• Journal of fish pathology
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• v.6 no.2
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• pp.93-101
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• 1993

The RNAI mutation of Saccharomyces cerevisia is a recessive and temperature sensitive lethal mutation which interferes with the production of mRNA, rRNA, and tRNA. However, the precise role of RNAI gene have not been revealed until yet. We have cloned rna1-1 mutant gene from rna1-1 mutant yeast strain(R49 ; trpl, ura3-52, rna1-1). The 3.4kb BglII fragment of wild type RNAI clone(81-2-6) contains whole RNAI gene. The genomic southern blotting with BglII digested R49 genomic DNA as a probe shows the unique and identical band with wild type 3.4kb BglII fragment. Therefore, We prepared partial BglII genomic library(3~4kb BglII fragments) into BamH I site of pUC19. The rna 1-1 mutant clone was screened with Digoxigenin(DIG)-lableled probe by high density colony hybridization. The 5'-flanking region of rna1-1 gene was sequenced by dideoxy chain termination method. The 5'-flanking sequence of RNAI gene contains three TATA-like sequence ; TAATA, TATA and TTTTAA at position of -67, -45, and -36 from first ATG codon respectively. The 5'-flanking region of wild type RNA I gene from ATG codon to -103nt was deleted with Bal31 exonuclease digestion, generating $pUC{\Delta}$/RNA I. After constructing $pYEP{\Delta}RNA$ I (consists of -103nt deleting RNA I gene, URA3 gene, $2{\mu}m$ rep. origin), pYEPrna1-1(consists of Xba I fragment of pUCrna1-1. URA3 gene, $2{\mu}m$ rep. origin), and pYEPRNAI. each plasmid was transformed into host strain(trpl, ura3-52, rna1-1) by electroporation, respectively. Yeast transformant carrying $pYEP{\Delta}RNA$ I did not complement the thermal sensitivity of rna1-1 gene. It means that TATA-like sequences in 5'-flanking region is not TATA sequence for transcribing RNAI gene and there may be other essential sequence in upstream region for the transcription of RNAI gene.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.2
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• pp.65-70
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• 2015

Antimicrobial agents or additives have commonly been used in domestic animals for the prevention and treatment of bacterial diseases. Unfortunately, this has resulted in the overgrowth of bacteria that is resistant to antimicrobial agents used by humans, and these might get disseminated to humans via the food. In this study, we investigated the prevalence of integrons, and characterized gene cassette arrays in Proteus mirabilis isolates obtained from chickens in Chungcheong province of Korea. Additionally, the correlation between gene cassette arrays and antimicrobial resistance rate was studied. A total of 26 Proteus mirabilis isolates were recovered from chickens in Chungcheong province in Korea. Antimicrobial susceptibility was determined by disk diffusion method. PCR and DNA sequencing were performed to characterize the gene cassette arrays. In addition, we employed repetitive extragenic palindromic sequence-based PCR (REP-PCR) method for clonality analysis of P. mirabilis strains. Of the 26 P. mirabilis isolates tested, 14 (53.8%) isolates carried class 1 integrons, while class 2 and class 3 integrons were not detected in our study. The class 1 integrons harbored genes encoding resistance to aminoglycosides (aacCA5, aadA2, aadA5 and aadA7), trimethoprim (dfrA17, and dfrA32), lincosamides (linF) and erythromycin (ereA). In particular, the presence of class 1 integron had a significant correlatation to a high resistance rate of aminoglycoside and trimethoprim. We confirmed that class 1 integrons are widely disseminated in P. mirabilis isolates from chickens, contributing to the resistance to diverse antimicrobial agents in Korea. To prevent further spreading of antimicrobial resistant genes among P. mirabilis isolates, constant monitoring and clinical policing will become necessary.

• Korean Journal of Clinical Laboratory Science
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• v.43 no.2
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• pp.68-74
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• 2011

