• Proceedings of the Korea Information Processing Society Conference
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• 2001.10a
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• pp.105-108
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• 2001

cDNA(complementary DNA)를 복제(cloneing)하여 염기 서열화 한 EST(Expressed Sequence Tag) 데이터는 여러 생물체들의 염기서열 정보들과 비교를 통해 유사점을 찾거나 기능적 부위 검색을 통해 유전자 기능을 추정한 수 있어 기능 유전체 연구에 많이 사용되고 있다. EST 데이터를 식물은 특정종(Species)별로, 동물의 경우 종의 조직별로 클러스터링 함으로써 아직 알려지지 않은 종의 유전자를 밝혀낼 수 있음은 물론 유전자의 발현에 따른 단백질의 기능도 알아낼 수 있다. 따라서 이 논문에서는 NCBI에서 flatfile 형태로 제공하는 EST 데이터를 분석하여 관계형 데이터베이스로 모델링하고 구축하였다. 또한 EST 데이터의 효율적인 사용을 위하여 데이터를 특정 종의 조직별로 클러스터링하여 제공하는 시스템을 설계하고 구현하였다.

• The Korean Journal of Food And Nutrition
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• v.15 no.4
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• pp.364-369
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• 2002

Streptomyces shows a eukaryotic characteristic that vegetative cell can grow into mycelial form and has morphological and physiological differentiation at a certain period during its life cycle. Streptomyces has been used for the production of many biologically active compounds, such as antibiotics and pronase. Production of second metabolites and differentiation of the vegetative cell share the certain period of its lift cycle. Therefore, second metabolites may affect the differentiation of the vegetative cell. One of the microbial hormone, called A-factor, regulates the production of second metabolites, sporulation and differentiation of the cells. Streptomyces griseus produces streptomycin as well as many different kinds of proteinase. As mentioned, period of proteinases production overlaps with the period of differentiation of the vegetative cells. Protease may play a important role for the differentiation of the cells. In this paper, function of the SGPD gene cloned from S. griseus IFO 13350 tested whether it affects for the differentiation of A-factor mutated S. griseus HH1 and S. griseus IFO13350. pWHM3 and pWHM3-sprD plasmid was transformed into S. griseus HH1 and S. griseus IFO13350. Chymotrypsin activity of the cultured medium of the transformants with pWHM3-sprD plasmid didn't show any change with that of the transformants with plasmid only. The transformants with pWHM3-sprD plasmid didn't show the increase of the production of actinorhodin as well as morphological change in S. griseus IFO 13350 and HH1, as well. The promoter sequences of the SGPA and SGPB gene which encode chymotrypsin-like protease, were compared with that of SGPD gene. Regulatory mechanism of gene expression of proteinase genes will be studied for the development of high production system for protease as well as the function of the proteases.

• The Korean Journal of Food And Nutrition
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• v.17 no.1
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• pp.47-52
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• 2004

In this study, I attempted to develope the expression and purification system of human mammaglobin proteins in Escherichia coli and to produce anti-human mammaglobin rabbit antibody for the detection of human mammaglobin protein in the peripheral blood of breast cancer patients. Human mammaglobin gene was cloned and sequenced from m-RNAs purified from donated breast cancer tissues using RT-PCR. The cloned gene was inserted into pET30, pET22, and pET32 plasmid. The cloned gene in pET30 yields insoluble proteins which was difficult to purify from the cells extracts. The mammaglobin gene in pET32 was strongly expressed soluble proteins which were isolated using Ni-NTA affinity chromagraphy and DEAE-ion exchange chromatography, followed by enterokinase digestion of the purified proteins. The isolated proteins had enough purity to use as a antigen for the production of anti-mammaglobin antibody in rabbits. The polyclonal antibody produced against the isolated mammaglobin showed a specificity to mammaglobin after Westernblot immuno assay. In conclusion, the isolated mammaglobin protein and the anti-mammaglobin rabbit antibody may be used for diagnosis of breast cancer as well as development of anti-breast cancer drug.

