• Journal of Animal Science and Technology
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• 제46권1호
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• pp.23-30
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• 2004

지방세포분화 과정에서 중추적인 역할을 한다고 보고되어지고 있는 PPAR$\gamma$ 유전자를 cloning 하기 위하여 한우 12개월 지방조직을 이용하여 Total RNA를 추출하고 RT-PCR을 수행하여 1515bp, 505 아미노산 서열을 가진 PPAR$\gamma$ 유전자를 cloning 하였다. bovine을 제외한 4종의 다른 동물과의 아미노산 서열을 비교한 결과 모두 90% 이상의 상동성을 나타내었다. 특히 NCBl에 보고된 Norwegian cattle PPAR$\gamma$ (NCBI Accession No. O18971)의 아미노산 서열과 비교한 결과 99.2%의 상동성을 나타내었으며 아미노산 서열중 81번째 이소루신, 140번째 아르기닌, 157번째 글루탐산, 486번째 리신이 각각 트레오닌, 트립토판, 글리신, 이소루신으로 치환된 상태의 아미노산 서열을 나타내었다. 특히 nuclear hormone receptor의 DNA binding domain 영역에 포함 되는 140번째, 157번째 아미노산의 치환은 앞으로 한우PPAR$\gamma$ 유전자가 조절하는 다른 유전자와의 관계를 밝히는데 매우 중요한 점이 될 것으로 사료되어진다. 한우 PPAR$\gamma$ 의 발현은 지방조직에서 매우 높은 발현을 보였으며, 한우 12개월령과 30개월령 지방조직에서의 PPAR$\gamma$ 발현을 비교 분석하였을 때 12개월령에서보다 30개월령 지방조직에서 PPAR$\gamma$ 발현이 약 6배정도 높았다. 이와 같은 결과는 한우 PPAR$\gamma$ 유전자도 사람과 생쥐에서와 같이 지방분화에 관여하고 있다는 보고와 일치하는 결과라고 사료된다.

• Genomics & Informatics
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• 제3권1호
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• pp.8-14
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• 2005

Although much is known about the molecular biology of platelets, the megakaryocytes' (MKs) molecular biology was not understood so well because of their rareness. By the cloning and characterization of thrombopoietin (TPO), which is the principal regulator of the growth and development of the MKs, researches on the MKs have been growing rapidly. To understand megakaryocytopoiesis, we investigated the gene expression profile of the MKs using oligonucleotide microarray where 10,108 unique genes were spotted. Comparing the fluorescence intensities of which ratio is $\ge$ ${\mid}2{\mid}$, 372 genes were up-regulated and 541 genes were down-regulated in MKs. For confirmatory expression, RNase protection assay (RPA) establishing abundant apoptotic gene expression was carried out. In MKs, many of the known genes, including several platelet related genes, GATA binding protein were highly expressed. Particularly, TGF beta, clusterin (complement lysis inhibitor), and thymosin beta 4 (actin-sequestering molecules) were expressed highly in MKs. As MKs specific expressed genes may regulate normal and pathologic platelet (and/or MK) functions, the transcript profiling using microarray was useful on molecular understanding of MKs,

• 대한바이러스학회지
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• 제26권2호
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• pp.173-179
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• 1996

The gene encoding gIII of bovine herpesvirus type 1 (BHV-1) PQ strain was cloned and expressed in baculovirus. Although the gIII gene is located in Hind III I fragment as the case of the other BHV-1 strains, differences in size and restriction endonuclease site within the fragment were identified. The gIII expression was predominantly detected on the surface on insect cells by indirect immunofluoresecnce assay using monoclonal antibody. The western blotting analysis also revealed the presence of expressed protein of a similar molecular size to the original gIII protein. The immunogenicity of expressed protein were tested in guinea pigs. The immunized guinea pigs with expressed protein developed the neutralizing antibodies against BHV-1.

