• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.619-624
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• 1992

Tn5 lac 삽입으로 채소입고병원균에 길항력이 약화된 T-67 및 고추역병균과 참깨역병균에 길항력이 약화된 T-81의 Tn5 lac 유전자 일부와 오른쪽 말단에 있는 길항관련 유전자의 flanking sequence가 cloning된 pAG67 및 pAG81 clone을 선발하였고, pAG67 및 pAG81 clone된 길항관련 유전자의 flanking sequence를 야생 길항균 Pseudomonas maltophilia B-14의 DNA를 probe로 사용하여 Southern hybridization으로 확인하였으며, 제한효소 지도를 작성하여 8Kb 및 4Kb 크기의 flanking sequence가 cloning되었음을 확인하였다. pAG6 및 pAG81의 flanking sequence를 EcoRi-BglII와 EcoRI-MpaI으로 분리하여 유전자 은행으로부터 길항관련 유전자가 cloning된 cosmid clone 7개주를 선발하였다.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.488-494
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• 1992

Three clones containing mouse CD4 gene were prepared using AKR genomic cosmid library. The role of 6, 500 bp 5' flanking region of the first exon of the AKR CD4 gene in tissue or developmental stage specific expression of CD4 has been studied. The deletion constructs containing various amounts of CD4 5' flanking sequences were prepared, and they were transfected into the cell lines representing different cell types or developmental stages of CD4 expression. Study of the reporter gene expression revealed that at least 1, 700 bp of 5' flanking region did retain promoter activity for CD4 expression. This area did not seem to contain enhancer activity for a full expression of CD4. However, the putative promoter interacted with other tissue specific enhancer sequence and showed the tissue specificity of the enhancer element.

• Korean Journal Plant Pathology
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• v.12 no.2
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• pp.169-175
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• 1996

콩 불마름병균 Xanthomonas campestris pv. glycines 8ra는 X. c. pv. vesicatoria에 길항력이 있는 bacteriocin인 glycinecin을 생성 분비한다. Bacteriocin 생성 분비 능력이 있는 콩 불마름병균을 효과적인 생물학적 방제원으로 활용하기 위해서는 좀더 체계적인 연구가 필요하여, bacteriocin 생성에 관계되는 유전자의 분리를 시도하였다. 약 2,000개의 Xanthomonas campestris pv. glycines 8ra cosmid library에서 bacteriocin의 생성 분비 능력을 조사하여 다섯 개의 clone을, pG011, pG0113, pG33과 pG35, 선발하였다. 그중 한 clone pG08을 임의로 선택하여 plasmid DNA를 분리하였다. Plasmid pG08에서 약 6.0 kb의 DNA를 떼어내어 다른 plasmid vector에 넣은 subclone pBL5는 bacteriocin의 생성 분비 능력이 있었다. Plasmid pG08을 제한효소 처리후 다시 접함시켜 만든 몇 개의 subclone과 pBL5의 제한효소 지도를 비교 분석한 결과 약 3.0 kb의 BamHI-HindIII 부분의 DNA가 bacteriocin의 생성에 관계함을 알았다.

• Korean Journal of Microbiology
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• v.29 no.4
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• pp.203-208
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• 1991

The dapA-complementing gene (L-2, 3-dihydrodipicolinate synthetase: DHDP synthetase, dapA) has been cloned by using a cosmid genomic bank of Corynebacterium glutamicum JS231 that is a lysine overproducer, AEC (s-(2-aminoethyl)-L-cysteine) resistant mutant. By enzymatic deletion analysis, the DNA region complementing the escherichia coli dapA host could be confined to 4.5kb SalI-generated DNA fragment. This DNA fragment was inserted into the C. glutamicum/E. coli shuttle vector pECCG117 to construct pDHDP5812. The specific activity of DHDP synthetase detected in C. glutamicum JS231/pDHDP5812 was increased about 10 fold above that of C. glutamicum JS231. The addition of leucine during growth did not repress the expressin of dapA, and the enzyme activity was not inhibited by lysine.

• Journal of Life Science
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• v.10 no.2
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• pp.50-54
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• 2000

The RAD4 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. A yeast RAD4 gene has been previously isolated by functional complementation. In order to identify the RAD4 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD4 DNA, and then isolated homologous C. cinereus chromosome. Here, we report the cloning and characterization of fungus C. cinereus homolog of yeast RAD4 gene. Southern blot analysis confirmed that C. cinereus contains the sequence homologous DNA to RAD4 gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from C. cinereus cells was hybridized with the 3.4 kb BglII DNA fragment of the S. cerevisiae RAD4 gene, a 2.5 kb of transcript was detected. The isolated gene encodes a protein of 810 amino acids.

• Applied Biological Chemistry
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• v.50 no.4
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• pp.262-267
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• 2007

To access the natural product antibiotics produced by uncultured microorganisms, six cosmid libraries of DNA extracted directly from soil samples (environmental DNA, eDNA) were constructed and screened for the production of antibacterial active molecules. Of the approximately 60,000 clones screened, one antibacterial clone (YS92B) was detected. Ethyl acetate extracts of clone YS92B showed antibacterial activity against various pathogenic bacteria (Listeria monocytogenes, Bacillus subtilis, Pseudomonas syringae, Xanthomonas campestris pv. oryzae, Staphylococcus epidemis). Active constituents from cultures of YS92B were isolated and purified using a bioassay-guided fractionation against B. subtilis through a series of procedures (ethyl acetate extraction, Sephadex LH20 column chromatography, High Performance Liquid Chromatography). NMR (Nuclear Magnetic Resonance) spectral analysis of a major antibacterial active YS92B-VII indicated that it is a lauric acid linked to tyrosine. This report describes the characterization of antibacterially active long chain N-acyl derivatives of tyrosine that are produced by eDNA clones hosted in Escherichia coli from Korean soils.