• Korean Journal of Microbiology
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• v.32 no.2
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• pp.120-125
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• 1994

The genes, trpB, trpA and 3’ trpC(F) of Vibrio metschnikovii strain RH530 were cloned and sequenced. The trpB and trpA genes had open reading frames of 1,173 bp and 804 bp encoding 391 and 268 amino acids, respectively. The trpB and trpA genes had conventional ribosome-binding sequences and overlapped with each other by one nucleotide, suggesting that these two genes are translationally coupled. 115 nucleotide upstream the trpB start codon, tjere was an incomplete open reading frame of the 3’-end of the trpC(F). The amino acid sequences of trpB, trpA and trpC(F) of V. metschnikovii RH530 had identities of 64.2%, 82.4% and 73.7% respectively, for those of V. parahaemolyticus; 58.7%, 72.3% and 54.9%, respectively, for Salmonella typhimurium; and 42.6%. 54.1% and 12.5%, respectively, for brevibacterium lactofermentum. The genetic organization of these genes, especially in the noncoding region between trpC(F) and trpB, was distinct from that of Enterobacteriaceae.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.5
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• pp.804-808
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• 1997

The genetic differentiation and characteristics of two oyster populations (Crassostrea gigas) in Korea were assessed based on the restriction fragment length polymorphisms (RFLP) analysis and the restriction patterns of subcloned mtDNA. The restriction fragments of twenty individuals in West Sea revealed an identical pattern, determined by 8 restriction enzymes. On the other hand, two haplotypes having variation at the HindIII site were shown in the specimens from South Sea; minor haplotypes (4 of 20) were similar to the results obtained from individuals in West Sea while major haplotypes were different from those in West Sea. It was suggested that oysters (C. gigas) of West Sea might have been introduced to South Sea. Each mitochondrial DNA from two oyster populations in Korea and from one in Japan was divided to three parts and subcloned into pUC19 to use in genetic studies effectively. Restriction map was constructed based on the cleavage pattern by multiple restriction enzymes.

• The Korean Journal of Mycology
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• v.23 no.2 s.73
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• pp.129-138
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• 1995

DNA fragment being able to restore in vitro activity of ${\beta}-1,3-glucan$ synthase was cloned by transformation of the Saccharomyces cerevisiae LP353 mutant strain with genomic library constructed in the YCp50. For the selection of transformants which showed no detectable phenotype linked to recovery of the defect in ${\beta}-1,3-glucan$ synthase activity, the colony autoradiography was succesfully applied. The restriction map of the cloned DNA fragment, which is 8.5-kb in length, was constructed. Both the YEplac195 and the YCp50 carrying the 8.5-kb fragment increased ${\beta}-1,3-glucan$ synthase activity of LP353 by two fold. Neither the YEplac195 nor the YCp50 carrying the 8.5-kb DNA fragment, however, complemented the temperature-dependent osmotic sensitivity which is another distinctive phenotype of LP353. Subcloning experiments indicated that a functional region was located in 4.8-kb BglII-KpnI fragment. The 4.8-kb fragment was also able to increase the level of ${\beta}-1,3-glucan$ content in cell wall as well as the resistance of cells to cell wall lytic enzyme, ${\beta}-1,3-glucanase$. The growth rate of the LP353 with 4.8-kb fragment was almost same as that of wild type strain in liquid medium with 1.2 M sorbitol at nonpermissive temperature. Taken these results together, the 4.8-kb fragment seemed to contain the BGS2 gene for ${\beta}-1,3-glucan$ synthase activity in yeast S. cerevisiae.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.81-86
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• 2004

Among the annotated ORFs of Streptomyces coelicolor, SCO7697 was supposed to encode for phytase (myo-inositol hexakisphosphate phosphohydrolase). The DNA fragment containing SCO7697 was cloned by the PCR from the chromosomal DNA of S.coelicolor A3(2)M. The cloned fragment was introduced into E. coli expres-sion vector, pET28a(+), to yield two recombinant plasmids, pET28-SP and pET28-LP, which were designed to encode different length of proteins. When the pET28-SP and pET28-LP were introduced into E. coli BL21, the transformants successfully overexpressed recombinant proteins, but the molecular weights of the expressed pro-teins were appeared bigger than those of expected in SDS-polyacrylamide gel electrophoresis. The shift of cul-tural temperature from 37 to $30^{\circ}C$ made most of expressed protein be solubilized. The expressed protein, however, did not show any phytase activity. When the DNA fragment with its own promoter placed on the E. coli-Streptomyces vector, pWHM3, and introduced into S. lividans, the phytase activity was not detected either. These results suggest that even though the SCO7697 was annotated as a probable phytase with high probability (E value is $6e^{-89}$), the real product doest not have phytase activity.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.103-110
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• 2003

