• 미생물학회지
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• 제25권4호
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• pp.262-273
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• 1987

pBR 322와 pBD9을 이용하여 Staphylococcus aureus에서 분리된 chloramphenicol 저항성(Cmr) plasmid인 pSBK 203상의 ori 부위를 cloning하였다. 또한 E. coli 내에서도 발현하는 pSBK 203상의 Cm 저항성 부위 및 cloning 된 ori 부위를 pBR 322에 재조합시켜 E. coli와 그람양성균인 Bacillus subtilis 양쪽 모두에서 복제되고 또 항생물질에 대한 저항성도 각각 발현되는 shuttle vector 구성을 시도하였다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.29-48
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• 2003

It is remarkable that nuclear transfer using differentiated donor cells can produce physiologically normal cloned animals, but the process is inefficient and highly prone to epigenetic errors. Aberrant patterns of gene expression in clones contribute to the cumulative losses and abnormal phenotypes observed throughout development. Any long lasting effects from cloning, as revealed in some mouse studies, need to be comprehensively evaluated in cloned livestock. These issues raise animal welfare concerns that currently limit the acceptability and applicability of the technology. It is expected that improved reprogramming of the donor genome will increase cloning efficiencies realising a wide range of new agricultural and medical opportunities. Efficient cloning potentially enables rapid dissemination of elite genotypes from nucleus herds to commercial producers. Initial commercialization will, however, focus on producing small numbers of high value animals for natural breeding especially clones of progeny-tested sires, The continual advances in animal genomics towards the identification of genes that influence livestock production traits and human health increase the ability to genetically modify animals to enhance agricultural efficiency and produce superior quality food and biomedical products for niche markets. The potential opportunities in animal agriculture are more challenging than those in biomedicine as they require greater biological efficiency at reduced cost to be economically viable and because of the more difficult consumer acceptance issues. Nevertheless, cloning and transgenesis are being used together to increase the genetic merit of livestock; however, the integration of this technology into farming systems remains some distance in the future.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.29-48
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• 2003

It is remarkable that nuclear transfer using differentiated donor cells can produce physiologically normal cloned animals, but the process is inefficient and highly prone to epigenetic errors. Aberrant patterns of gene expression in clones contribute to the cumulative losses and abnormal phenotypes observed throughout development. Any long lasting effects from cloning, as revealed in some mouse studies, need to be comprehensively evaluated in cloned livestock. These issues raise animal welfare concerns that currently limit the acceptability and applicability of the technology. It is expected that improved reprogramming of the donor genome will increase cloning efficiencies realising a wide range of new agricultural and medical opportunities. Efficient cloning potentially enables rapid dissemination of elite genotypes from nucleus herds to commercial producers. Initial commercialisation will, however, focus on producing small numbers of high value animals for natural breeding especially clones of progeny-tested sires. The continual advances in animal genomics towards the identification of genes that influence livestock production traits and human health increase the ability to genetically modify animals to enhance agricultural efficiency and produce superior quality food and biomedical products for niche markets. The potential opportunities inanimal agriculture are more challenging than those in biomedicine as they require greater biological efficiency at reduced cost to be economically viable and because of the more difficult consumer acceptance issues. Nevertheless, cloning and transgenesis are being used together to increase the genetic merit of livestock; however, the integration of this technology into farming systems remains some distance in the future.

• 한국미생물·생명공학회지
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• 제28권1호
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• pp.14-20
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• 2000

The transformation of Bacillus subtilis K-54 and J-10 was carried out with constructed vectors containing structure and enhancer genes of aprN and prtR, to increase their fibrinolytic enzyme activity. Bands for the aprN and prtR genes were identified from B. subtilis J-10 by PCR that was carried out with the constructed primers for the genes. In addition, the gene fragments contained promoter site based on the results of analysing their nucleotide sequence. The two gene fragments, aprN and prtR, obtained by the PCR, were, then, inserted to vector such as T-vector and E.coli/Bacillus shuttle vector. The constructed vector were designated as pAPR2 (aprN), pENC2 (prtR) and pFLA1 (aprN and prtR), respectively. The constructed vector was used for transformation of the strains of B.subtilis J-10 and B. subtilis K-54 and the fribrinolytic activity of the transformed strains was investigated. The introduction of the vector, pAPR2 and the fibrinolytic activity of the transformed strains was investigated. The introduction of the vector, pAPR2 and pFLA1, resulted in the increase of fibrinolyitic enzyme activity in B. subtilis J-10 by 27.3% and 16%, respectively. However, the introduction of pENC2 to B. subtilis J-10 did not seem to induce increase of the enzyme activity. The strain of B.subtilis K-54 transformed with pENC2 showed an increased fibrinolytic activity by 5 folds compared with that of the original strain of B. subtilis K-54.