Pseudomonas aeruginosa is an aerobic, Gram-negative, glucose-nonfermenting bacterium, which has emerged as a serious opportunistic pathogen. Recently, outbreaks of carbapenem resistant P. aeruginosa give rise to significant therapeutic challenges for treating nosocomial infections. The genes of metallo-${\beta}$-lactamase (MBL), a powerful carbapenemase, are carried as a part of the mobile gene cassettes inserted into integrons playing an important role in rapid dissemination of antibiotic resistance genes among bacterial isolates. In this study, we investigated the prevalence of integron in imipenem resistant P. aeruginosa isolates. A total of 61 consecutive, non-duplicate, and imipenem resistant P. aeruginosa strains were isolated from a university hospital in the Chungcheong province of Korea. We employed repetitive extragenic palindromic sequence-based PCR (rep-PCR) method for the selection of clonally different P. aerusinosa strains. PCR and DNA sequencing were conducted for the detection of integrons. Twenty-one clonally different P. aeruginosa strains were isolated. Only one (P28) of the strains harbored $bla_{VIM-2}$ that was found as gene cassettes in class 1 integrons. Four of 21 carbapenem resistant P. aeruginosa strains harbored class 1 integron containing aminoglycoside resistance determinant. All of the integrons detected in the study contained more than one resistance gene cassette, which can mediate resistance to multiple antibiotics. To prevent further spreading of the multi-drug resistant P. aeruginosa, conseguent monitoring and clinical polices are required.

• Journal of Microbiology
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• v.45 no.4
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• pp.344-349
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• 2007

We have isolated previously three synthetic lethal mutants in Schizosaccharomyces pombe, which genetically interact with mex67, in order to identify the genes involved in mRNA export. A novel nup97 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex3. The nup97 gene contains one intron and encodes an 851 amino-acid protein that is similar to nucleoporins, Nppl06p in S. pombe and Nic96p in Saccharomyces cerevisiae. The nup97 gene is essential for vegetative growth, and nup97 null mutant harboring pREP41X-Nup97 showed $poly(A)^+$ RNA export defect when expression of nup97 is repressed in the presence of thiamine. These results suggest that nup97 is involved in mRNA export from the nucleus to cytoplasm.

• Korean Journal of Microbiology
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• v.49 no.3
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• pp.228-236
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• 2013

Campylobacter jejuni is one of important food-borne pathogens causing human gastroenteritis. We isolated 42 strains of C. jejuni from diarrhea patients and 4 food-poisoning outbreaks in 2010, Gyeonggi-do. In this study, 42 strains were tested for genetic characteristics, the serotype distribution and antimicrobial resistant rate. The presence of hipO (100%), cdtB (100%), and mutated gyrA (95.2%) genes was detected in C. jejuni by polymerase chain reaction (PCR). Detection of mutated gyrA gene correlated with ciprofloxacin resistance. Forty isolates had mutated gyrA gene and were actually resistant to ciprofloxacin. Furthermore, comparing the gyrA DNA sequence data, ciprofloxacin-resistant isolates had a mutation of the DNA sequence from ACA (threonine) to ATA (isoleucine). But 41 strains (97.6%) of patient isolates were susceptible to erythromycin and azithromycin. A total of 35.7% among 42 C. jejuni isolates were identified into 4 different serotypes. The serotype distribution of C. jejuni strains were shown to be HS2(B), HS3(C), HS4(D), HS19(O). To investigate the genotypes of C. jejuni isolated in Gyeonggi province, repetitive sequence polymerase chain reaction (rep-PCR) analysis and SmaI-digested pulsed-filed gel electrophoresis (PFGE) profile analysis were performed. From the PFGE analysis of 42 C. jejuni strains, 12 clusters of PFGE profile were obtained. On the other hand, 11 clusters of rep-PCR profile were obtained from 42 strains of C. jejuni.

• Food Science of Animal Resources
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• v.38 no.4
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• pp.816-822
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• 2018

Bifidobacterium is recognized as one of the most beneficial microorganisms in our gut. Many researches on bifidobacteria have been done to understand their roles in the gut. The objective of the present study was to develop a bioluminescent labelling plasmid vector for bifidobacteria to facilitate their visualization in vitro, in situ, and in vivo. A plasmid replicon (2.0 kb) of plasmid pFI2576 previously identified from B. longum FI10564 was amplified by PCR and cloned into pUC19 plasmid vector (2.68 kb). The cloned replicon was subcloned into pTG262 ($luc^+$) recombinant plasmid vector (7.4 kb) where a luciferase gene ($luc^+$) from pLuc2 (8.5 kb), an Escherichia coli and lactobacilli shuttle vector, was inserted into pTG262 plasmid vector. The final recombinant DNA, pTG262::pFI2576 rep ($luc^+$), was transferred into a B. catenulatum strain. This recombinant strain showed 3,024 relative luminescence units at $OD_{600}$ value of 0.352. Thus, this recombinant plasmid construct can be broadly used for labelling bifidobacteria.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.169-178
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• 2010