• BMB Reports
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• 제37권5호
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• pp.574-581
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• 2004

The full-length cDNA of the lumbrokinase fraction 6 (F6) protease gene of Lumbricus rubellus was amplified using an mRNA template, sequenced and expressed in E. coli cells. The F6 protease gene consisted of pro- and mature sequences by gene sequence analysis, and the protease was translated and modified into active mature polypeptide by N-terminal amino acid sequence analysis of the F6 protease. The pro-region of F6 protease consisted of the 44 residues from methionine-1 to lysine-44, and the mature polypeptide sequence (239 amino acid residues and one stop codon; 720 bp) started from isoleucine-45 and continued to the terminal residue. F6 protease gene clones having pro-mature sequence and mature sequence produced inclusion bodies in E. coli cells. When inclusion bodies were orally administrated rats, generated thrombus weight in the rat' venous was reduced by approximately 60% versus controls. When the inclusion bodies were solubilized in pepsin and/or trypsin solutions, the solubilized enzymes showed hemolytic activity in vitro. It was concluded the F6 protease has hemolytic activity, and that it is composed of pro- and mature regions.

• 한국식물학회:학술대회논문집
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• 한국식물학회 2002년도 춘계학술발표대회:발표눈문요지록
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• pp.62-72
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• 2002

This study has investigated the biosynthesis and function of the heavy metal binding peptides, the phytochelatins, in plants. PCs are synthesised enzymatically from glutathione by the enzyme PC synthase in the presence of heavy metal ions. Using Arabidopsis thaliana as a model organism cadmium-sensitive, phytochelatin-deficient mutants have been isolated and characterised in previous studies. The cadl mutants have wildtype levels of glutathione, are PC deficient and lack PC synthase activity. Thus, the CADl gene has been proposed to encode PC synthase. The CADl gene was isolated by a positional cloning strategy The gene was mapped and a candidate identified. Each of four cadl mutants had a single base pair change in the candidate gene and the cadmium-sensitive, cadl phenotype was complemented by the candidate gene. This demonstrated the CADl gene had been cloned. A homologous gene in the fission yeast, Schizosaccharomyces pombe was identified through database searches. A targeted-deletion mutation of this gene was constructed and the mutant, like cadl mutants of Arabidopsis, was cadmium-sensitive and PC-deficient. A comparison of the redicted amino acid sequences reveals a highly conserved N-terminal region Presumed to be the catalytic domain and a variable C-terminal region containing multiple Cys residues proposed to be involved in activation of the enzyme by metal ions. Similar genes were also identified in animal species. The Arabidopsis CADl/AtPCSl and S. pombe SpbPCS genes were expressed in E. coli and were shown to be sufficient for glutathione-dependent, heavy metal activate PC synthesis in vitro, thus demonstrating these genes encode PC synthase enzymes. Using RT-PCR, AtPCSl expression appeared to be independent of Cd exposure. However, at higher levels of Cd exposure a AtPCSl-CUS reporter gene construct appeared to be more highly expressed. Using the reporter gene construct, AtPCSl was expressed most tissues. Expression appeared to be greater in younger tissues and same higher levels of expression was observed in some regions, including carpels and the base of siliques. AtPCS2 was a functional gene encoding an active PC synthase. However, its Pattern of expression and the phenotype of a mutant (or antisense line) have not been determined. Assuming the gene is functional then it has clearly been maintained through evolution and must provide some selective advantage. This implies that, at least in some cells or tissue, it is likely to be the dominant PC synthase expressed. This remains to be determined

• 미생물학회지
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• 제22권3호
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• pp.167-173
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• 1984

The cellabiase (${\beta}$-glucosidase) gene in a Cellulomonas sp. CS1-1 was cloned into E. coli HB101 using the vector plasmid pBR322, and the expression of the gene in E. coli studied. The chromosomal DNA of the cellulomonas was digested by seveal restriction enzymes, each of which has only one cleaving site in plasmid pBR322. The recombinant plasmid, pSB2, created with Sal I frament, was expressed for the cellobiase gene in E. coli. The recombiant plasmid was estimated to contain 6.4 Kb foreign DNA at the Sal I site of plasmid pBR322 and the inserted DNA was mapped by single and double digestion with several enzymes. E. coli HB101(pSB2) has slowly grown in a mineral liquid medium containing cellobiose as a sole carbon source. The cellobiase activity in the transformed E. coli was 132 units per liter, which is equivalent to one twenty fifth of that in doner strain Cellulomonas sp. CS1-1. The transforned cell with plasmid containing cellulase gene grow well in the LB mediuns. The synthesis of cellobiase in the strain, E. coli HB101 (pSB2), was inhibited by glucose and at high concentration of cellobiose, and induced by cellobiose at low concentration.