• Applied Biological Chemistry
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• v.48 no.4
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• pp.345-351
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• 2005

To access the natural products of uncultured microorganisms, we constructed and screened the metagenomic DNA libraries by using a cosmid vector and DNA inserts isolated directly from soil. Initial screening of the libraries in Escherichia coli resulted in the isolation of several clones that produce a dark brown color when grown in LB medium. One of the positive clones, designed pYS85C, was transposon mutagenized and the DNA surrounding the transposon insertions in cosmids that no longer conferred the production of brown pigment to E. coli was sequenced. Annotation of the pYS85C sequence obtained from the transposon mutagenesis experiment indicated a single 393 amino acid open reading frame (ORF) with a molecular mass of about 44.5 kDa, predicted to be a 4-hydroxyphenylpyruvate dioxygenases (HPPDs), was responsible for the observed brown pigment. In a BLAST search against deposited sequence, the translated protein from this ORF showed moderate-level identity (>60%) to the other known HPPDs and was most conserved in the C-terminal region of the protein. These results show that genes involved in natural product synthesis can be cloned directly from soil DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.

• Applied Biological Chemistry
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• v.38 no.4
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• pp.313-319
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• 1995

The involvement of the cell-wall degrading enzymes in Rhizobium has long been an unsolved question about the infection process in the formation of root nodule. To assess the contribution of the cellulase to the nodulation of rhzobia, here we report the production of cellulase from R. meliloti TAL1372 which degrade carboxymethylcellulose (CMC) model substrate with CMC-plate method. We constructed a genomic library by cloning Sau3A-digested genomic DNA from R. meliloti TAL1372 into the BamHI site of the cosmid vector pLAFR3. Out of more than one thousand transductants of E. coli, one clone (pRC8-71) had CM-cellulase activity and contained pLAFR3 cosmid with 30 kb insert of R. meliloti DNA The product of CM-cellulase gene was analyzed by native PAGE. About 45 kD protein was considered to be a product of the gene. Tn5 mutagenesis reveals that the structural gene located in a ca. 3 kb KpnI fragment. The cellulase-minus mutants of R. meliloti TAL1372 were obtained by Tn5 mutagenesis of pRC8-71 and marker exchange techniques. Analyses of the nodulation ability of these Tn5 mutants showed that the CM-cellulase gene of R. meliloti TAL1372 may be involved in early nodulation development on alfalfa (Medicago satiua).

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.47-52
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• 1994

The cells of Campylobacter jejuni heat-shocked at 48${\circ}C$ for 30 min synthesized the heat shock proteins of HSP90, HSP66 and HSP60. Those heat shock proteins were found to correspond to the heat shock proteins of HSP87, HSP66 (DnaK), and HSP58 (GroEL) of E. coli, respectively. By Southern blot analysis of the chromosomal DNAs of C. jejuni with groESL and dnaK genes of E. coli as DNA probes, the heat shock genes of C. jejuni which are homologous to the E. coli groESL and dnaK genes were found to exist in the chromosomal DNA. The genomic libraries of C. jejuni were constructed with the cosmid vector pWE15 and the groEL gene of C. jejuni were cloned in E. coli B178 groEL44 temperature senstive mutant. The hybrid plasmid (pLC1) was inserted with the DNA fragment (about 5.7kb in size) containing the groEL gene. E. coli groEL44 mutant cell transformed with the pLC1 could grow at 42${\circ}C$ by synthesizing the HSP60 of C. jejuni and regained the susceptibility to the ${\lambda}$ vir phage by expression of the groEL gene in the cloned cells. These indicated that the groEL products of C. jejuni had chaperon effects by synthesizing the heat shock proteins in the cloned cells of E. coli.

• Applied Biological Chemistry
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• v.41 no.2
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• pp.125-129
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• 1998

Xanthomonas sp. isolated from soil exhibited cell wall lytic activity of Candida albicans and secreted chitinase in chitin media. Especially, the chitinase activity was induced by chitin and reached a maximum level at 3 days culture in chitin media. We constructed genomic library of Xanthomonas sp. using cosmid vector in E. coli. Oligonucleotide probe was synthesized from the consensus sequence corresponding to chitinase active site, which was derived from the comparison of amino acid sequences of bacterial chitinase genes. Using this oligonucleotide probe, we screened the genomic library. By restriction enzyme mapping of the positive clones, we identified 4 independent clones which may contain the chitinase gene. One of the clones, named pXCH1 (1.2 kb insert), was further analyzed. Northern blot analysis indicated that is transcripts, 1 kb and 0.8 kb, were induced by chitin. When the cloned gene was induced by IPTG in E.coli cell, chitinase activity which was secreted onto culture media was not observed. However, when the cell was disrupted by using sonicator and then centrifuged, the supernatant exhibited chitinase activity. SDS-PAGE of the supernatant indicated that about 35 kDa protein was induced by IPTG. From these results, it was concluded that the cloned DNA was one of the chitinase genes of Xanthomonas sp.