• KSBB Journal
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• 제11권1호
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• pp.37-45
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• 1996

한국의 남해안에서 한천 분해능이 뛰어난 해양 미 생물을 분리하여 통정한 결과 Pseudomonas 속으로 판명되었으며, 본 연구에서는 이 균을 Halophilic P Pseudomonas sp. W7이라 명영하였다. 이 균주는 호염성 세균으로, 한천의 존재 하에서 높은 효소활성 을 가지는 extracellular agarase를 생산해 내었다. 이 extracellular agarase를 DEAE-Cellulose ani- on exchange chromatography와 gel filtration을 통해 정제하였으며, 정제된 agarase는 SDS-PAGE 를 통해 약 89KDa의 분자량을 지니는 single protein band염을 확인하였다. 한편 agarase의 대량생 산을 위하여 host cell Eo coli JM83과 vector pUC19를 이용하여 gene cloning을 행하였다. Pseudomonas sp. W7의 chromosomal DNA가 삽 입 된 균주 중에 서 agarase activity를 나타내는 E. coli JM83/pSWl과 Eo coli JM83/pSW3를 선멸하 였으며, 전기영통 실험결과 이들은 각각 307Kb, 3.0 K Kb의 chromosomal 단편을 지니고 있음을 확인하였 다. 또한 agarase 유전자가 압입된 변이 균주(B)는 inclusion body 형태의 interacellular agarase를 세포 내에 축적하고 있음이 확인되었다.

• Journal of Microbiology
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• 제34권4호
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• pp.355-362
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• 1996

Complementation cloning of the argC, E, B, D, F, and G genes in Corynebacterium glutamicum was done by transforming the genomic DNA library into the corresponding arginine auxotrophs fo Escherichia coli. Recombinant plasmids containing 6.7 kb and 4.8kb fragments complementing the E. coli argB mutant were also able to complement the E. coli argC, E, A, D, and F mutants, indicating the clustered organization of the arginine biosynthetic genes within the cloned DNA fragments. The insert DNA fragments in the recombinant plasmids, named pRB1 AND pRB2, were physically mapped with several restriction enzymes. By further subcloning the entire DNA fragment containing the functions and by complementation analysis, we located the arg genes in the order of ACEBDF on the restriction map. We also determined the DNA nucleotide sequence of the fragment and report here the sequence of the argB gene. When compared to that with the mutant strain, higher enzyme activity of N-acetylglutamate kinase was detected in the extract of the mutant carrying the plasmid containing the putative argB gene, indicating that the plasmid contains a functional argB gene. Deduced amino acid sequence of the argB gene shows 45%, 38%, and 25% identity to that from Bacillus strearothermophilus, Bacillus substilus, and E. coli respectively. Our long term goal is genetically engineering C. glutamicum which produces more arginine than a wild type strain does.

• Journal of Microbiology and Biotechnology
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• 제23권3호
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• pp.430-435
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• 2013

Multidrug resistance, especially multidrug efflux mechanisms that extrude structurally unrelated cytotoxic compounds from the cell by multidrug transporters, is a serious problem and one of the main reasons for the failure of therapeutic treatment of infections by pathogenic microorganisms as well as of cancer cells. Streptococcus mutans is considered one of the primary causative agents of dental caries and periodontal disease, which comprise the most common oral diseases. A fragment of chromosomal DNA from S. mutans KCTC3065 was cloned using Escherichia coli KAM32 as host cells lacking major multidrug efflux pumps. Although E. coli KAM32 cells were very sensitive to many antimicrobial agents, the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetracycline, kanamycin, rhodamin 6G, ampicillin, acriflavine, ethidium bromide, and tetraphenylphosphonium chloride. This suggested that the cloned DNA fragment carries a gene encoding a multidrug efflux pump. Among 49 of the multidrug-resistant transformants, we report the functional gene cloning and characterization of the function of one multidrug efflux pump, namely MdeA from S. mutans, which was expressed in E. coli KAM32. Judging from the structural and biochemical properties, we concluded that MdeA is the first cloned and characterized multidrug efflux pump using the proton motive force as the energy for efflux drugs.