The microbial community structure in Thailand soils contaminated with low and high levels of arsenic was determined by denaturing gradient gel electrophoresis. Band pattern analysis indicated that the bacterial community was not significantly different in the two soils. Phylogenetic analysis obtained by excising and sequencing six bands indicated that the soils were dominated by Arthrobacter koreensis and $\beta$-Proteobacteria. Two hundred and sixty-two bacterial isolates were obtained from arsenic-contaminated soils. The majority of the As-resistant isolates were Gramnegative bacteria. MIC studies indicated that all of the tested bacteria had greater resistance to arsenate than arsenite. Some strains were capable of growing in medium containing up to 1,500 mg/l arsenite and arsenate. Correlations analysis of resistance patterns of arsenite resistance indicated that the isolated bacteria could be categorized into 13 groups, with a maximum similarity value of 100%. All strains were also evaluated for resistance to eight antibiotics. The antibiotic resistance patterns divided the strains into 100 unique groups, indicating that the strains were very diverse. Isolates from each antibiotic resistance group were characterized in more detail by using the repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique with ERIC primers. The PCR products were analyzed by agarose gel electrophoresis. The genetic relatedness of 100 bacterial fingerprints, determined by using the Pearson product-moment similarity coefficient, showed that the isolates could be divided into four clusters, with similarity values ranging from 5-99%. Although many isolates were genetically diverse, others were clonal in nature. Additionally, the arsenic-resistant isolates were examined for the presence of arsenic resistance (ars) genes by using PCR, and 30% of the isolates were found to carry an arsenate reductase encoded by the arsC gene.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.448-456
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• 2012

Thirty-seven carbofuran-degrading bacteria were isolated from agricultural soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize carbofuran as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that the isolates were related to members of the genera Rhodococcus, Sphingomonas, and Sphingobium, including new types of carbofuran-degrading bacteria, Bosea and Microbacterium. Among the 37 isolates, 15 different chromosomal DNA patterns were obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences. Five of the 15 representative isolates were able to degrade carbofuran phenol, fenoxycarb, and carbaryl, in addition to carbofuran. Ten of the 15 representative isolates had 1 to 8 plasmids. Among the 10 plasmid-containing isolates, plasmid-cured strains were obtained from 5 strains. The cured strains could not degrade carbofuran and other pesticides anymore, suggesting that the carbofuran degradative genes were on the plasmid DNAs in these strains. When analyzed with PCR amplification and dot-blot hybridization using the primers targeting for the previously reported carbofuran hydrolase gene (mcd), all of the isolates did not show any positive signals, suggesting that their carbofuran hydrolase genes had no significant sequence homology with the mcd gene.

• Journal of Life Science
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• v.17 no.1 s.81
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• pp.39-44
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• 2007

The Rqh1 gene is essential for vegetative growth in fission Yeast. The rqh1 mutant showed that sensitivity of DNA damaging agent, a wild range of phenotype including abnormal gene expression and cell elongation. This result showed that the rqhl-overexpression cell was sensitivity to DNA damaging agent like rqhl mutant. When Rqh1 have an over-expression by $nmt1^+$ promoter of pREP vector, rqh1 mutant DNA damaging agent sensitivity could be compensated. We isolated two strong mutant containing complementation gene, rqh156 and rqh172, respectively. This result observed that the DNA damaging agent sensitivity of rqhl mutant was complemented by the expression of rqh156 and rqh172. They induced mRNA expression in a dose-dependent manner HU, MMS and UV. The HU sensitivity of the rqhl was complemented by the expression of rqh156 and rqh172. The mRNA expression of rqh156 decreased on HU dose dependent but the mRNA expression of rqh172 did not decrease on HU dose dependent. The MMS and W sensitivity of the rqhl was complemented by the expression of rqh156 and rqh172. These results indicate that the isolated rqhl gene may play an important role in DNA metabolism.

• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.796-801
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• 2011

Corynebacterium tuberculostearicum B146, a strain derived from healthy human skin, contains a medium copy plasmid, p1B146. This plasmid was cloned and its complete nucleotide sequence determined. As a result, p1B146 was found to be 4,2 kb in size with a 53% G+C content, plus six open reading frames (ORFs) were distinguished. According to a computer-assisted alignment, two of the ORFs exhibited significant similarities to already-known common plasmid proteins, the first being the RepA gene, responsible for plasmid replication via a rolling-circle mechanism, and the second being an FtsK-like protein, the function of which remains unclear. The presence and quantity of RNA fragments in the putative ORFs were also evaluated.