• Journal of the Korea Organic Resources Recycling Association
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• v.16 no.4
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• pp.64-73
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• 2008

The cDNA of CCF (coelomic cytolytic factor)-like gene (EC 3.2.1.16), a kind of glycosyl hydorlase, was isolated and cloned from the midgut of the earthworm Eisenia anderi. The size of nucleotide sequence appeared to be 1,152 bp and its predicted coding region was composed of 384 amino acid residues including the initiation methionine. The 17 residues at N-terminal end in the deduced amino acid sequence were regarded to be a signal peptide. Based on the amino acid sequence analysis, it appeared that this CCF-like protein could belong to glycosyl hydrolase family 16 (GHF16) and showed a high sequence homology of about 79~99% with CCF and CCF-like proteins from other earthworm species. The CCFs and CCF-like proteins from various earthworm species exhibited a 100% homology in the polysacchride-binding motif and glucanase motif. It has been reported that the CCFs isolated from E. fedita appeared to show a broader pattern recognition specificity than those from other earthworm species because this species resides in decaying organic matter showing very high microbial activity, implying that CCF-like protein isolated in this study from E. andrei might exhibit a broad substrate specificity that is a useful characteristic for industrial application. A phylogenetic analysis using the deduced amino acid sequences of CCF-related proteins through the BLASTX revealed that GHF16 families could be divided into three groups of metazoa, viriplantae and eubacteria subfamily. Subsequently the CCF-related proteins of metazoa subfamily could clearly be subgroup into lophotrochozoan and edysozoan type including a deuterostome origin. Further understanding of the biological properties of E. andrei CCF-like protein should be addressed to regulate the ${\beta}$-D-glucan hydrolysis and production for the industrial uses.

• Journal of Environmental Science International
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• v.17 no.10
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• pp.1121-1127
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• 2008

The free-living amoeba and Acanthamoeba sp. are widely distributed in fresh water, soil, air and dust in the world. We studied distribution of amoeba from low Nakdong River(Mulgum and Maeri) and removal efficiency in water treatment process of Busan metropolitan city. During this investigation, water quality showed pH $7.4{\sim}9.6({\pm}1.1)$, water temperature $2.0{\sim}29.0({\pm}17)^{\circ}C$, turbidity $4.8{\sim}27.4({\pm}11.0)$ NTU, chlorophyll-a $10.3{\sim}109.0({\pm}44.3)\;mg/m^{3}$, BOD $1.7\sim4.9({\pm}2.6)$ mg/L, COD $3.1\sim-6.9({\pm}5.0)$ mg/L and total coliform $17\sim920({\pm}200.5)$ MPN/100 mL. The free-living amoeba were detected highly than Acanthamoeba sp., 11 out of 22 in raw water samples were positive (50%) for Acanthamoeba sp. from February 2005 to December 2005. The seasonal characteristics of tree-living amoeba and Acanthamoeba sp. in raw water were mainly distributed through the spring to the early fall. When tree-living amoeba and Acanthamoeba sp. were passed through the water treatment of pilot-plant, approximately 80% was sure to be removed through pre-ozonation, sedimentation, send filtration. 100% was removed after post-ozonation process. All of the isolated amoebas from Nakdong River were Acanthamoeba sp. AC311 18S ribosomal RNA gene with 98% nucleotide sequence homology.

• Korean Journal of Medicinal Crop Science
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• v.13 no.5
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• pp.276-283
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• 2005

Ascorbic acid is a great antioxidant and helps protect the body against pollutants. GDP-mannose pyrophosphorylase (GMPase) is a key enzyme in manufacturing GDP-mannose, a glycosyl donor for ascorbate and cell wall biosynthesis as well as for protein glycosylation. In this study, we described molecular cloning of a full-length cDNA from Potato (Solanum tuberosum L. cv. Jasim), using tuber. The cDNA isolated encoded a GDP-mannose pyrophosphrylase. The nucleotide sequence of pGMPC showed about 95%, 89% and 80% homology with S. tuberosum (AF022716), N. tabacum (AB066279) and A. thaliana (AF076484) cDNAs clone known as GMPase, respectively. We detected the expression of GMPase using RT-PCR. The highest expression of GMPase was found in stems, and the largest amount of ascorbic acid was also presented in stems. In contrast, the leaf showed minimal level of GMPase transcript and ascorbic acid content. We propose that GMPase expression patterns were similar to the changes of ascorbic acid content in the leaves treated with diverse stresses.

• Journal of fish pathology
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• v.10 no.1
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• pp.39-44
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• 1997

Metallothionein is an important and essential protein to control the intracellular concentration of heavy metals, which exist in all organisms from bacteria to vertebrates. Although the detailed functions and induction mechanisms of metallothionein gene have not been clearly characterized until yet, the structure of several metallothionein genes has been revealed. Especially, piscine metallothionein is regarded as an important protein because it is induced by several heavy metal pollutants and environmental stress and it could be determined the comparative amount of heavy metals and the extent of environmental stress by assaying the RNA transcript of metallothionein gene in the method of the quantitative RT-PCR(Reverse Transcriptase Polymerase Chain Reaction). In this study I have characterized the 450 bp PCR fragment of metallothionein gene amplified by using the mixture of internal specific primers and universal 3' end primer. The nucleotide sequence analysis of 450 bp PCR fragment amplified in cDNA library of channel catfish did not show strong homology to other piscine metallothionein genes.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.153-159
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• 2001