• 대한바이러스학회지
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• 제26권1호
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• pp.77-90
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• 1996

Nucleocapsid protein (NP)which exists in the particle of hantavirus and surrounds the viral RNA genome is one of the major structural proteins and plays role of antigen to elicit the antibody detected predorminantly right after infection of the virus in the patients of hemorragic fever with renal syndrome (HFRS)or experimental animals. NP is important target antigen in serological diagnostic system of HFRS utilizing whole antigens from the native virus particle, such as IFA, ELISA and Western blotting. Therefore, the preparation of this protein in the level of higher quantity and purity is desirasble for developed dianosis of the disease. The purpose of this study is the cloning of NP gene which exists in the S genome segment of Maaji (MAA) virus and expression of the gene to obtain qualified, genetically engineered NP to be utilized as an immunodiagnostic antigen. First of all, for the purpose of amplifing the MAA-NP gene by PCR, the specific primers were built from the known nucleotide sequence of Hantaan viral NP gene. The viral cDNA of the NP gene was synthesized by using the primers and RNase $H^-$ AMV reverse transcriptase. Thereafter, using this cDNA as a template, the NP gene was amplified specifically by Taq DNA polymrerase. The pT7blue (R)T-overhang vector systems were used for cloning of the amplified NP gene. The expression system was consisted of BL21 (DE3)pLysS and pET16b as a host and a plasmid repectively. Into Ndel site of pET16b, NP gene was ligated with cohesive end for the expression. Insertion of NP gene in the plasmid was confirmed by PCR and mini prep methods. For expression, IPTG was used and the expressed protein was characterized by Western blotting. The MAA-NP was expressed as the form of inclusion body (insoluble fraction)and the protein purified by affinity and metal chealating columns reacted specifically with the sera from patients of HFRS as to be tested by ELISA and Western blotting.

• 농업과학연구
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• 제39권1호
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• pp.23-28
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• 2012

In order to cloning of PPO gene as a melanin formation related genes involved in hardening and pigmentation of insect integument or wing, we cloned cDNA and analyzed the sequence of PPO gene of H. axyridis. PPO2 primer were designed based on the sequences of PPO genes of Tribolium castaneum and Drosophila melanogaster, and then plasmid DNA were cloned from PCR products obtained from different two color patterns. When the plasmid DNA band pattern were digested by restriction enzymes, BamH1, Xba1, and EcoR1, we found same size band pattern. However, this sequence was not homologous to sequence of T. castaneum PPO gene. Using the primer designed based on the sequence of D. melanogaster, 209 bp PCR product was observed.

• Asian-Australasian Journal of Animal Sciences
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• 제12권1호
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• pp.77-83
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• 1999

Molecular biological techniques that recently developed, have made it possible to realize some of new attempts in the research field of rumen microbiology. Those are 1) cloning of genes from rumen microorganisms mainly in E. coli, 2) transformation of rumen bacteria and 3) ecological analysis with nonculturing methods. Most of the cloned genes are for polysaccharidase enzymes such as endoglucanase, xylanase, amylase, chitinase and others, and the cloning rendered gene structural analyses by sequencing and also characterization of the translated products through easier purification. Electrotransformation of Butyrivibrio fibrisolvens and Prevotella ruminicola have been made toward the direction for obtaining more fibrolytic, acid-tolerant, depoisoning or essential amino acids-producing rumen bacterium. These primarily required stable and efficient gene transfer systems. Some vectors, constructed from native plasmids of rumen bacteria, are now available for successful gene introduction and expression in those rumen bacterial species. Probing and PCR-based methodologies have also been developed for detecting specific bacterial species and even strains. These are much due to accumulation of rRNA gene sequences of rumen microbes in databases. Although optimized analytical conditions are essential to reliable and reproducible estimation of the targeted microbes, the methods permit long term storage of frozen samples, providing us ease in analytical work as compared with a traditional method based on culturing. Moreover, the methods seem to be promissing for obtaining taxonomic and evolutionary information on all the rumen microbes, whether they are culturable or not.