• 한국식품영양학회지
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• 제6권4호
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• pp.314-321
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• 1993

We have cloned the Bacillus cellulyticus K-12 avicelase (Avi, E.C.3.2.1.4) gene (ace A) In E. coli. This was accompanied by using the vector PT7T3U 19 and Hind W -Hind m libraries of Bacillus cellulyticus K-12 chromosomal inserts created in 5.cofi. The Libraries were screened for the expression of avicelase by monitoring the immunoreaction of the anti-avicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5kb Hind III fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constituvely using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide which showed a CMCase activity with an Mr of 54000 was detected.

• International Journal of Industrial Entomology and Biomaterials
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• 제17권2호
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• pp.189-195
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• 2008

The sequence of hydroxypyruvate isomerase gene was obtained in NCBI. In this study, the hydroxypyruvate isomerase gene of Bombyx.mori was identified and annotated with bioinformatics tools. The result was confirmed by RT-PCR, prokaryotic expression, mass spectrographic analysis and sub-cellular localization. The hydroxypyruvate isomerase cDNA comtains a 783bp ORF, and has 4 exons. The deduced protein has 260 amino acid residues with the predicted molecular weight of 29169.30 Da, isoelectric point of 6.10, and contains conserved PRK09997 and Hfi domains. The hydroxypyruvate isomerases of Nasonia vitripennis and Bombyx mori have a high homology. Through RTPCR analysis, we found that this transcript was present in testis, ovary, blood-lymph, fat body, midgut, silk gland and tuba Malpighii. This protein was located in cytoplasm through immunohistochemistry. We submitted the cloned gene under the accession number EU344910. The enzyme has been classified under accession number EC 5.3.1.22.

• Journal of Plant Biotechnology
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• 제36권1호
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• pp.45-52
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• 2009

In order to understand the molecular interactions that occur between rice and the rice blast fungus during infection, we previously identified a number of rice blast fungal elicitor-responsive genes from rice (Oryza sativa cv. Milyang 117). Here, we report the cloning and characterization of the rice fungal elicitor-inducible gene Oshin1 (GenBank Accession Number AF039532). Sequence analysis revealed that the Oshin1 cDNA is 1067 bp long and contains an open reading frame encoding 205 amino acid residues. The Oshin1 gene shows considerable sequence similarity to the tobacco hin1 and hin2 genes. The predicted Oshin1 protein has a cysteine-rich domain at the N-terminus and is rich in leucine, serine, and alanine residues. Southern blot analysis suggests that Oshin1 gene is a member of a small gene family in the rice genome. To examine the expression of Oshin1, Northern blot analysis was conducted. Expression of the Oshin1 transcript is rapidly induced in suspension-cultured rice cells treated with fungal elicitor, salicylic acid or hydrogen peroxide. In addition, Oshin1 transcript levels are rapidly increased by treatment with $Ca^{2+}$/A23187. The expression of Oshin1 was also elevated in 3-week old leaf tissues upon ethephon application or fungal elicitor treatment. Our results suggest that the Oshin1 gene is involved in plant defense responses to environmental stresses.

• Journal of Life Science
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• 제9권1호
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• pp.54-57
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• 1999

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in E. coli TP2139 (${\Delta}$lac, ${\Delta}$crp). One of the cloned genes, pCKB13, was further analyzed. In order to find whether the increased expression of the gene under the direction of maltose metabolism, we constructed several recombinant subclones. We have confirmed that the clone, pCKB13 codes Coenzyme A transferase gene by partial nucleotide sequencing in the terminal region. The enzyme activity of Coenzyme A transferase increased after introduction of the multicopy of the cloned gene in E. coli. The recombinant proteins expressed by multicopy and induction with IPTG, two polypeptide of 26-and 28-kDa, were confirmed by SDS-PAGE. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.