-Soo ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 269~274
We evaluated the PCR assay and two commercialized PCR kits for the detection of mycoplasma in veterinary via live vaccines. The PCR assay could specifically detect all the tested Mycoplasma spp. and Acholeplasma spp., whereas two commercialized PCR kits did not. Also, the specificity of the PCR assay showed that 4 reference strains and 7 field isolates belonging to avian mycoplasma species could be all detected. The sensitivity of the PCR assay was determined using pure cultured Mycoplasma spp. and Acholeplasma spp. with a range of 1 to 100 colony forming units/ml in 9 CFR Mycoplasma broth. To test the availability of the PCR assay for veterinary live viral vaccines, A. laidlawii was artificially inoculated into the swine transmissible gastroenteritis-rota virus combined vaccine and canine parvovirus vaccine, respectively and the sensitivity of the PCR assay was similar with the result of cultured samples. In this study, the PCR assays could be used as rapid and sensitive methods for the detection of mycoplasma in veterinary live viral vaccines.
Expression of Anthrax Lethal Factor, a Major Virulence Factor of Anthrax, in Saccharomyces cerevisiae
Hwang Hyehyun ; Kim Joungmok ; Choi Kyoung-Jae ; Chung Hoeil ; Han Sung-Hwan ; Koo Bon-Sung ; Yoon Moon-Young ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 275~280
Anthrax is an infectious disease caused by the gram-positive bacterium, Bacillus anthracis. Anthrax toxin is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF onto the cytosol. LF is a zinc-dependent metalloprotease, which is a critical virulence factor in cytotoxicity of infected animals. Therefore, it is of interest to develop its potent inhibitors for the neutralization of anthrax toxin. The first step to identify the inhibitors is the development of a rapid, sensitive, and simple assay method with a high-throughput ability. Much efforts have been concentrated on the preparation of powerful assays and on the screening of inhibitors using these system. In the present study, we have tried to construct anthrax lethal factor in yeast expression system to prepare cell-based high-throughput assay system. Here, we have shown the results covering the construction of a new vector system, subcloning of LF gene, and the expression of target gene. Our results are first trial to express LF gene in eukaryote and provide the basic steps in design of cell-based assay system.
Analysis of Microbial Community During the Anaerobic Dechlorination of Perchloroethylene and Trichloroethylene
Lee Jae-Won ; Kim Byung-Hyuk ; Ahn Chi-Yong ; Kim Hee-Sik ; Yoon Byung-Dae ; Oh Hee-Mock ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 281~286
In this study, the anaerobic enrichment cultivation was performed with the sediments and the dredged soils from the cities of Ulsan, Masan, Yeosu, Gwangyang, Ansan and Seongnam. Acetate as an electron donor and PCE (perchloroethylene) or TCE (trichloroethylene) as an electron acceptor were injected into the serum bottle with an anaerobic medium. After the incubation of 12 weeks, the removal efficiency of PCE was highest at
in the treatment with the sediment of Ulsan. Also, the bacterial community structure was analyzed by D-DGGE (double denatured gradient gel electrophoresis) through PCR-based 16S rDNA approaches. The dominant species id the anaerobic enrichment were found to belong to the genus of Desulfovibrio.
Cyanobacterial Diversity Analysis Using cpcBA-Intergenic Spacer Region
Choi Gang-Guk ; Park Yong-Ha ; Ahn Chi-Yong ; Bae Myoung-Sook ; Oh Hee-Mock ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 287~292
The cyanobacterial diversity was analyzed by restriction fragment length polymorphism (RFLP) of PCR-amplified rpcBA-Intergenic Spacer (IGS) genes and cpcBA-IGS gene sequencing with a sample collected at Chuso-ri in Daechung Reservoir on March 15, 2005, The Shannon-Weiner diversity index was 0.65, indicating that the cyanobacterial community structure was simple. PCR-RFLP profiles obtained were Phormidium spp. (58 clones), Anabaena spp. (14 clones), Microcystis spp. (4 clones), Spirulina sp. (1 clone) and uncultured cyanobacteria (2 clones). The PCR-RFLP of cpcBA-IGS revealed that Phormidium spp. and Anabaena spp. dominated in the invested sample. As a consequence, it seems that the analysis of functional genes such as cpcBA-IGS can be used for the species identification and community analysis of cyanobacteria.