The regulation of pyrimidine nucleotide synthesis has been proved to be controlled by a regulatory protein PyrR-mediated attenuation in the Gram-positive bacteria. After several bacterial genome sequencing projects, we have discovered the PyrR orthologues in the databases for Haemophilus influenzae and Synechocystis and sp. PCC6803 genome sequences. To investigate whether these PyrR orthologue proteins regulate pyrimidine nucleotide synthesis as well as the cases of Bacillus, the PyrR regions of each strains were amplified by PCR and cloned with pUC19 or T-vector in Escherichia coli and with a shuttle vector pHPS9 for E. coli and B. subtilis. For the regulation test of the PyrR orthologues, the aspartate-transcarbamylase (ATCase) assay was carried out. From the results of the ATCase assay, it was confirmed that Synechocystis sp. PCC6803 could not restore by pyrimidines to a B. subtilis, PyrR but H. influenzae PyrR could. For Purification of PyrR orthologue proteins, PyrR orthologue genes were cloned into the expression vector (pET14b). Over-expressed product of PyrR orthologue genes was purified and analyzed by the SDS-PACE. The purified PyrR orthologue proteins from H. influenzae and Synechocystis sp. PCC6803 turned out to be molecular mass of 18 kDa and 21 kDa, respectively. The result of uracil phosphoribosyl transferase (UPRTase) assay with purified PyrR orthologue proteins showed that H. influenzae PyrR protein only has UPRTase activity. In addition, we could predict several regulatory mechanisms that PyrR orthologue proteins regulate pyrimidine de novo synthesis in bacteria, through phylogenetic analysis for PyrR orthologue protein sequences.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.1-7
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• 2002

Phytase from Escherichia coli WC7 was purified from cell extracts and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for phytate hydrolysis was 6$0^{\circ}C$ and pH 5.0, respectively. The enzyme was stable up to 6$0^{\circ}C$ and over broad pH range (pH 2-12). The enzyme had higher affinity for sodium phytate than p-nitrophenylphosphate (pNPP). That is, the apparent Km value for sodium phytate and pNPP were $0.15\pm$0.02 mM and 2.82$\pm$0.05 mM, respectively. The gene encoding the phytase was cloned in E. coli XL1-Blue. Sequence analysis showed an open reading frame of 1241 Up encoding a signal peptide (22 aa) and a mature enzyme (410 aa). WC7 phytase was expressed up to 17.5 U/ml in the transformed E. coli XL1-Blue/pUEP, which was 23-fold higher than the activity from wild strain.

• Journal of Life Science
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• v.22 no.4
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• pp.437-446
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• 2012

A metagenomic library of cow rumen in the pCC1FOS phage vector was screened in $E.$ $coli$ EPI300 for cellulase activity on carboxymethyl cellulose agar plates. One clone was partially digested with $Sau$3AI, ligated into the $Bam$HI site of the pBluescript II SK+ vector, and transformed into $E.$ $coli$ $DH5{\alpha}$. We obtained a 1.5 kb insert DNA, designated $cel$5C, which hydrolyzes carboxymethyl cellulose. The cel5C gene has an open reading frame (ORF) of 1,125 bp encoding 374 amino acids. It belongs to the glycosyl hydrolase family 5 with the conserved domain LIMEGFNEIN. The molecular mass of the Cel5C protein induced from $E.$ $coli$ $DH5{\alpha}$, as analyzed by CMC SDS-PAGE, appeared to be approximately 42 kDa. The enzyme showed optimum cellulase activity at pH 4.0, and $50^{\circ}C$. We examined whether the $cel$5C gene comes from the 49 identified cow rumen bacteria using PCR. No PCR bands were identified, suggesting that the $cel$5C gene came from the unidentified cow rumen bacteria.