In order to clone the peptide synthetase gene form Lysobacter lactamgenus IFO 14,288, the gene fragments were amplified using primers for the adenylation domain and the thionylation domain of the peptide synthetase genes in other organisms by polymerase chain reaction (PCR). The resulting 0.5-kb fragment was cloned in a pGEM-T vector, and the nucleotide sequences were determined. Six different PCR products were obtained; three were identified to be a part of L-$\alpha$-aminoadipyl-L-cysteinyl-D-valine (ACV) synthetase and three to be other peptide synthetases. Using each of the two different classes of PCR products as mixed probes, a cosmid library of L. lactamgenus chromosomal DNA constructed in a pHC79 vector was screened by an in situ hybridization procedure, and one positive clone was selected which was bound by peptide synthetase gene fragments as well as ACV synthetase gene fragments. The partial sequence analysis formt he obtained pPTS-5 cosmid showed th presence of more than two open reading frames. These were for two putative membrane transporters, which were homologous with several integral membrane proteins including the ABC transporter ATP-binding protein of E. coli (YbjZ) and the metal ion uptake protein of Bacillus subtilis (YvrN). A 45% homology was also found between the two transporter proteins at the carboxy terminus. Through a hydropathy analysis and transmembrane analysis. 4-5 transmembrane domains were found in these two proteins. When the genes were expressed in Escherichia coli, the gene products inhibited the hose cell growth, probably due to the disturbance of the membrane transport system.

• The Plant Pathology Journal
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• v.30 no.4
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• pp.375-383
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• 2014

Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC) transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.). Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1) gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5'-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.77-83
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• 2006

A gene encoding a $\gamma-butyrolactone$ autoregulator receptor was cloned from Saccharopolyspora erythraea, and the biochemical characteristics, including the autoregulator specificity, were determined with the purified recombinant protein. Using primers designed for the conserved amino acid sequence of Streptomyces $\gamma-butyrolactone$ autoregulator receptors, a 120 bp S. erythraea DNA fragment was obtained by PCR. Southern and colony hybridization with the 120 bp fragment as a probe allowed to select a genomic clone of S. erythraea, pESG, harboring a 3.2 kb SacI fragment. Nucleotide sequencing analysis revealed a 615 bp open reading frame (ORF), showing moderate homology (identity, $31-34\%$; similarity, $45-47\%$) with the $\gamma-butyrolactone$ autoregulator receptors from Streptomyces sp., and this ORF was named seaR (Saccharopolyspora erythraea autoregulator receptor). The seaR/pET-3d plasmid was constructed to overexpress the recombinant SeaR protein (rSeaR) in Escherichia coli, and the rSeaR protein was purified to homogeneity by DEAE-Sephacel column chromatography, followed by DEAE-ion-exchange HPLC. The molecular mass of the purified rSeaR protein was 52 kDa by HPLC gel-filtration chromatography and 27 kDa by SDS-polyacrylamide gel electrophoresis, indicating that the rSeaR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that rSeaR has clear binding activity with a VB-C-type autoregulator as the most effective ligand, demonstrating for the first time that the erythromycin producer S. erythraea possesses a gene for the $\gamma-butyrolactone$autoregulator receptor.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.483-489
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• 2004

Pseudomonas sp. S-47 is a bacterium capable of degrading benzoate as well as 4-chlorobenzoate (4CBA). Benzoate and 4CBA are known to be degraded via a meta-cleavage pathway characterized by a series of enzymes encoded by xyl genes. The meta-cleavage pathway operon in Pseudomonas sp. S-47 encodes a set of enzymes which transform benzoate and 4CBA into TCA cycle intermediates via the meta-cleavage of (4-chloro )catechol to produce pyruvate and acetyl-CoA. In the current study, the meta-pathway gene cluster was cloned from the chromosomal DNA of S-47 strain to obtain pCS1, which included the degradation activities for 4CBA and catechol. The genetic organization of the operon was then examined by cloning the meta-pathway genes into a pBluescript SKII(+) vector. As such, the meta-pathway operon from Pseudomonas sp. S-47 was found to contain 13 genes in the order of xylXYZLTEGFlQKIH. The two regulatory genes, xylS and xylR, that control the expression of the meta-pathway operon, were located adjacently downstream of the meta-pathway operon. The xyl genes from strain S-47 exhibited a high nucleoside sequence homology to those from Pseudomonas putida mt-2, except for the xylJQK genes, which were more homologous to the corresponding three genes from P. stutzeri AN10. One open reading frame was found between the xylH and xylS genes, which may playa role of a transposase. Accordingly, the current results suggest that the xyl gene cluster in Pseudomonas sp. S-47 responsible for the complete degradation of benzoate was recombined with the corresponding genes from P. putida mt-2 and P. stutzeri AN10.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1237-1244
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• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• Journal of fish pathology
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• v.5 no.2
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• pp.143-152
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• 1992

Metallothionein is an essential and common protein to regulate the intracellular concentration of heavy metals, which exist in most organisms from bacteria to vertebrates. Although the detailed function of metallothianein has not been fully identified until yet, it may be involoved in the cellular protection against the heavy metal toxicity and in the global regulation of several other genes and the expression of metalloproteins. We have cloned the full cDNA clone of metallothionein gene in Channel Catfish by Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR) starting from poly(A)-containing mRNAs. All PCR fragments have been subcloned into EcoRV site of pBluescript SK+ and dT-tailed at Smal site of pUC19, then PCR products are recovered by the double digestion of recombinant plasmids wiht EcoRI and HindIII, which are adjacent to EcoRV site in multicloning sites or by rapid PCR screening. The nucleotide sequence analysis of pMT150(one of the PCR clones) showed high homology with several other piscine metallothionein cDNA genes.