Isolation and Phylogenetic Characterization of Chitinase Producing Oligotrophic Bacteria
Kim Soo-Jin ; Kim Min-Young ; Koo Bon-Sung ; Yoon San-Hong ; Yeo Yun-Soo ; Park In-Cheol ; Kim Yoon-Ji ; Lee Jong-Wha ; Whang Kyung-Sook ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 293~299
Many isolates from soil of Korean ginseng rhizosphere did not show remarkable growth on full strength of the conventional nutrient broth (NB medium) but grew on its 100-fold dilution (DNB medium). Six hundred-forty strains were isolated as oligotrophic bacteria. In the course of screening for new bioactive compounds from oligotrophic bacteria from soil, 8 strains which had appeared to form of clear zone on a medium containing colloidal chitin as a sole carbon source were selected for further studies. Strain CR42 hydrolyzed a fluorogenic analogue of chitin, 4-methylumbelliferyl-D-glucosaminide (MUF-NAG) . Mo st of the culture supernatant of these isolates hydrolyzed 4-methylumbelliferyl-D-N,N'-diacetylchitobioside (MUF-diNAG). The isolates were heterogeneous and categorized to gamma- and beta-proteobacteria, Bacillaceae, Actinobactepia, and Bacteroides by 16S rRNA analysis. Two strains, WR164 and CR18, had a 16S rRNA sequence of
identical to uncultured bacteria. It was observed that CR2 and CR75 could inhibit the growth of Colletotrichum gloeosporioides with hyphal extention-inhibition assay on PDA plate supplemented with
Derepression of a Methionine Biosynthetic Gene by Utilizing a Promoter Isolated from Corynebacterium glutamicum
Park Soo-Dong ; Park Ik-Hyun ; Choi Jong-Soo ; Kim Il-Kwon ; Kim Younhee ; Lee Heung-Shick ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 300~305
A transcriptionally active fragment
isolated by utilizing the promoter-probe shuttle vector pSK1Cat was analyzed. By subcloning analysis, the 180 bp region
responsible for the activity was determined. Transcriptional fusion of the C. glutamicum metX gene to
resulted in a 24-fold increase in MetX activity in a complex medium, while a 13-fold increase was observed with the
promoter. Additionally, the expression conferred by
was not affected by methionine added to the growth medium, suggesting that the
clone is useful for the deregulated expression of biosynthetic genes in C. glutamicum during amino acid fermentation. Introduction of
into a lysine-producing C. glutamicum resulted in the production of methionine to 0.8 g/l.
Generation and Expression of Amino-Terminal Domain of the Gene Coding for the Lumazine Protein from Photobacterium phosphoreum
Woo Young-Eun ; Kim So-Young ; Lee Chan-Yong ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 306~311
In this study, the amino-terminal half truncated lump and the whole lump genes from Photobacterium phosphoreum coding for the lumazine protein were cloned by polymerase chain reaction and expressed in Escherichia coli. To identifiy of the binding site of the ligand or substrate, the amino acid identities from the sequences of the lumazine protein, yellow fluorescent protein, and riboflavin synthase from different organisms were also compared and analyzed.
Ultrastructural Studies on the Autolysis of Coprinellus congregatus
Choi Hyung-Tae ; Cho Chung-Won ;
The Korean Journal of Microbiology, volume 41, issue 4, 2005, Pages 312~315
Coprinellus congregatus, known as an inky cap, is autolysed into ink soon after the maturation of the mushrooms. Electron microscopy was used to examine the ultrastructural changes associated with the autolysis as an initial step to understand the role of hydrolytic enzymes in this process. During the early stages of maturation of the mushrooms, most of cytoplasm of hymenial and subhymenial tissues seemed to be transported to the developing basidiospores. The depletion of cytoplasm within the tissues and the maturation of the basidiospores may initiate the degradation of the cell walls of the tissues. Both hymenial and subhymenial tissues seemed to degraded at the same time. This study suggested that the critical steps in the autolysis of mushrooms is not the degradation of the cytoplasm, but the degradation of the cell wall by hydrolytic enzymes such as chitinases.