• Environmental Analysis Health and Toxicology
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• v.18 no.4
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• pp.287-294
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• 2003

출아형 효모 Saccharomyces cerevisiae RAD3 유전자는 절제회복 및 세포의 생존에 필수적이며, DNA dependent ATPase와 DNA-RNA helicase활성을 가지고 있는 것으로 알려져 있다. 본 연구는 분열형 효모 Schizosaccharomyces pombe에서 절제회복과 세포의 생존에 필수적인 출아형 효모 RADS유전자와 유사한 유전자를 S. pombe genomic DNA library에서 분리하여 그 특성을 연구하였다. 분리한 RADS 유사유전자를 HRD3 유전자라 명명하였다. 발현 vector pET3a를 이용하여 분리한 HRD3 유전자를 과 발현하였을 때 HRD3단백질은 숙주단백질의 합성 억제 또는 분해 촉진을 유발하여 숙주세포인 대장균에 독성 효과를 나타냄이 관찰되었다. HRD3유전자와 lacZ유전자를 융합시킨 여러 가지 재조합 vector를 만들어 이들 융합단백질을 분리하였다. 이 결과 HRD3단백질의 카르복실 말단 부위가 DNA회복기능과 대장균에서의 독성효과를 나타내는 중요한 부위로 생각된다.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.30-36
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• 1992

nif (nitrogen fixation)-H.D, K genes of Frankia sp. SNU 014201. a symbiotic strain isolated from root nodule of Alnus hirsura, were found to be located in the genome on 13.5 kb of EcoRI, 18.0 kb of BamHI, 10.5 kb of BglII and 4.5 kb of KpnI fragments. Using EMBL-3 BamHI arms of bacteriophage lambda. the genomic library was constructed. from which fourteen recombinant phage nif-clones were selected. Among them, Ahnif-I2 had insert DNA of 18 kb, in which 7.9 kb of BamHl fragment contained nif-H, D, K and 3.6 kb of HindlIl/KpnI had nif-H and partial -D. Therefore, the 7.9 kb and 3.6 kb fragments were subcloned and partial restriction maps were constructed. As the results, nif-F1, D.K genes were found to be located continuously on the 6.5 kb of HindII/BamHI and 5.2 kb of SalIIBamHI fragment in the genome of Frankia sp. SNU 014201.

• The Korean Journal of Mycology
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• v.22 no.3
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• pp.247-253
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• 1994

This study was carried out to isolate and characterize $A{\alpha}$ mating locus controlling fruiting body formation directly in the Basidiomycete Schzophyllum commune growing in the North America. Total numbers of genomic library of S. commune UVM1-34 was about $2{\times}10^4$ cells. About 90% library was appeared to have about 35 kb inserted genome DNA in cosmid pTC20 vector. 6 clones were proved to have positive signal to probes within Z and Y region in colony and southern hybridization. In the mating activity test, all the 6 positive clones were appeared to have $A{\alpha}3$ mating activity although they had two different restriction patterns. pSC13 containing 5.7 Kb PstI-fragment of UVM 1-34 $A{\alpha}3$ allele showed about 50% clamp cell formation indicating mating activity when cotransformation was done together with cosmid pTC20.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.153-156
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• 2000

Xylan, the major hemicellulose component of many plants, occurs naturally in a partially acetylated form and lignin, the most resistant component in plant cell wall degradation, is also attached to ${\beta}-1,4-linked-D-xylose$ backbone through the ester linkage. Esterases are required to release the esterified substituent and acetyl esterases are important in the complete degradation of acetylated polysaccharides, like pectins and xylans. The gene(Axe) encoding acetyl xylan estarase(AXE) was isolated from genomic ${\lambda}$ library from Aspergillus ficuum. Nucleotide sequencing of the Axe gene indicated that the gene was separated with two intervening sequences and the amino acid sequence comparison revealed that it was closely related to that from A. awamori with the 92 % indentity. Heterologous expression of AXE was conducted by using YEp352 and Saccharomyces cerevisae 2805 as a vector and host expression system, respectively. The Axe gene was placed between GAL1 promoter and GAL7 terminator and then this recombinant vector was used to transform S. cerevisiae 2805 strain. Culture filtrate of the transformed yeast was assayed for the presence of AXE activity by spectrophotometry and, comparing with the host strain, four to five times of enzyme activity was detected in culture filtrate of transformed yeast.

• Journal of Life Science
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• v.27 no.4
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• pp.398-407
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• 2017

BTG 1 (B-cell translocation gene 1) gene was first identified as a translocation gene in a case of B-cell chronic lympocytic leukemia. BTG1 is a member of the BTG/TOB family with sharing a conserved N-terminal region, which shows anti-proliferation properties and is able to stimulate cell differentiation. In this study, we identified and characterized the pacific oyster Crassostrea gigas BTG1 (cg-BTG1) gene from the gill cDNA library by an Expressed Sequence Tag (EST) analysis and its nucleotide sequence was determined. The cg-BTG1 gene encodes a predicted protein of 182 amino acids with 57% 56% identities to its zebrafish and human counterparts, and is an intron-less gene, which was confirmed by PCR analysis of genomic DNA. Maximal homologies were shown in conserved Box A and B. The deduced amino acid sequence shares high identity with other BTG1 genes of human, rat, mouse and zebrafish. The phylogenic analysis and sequence comparison of cg-BTG1 with other BTG1 were found to be closely related to the BTG1 gene structure. In addition, the predicted promoter region and the different transcription-factor binding site like an activator protein-1 (AP-1) response element involved in negative regulation and serum response element (SRE) were able to be identified by the genomic DNA walking experiment. The quantitative real-time PCR analysis showed that the mRNA of cg-BTG1 gene was expressed in gill, heart, digestive gland, intestine, stomach and mantle. The cg-BTG1 gene was expressed mainly in heart and mantle.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.495-500
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• 1997

To isolate a gene for cyclodextrin glycosyltransferase (CGTase) from alkalophilic Bacillus sp. E1, polymerase chain reaction (PCR) amplification was carried out. Direct molecular cloning of 1.2 kbp fragment and partial nucleotide sequence analysis of the PCR amplified clone, pH12, showed close homology with CGTases from Bacillus species. To investigate the genomic structure of the gene, Southern blot analysis of genomic DNA was carried out with the clone pH12 as a molecular probe. It showed that 5.3 kbp XbaI fragment was hybridized with the probe pH12. To isolate a genomic clone, genomic DNA library was constructed and a genomic clone for CGTase, pCGTE1, was isolated. Nucleotide sequence analysis of the clone pCGTE1 revealed that BCGTE1 contained 2,109 bp open reading frame encoding a polypeptide of 703 amino acids and showed over 94.3% amino acid sequence homology with CGTase of ${\beta}-cyclodextrin$ producer, Bacillus sp. KC201.(Received October 7, 1997; accepted October 20, 1997)

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.851-856
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• 2005

Although valuable microbes have been isolated from the soil for the various productions of useful components, the microbes which can be cultivated in the laboratory are only $0.1-1\%$ of all microbes. To solve this problem, the study has recently been tried for making the valuable components from the environment by directly separating unculturable micrbial DNA in the soil. But it is known that humic acid originated from the soil interrupts various restriction enzymes and molecular biological process. Thus, in order to prevent these problems, this study modified the method separated soil DNA with phenol, CTAB and PEG. In order to compare the degree of purity for each DNA and the molecular biological application process, $A_{260}/A_{280}$ ratio, restriction enzymes, and PCR were performed. In case of DNA by the modified method, total yield of DNA was lower but $A_{260}/A_{280}$ ratio was higher than the previously reported methods. It was confirmed that the degree of purity is improved by the modified method. But it was not cut off by all kinds of tested restriction enzymes because of the operation of a very small amount of interrupting substances. When PCR was operated with each diluted DNA in different concentrations and GAPDH primer, the DNA by the modified method could be processed for PCR in the concentration of 100 times higher than by the previously reported separation method. Therefore, this experiment can find out the possibility of utilization for the unknown substances by effectively removing the harmful materials including humic acid and help establishing metagenomic DNA library from the soil DNA having the high degree of purity.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.65-72
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• 2004

Instability of some eukaryotic sequence propagated in prokaryotic hosts is a frequently observed phenomenon. It is well documented that long inverted repeats, AT-rich sequences with structures like Z-DNA are extremely unstable in E. coli. These sequences may either be under-represented or even lost when cloned in E. coli. When we analyzed the polymorphic pattern for several tandom repeat (TR) in human SCKI gene, we found some TR regions were frequently deleted from plasmids and had difficult problem for their sequencing. These regions may result in non-clonability of the DNA sequence. Here we have cloned two difficult TR regions under low temperature and made two library for DNA sequencing using a nebulizer or sonicator. This study will help to determine the unstable genomic elements in complex mammalian genome.

• Animal cells and systems
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• v.7 no.2
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• pp.159-164
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• 2003

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. The RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA-RNA helicase activities. To examine the extent of conservation of structure and function of a S. pombe RAD3 during eukaryotic evolution, the RAD3 homolog gene was isolated by screening of genomic DNA library. The isolated gene was designated as HRD3 (homolog of RAD3 gene). Southern blot analysis confirmed that S. pombe chromosome contains the same DNA as HRD3 gene and this gene exists as a single copy in S. pombe. The transcript of 2.8 kb was detected by Northern blot analysis, The level of transcripts increased by ultraviolet (UV) irradiation, indicating that HRD3 is one of the UV-inducible genes in S. pombe. Furthermore, the predicted partial sequence of HRD3 protein has 60％ identity to S. cerevisiae RAD3 gene. This homology was particularly striking in the regions identified as being conserved in a group of DNA helicases. Gene deletion experiments indicate that the HRD3 gene is essential for viability and DNA repair function. These observations suggest evolutionary conservation of other protein components with which HRD3 might interact in mediating its DNA repair and viability functions.

• Korean Journal Plant Pathology
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• v.14 no.2
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• pp.177-183
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• 1998

A DNA fragment which is involved in tolassin production was cloned to obtain a molecular marker of Pseudomonas tolaasii, a casual agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). Tolaasin is a lipodepsipeptide toxin and known as a primary disease determinant of the P. tolaasii. It is responsible for formation of white line in agar when P. tolaasii were cultured against white line reacting organisms (WLROs). White line negative mutants (WL-) were generated by conjugation between rifampicin resistant strain of P. tolaasii and E. coli carrying suicidal plasmid pSUP2021 : : Tn5. The ability of tolaasin production of the WL- mutants was examined by hemolysis test, pathogenicity test, and high pressure liquid chromatography (HPLC) analysis of culture filtrate. All of the WL- mutants were lost the ability of tolaasin production (Tol-). Genomic library of the Tol- mutant was constructed in pLAFR3 and the cosmid clone containing Tn5 was selected. DNA fragment fro franking region of Tn5 was cloned from the plasmid and used as a probe in Southern blot. DNA-DNA hybridization with the probe to total DNA from group of bacteria ecologically similar to P. tolaasii including WLORs, fluorescent Pseudomonads isolated from oyster mushroom, P. agarici, P. gingeri, and some of other species of Psedomonas showed that some of the tested bacteria do not have any hybridized band and others have bands sowing RFLP. The cloned DNA fragment or its nucleotide sequence will be useful in detection and identification of the P. tolaasii.