• Journal of Microbiology
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• 제35권4호
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• pp.327-333
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• 1997

Although extensively characterized in human cells, no heterogeneous nuclear ribonucleoprotein(hnRNP) has been found in the fission yeast Schizosaccharomyces pombe which is amenable to genetic studies and more similar to mammals than Saccharomyces cerevisiae is in terms of RNA processing. As a first step to characterize hnRNPs from S. pombe, attempt was made to find human hnRNP A1 homologs from S. pombe. The RNA molecule (A1 winner) containing the consensus high-affinity hnRNP A1 binding site (UAGGGA/U) was synthesized in vitro and used in an ultraviolet(UV) light-induced protein-RNA cross-linking assay. A number of S, pombe proteins bound to the A1 winner RNA. An approximately 50-kDa protein(p50) cross-linked more efficiently to the A1 winner RNA than other proteins. The p50 protein did not cross-link to a nonspecific RNA, but rather to the A1-5’ SS RNA in which the consensus 5’ splice junction sites of S. pombe introns were abolished. This suggests that the p50 protein, however, did not bind to the single-stranded DNA to shich the human hnRNP A1 could bind and be eluted with 0.5M NaCl. Further analysis should reveal more features of this RNA-binding protein.

• Journal of Microbiology
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• 제44권5호
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• pp.577-580
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• 2006

GST pull-down assays were used to characterize the SARS-CoV membrane (M) and nucleocapsid (N) interaction, and it was found that the amino acids 211-254 of N protein were essential for this interaction. When tetrad glutamines (Q) were replaced with glutamic acids (E) at positions of 240-243 of the N protein, the interaction was disrupted.

• Journal of Microbiology
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• 제44권5호
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• pp.556-561
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• 2006

A differential display for the expression profiles of wild-type Cryphonectria parasitica and its virally-infected isogenic hypovirulent strain revealed several transcripts of interest, which evidenced significant matches with fungal genes of known function. Among which, we have further analyzed an amplified PCR product with significant sequence similarity to the known fungal stress-responsive thioredoxin gene from Neurospora crassa. The product of the cloned thioredoxin gene, CpTrx1, consists of 117 amino acids, with a predicted molecular mass of 13.0 kDa and a pI of 5.4. Sequence comparisons demonstrated that the deduced protein sequence of the CpTrx1 gene evidenced a high degree of homology to all known thioredoxins, with the highest degree of homology with trx1, a thioredoxin gene from Saccharomyces cerevisiae, and evidenced a preservation of the conserved hall markresidues (Trp-Cys-Gly-Pro-Cys) at the active site of thioredoxin. The E. coli-generated CpTRX1 manifested thioredoxin activity, according to the insulin reduction assay, which indicates that the cloned gene does indeed encode for the C. parasitica thioredoxin.

• Journal of Microbiology
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• 제45권2호
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• pp.153-157
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• 2007

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.

• Journal of Microbiology
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• 제45권6호
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• pp.572-577
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• 2007

To enhance therapeutic potential of murine monoclonal antibody, humanization by CDR grafting is usually used to reduce immunogenic mouse residues. Most humanized antibodies still have mouse residues critical for antigen binding, but the mouse residues may evoke immune responses in humans. Previously, we constructed a new humanized version (AKA) of mouse CC49 antibody specific for tumor-associated glycoprotein, TAG-72. In this study, to select a completely human antibody light chain against TAG-72, guided selection strategy using phage display was used. The heavy chain variable region (VH) of AKA was used to guide the selection of a human TAG-72-specific light chain variable region (VL) from a human VL repertoire constructed from human PBL. Most of the selected VLs were identified to be originated from the members of the human germline VK1 family, whereas the VL of AKA is more homologous to the VK4 family. Competition binding assay of the selected Fabs with mouse CC49 suggested that the epitopes of the Fabs overlap with that of CC49. In addition, they showed better antigen-binding affinity compared to parental AKA. The selected human VLs may be used to guide the selection of human VHs to get completely human anti-TAG72 antibody.

• 한국환경보건학회지
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• 제42권4호
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• pp.274-279
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• 2016

Objectives: We investigated bacterial diversity in the Han River water resource protection area in order to provide basic microbiological information on the drinking water safety of the Seoul metropolitan region. Methods: Samples were collected in the spring and winter, but not during the rainy season. Pyrosequencing, gene amplification, and extraction of nucleic acids were employed in this study. Results: In total, 57 and 48 operational taxonomic units were respectively analyzed in samples collected during spring and winter. Proteobacteria were predominant in all samples. The samples contained phylogenetically diverse bacterial communities, with eleven major phyla and 36 genera. Cyanobacteria were predominant in the spring samples, but not in the winter samples. The predominant species in the samples collected during both seasons belonged to the genus Aquamicrobium and Bradyrhizobium. Moreover, no pathogenic bacteria were detected in the samples. Conclusion: Proteobacteria were predominant in the samples from the Han River water source protection area. Cyanobacteria were more predominant in the spring samples than in the winter samples, but Aquamicrobium and Bradyrhizobium were predominant in both sampling seasons.

• Journal of Microbiology
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• 제44권1호
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• pp.42-49
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• 2006

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the generated DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was' achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the disagnosis of mycoplasmal contamination in cell culture systems.

• 미생물학회지
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• 제30권2호
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• pp.108-112
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• 1992

To investigate the expression and the secretion of heterologous proteins in yeast, we constructed an yeast secretion vector and produced a human secretory protein, .alpha.-1-antitrypsin (.alpha.-1-AT), from yeast cells. The secretion vector pGAT8 was constructed by inserting the signal sequence of yeast acid phosphatase gene (PH05) into the .alpha.1-AT expression vector pGAT6 which contained .alpha.-1-AT cDNA fused to GAL10-CYC1 promotor. The .alpha.-1-AT was produced efficiently in the yeast cells transformed with plasmid pGAT8, which was onfirmed both by the .alpha.-1-AT activity assay and by the immunoblot method using .alpha.-1-AT antibody. We also showed the secretion of .alpha.-1-AT into the culture media and into the periplasmic space by immunoblot.

• Journal of Microbiology
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• 제37권3호
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• pp.148-153
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• 1999

Laccase produced by Coriolus hirsutus was purified to electrophoretic homogeneity by acetone precipitation, Sephacryl S-2000 HR chromatography, DEAE Sepharose CL-6B chromatography, and Mono Q HR 5/5 chromatography. The purification of laccase was 46.6-fold with an overall yield of 23.7%. Laccase from this fungus was a monomeric glycoprotein with 16% carbohydrate content, and has an isoelectric point of 4.2, and molecular mass of 78 kDa, respectively. The N-terminal amino acid sequence of the enzyme showed significant homology to hoste of laccases from Coriolus versicolor, Pycnoporus cinnabarius, and an unidentified basidiomycete, PM1. The highest rate of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation by laccase was reached at 45$^{\circ}C$, and te pH optima of the enzyme varied depending on the substrate in the range of 2.0 to 4.5. The enzyme was stable at 60$^{\circ}C$ for 5 h and lost 80% activity at 80$^{\circ}C$ in 30 min. The enzyme oxidized a variety of usual laccase substrates including lignin-related phenol, and had the highest affinity toward ABTS. Under standard assay conditions, the apparent Km value of the enzyme toward ABTS was 8.1 ${\mu}$M. The enzyme was completely inhibited by L-cysteine and sodium azide, but not by potassium cyanide, SDS, ad thiourea.

• 미생물학회지
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• 제30권3호
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• pp.199-206
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• 1992

Six isolated strains degrading aniline were selected, identified and designated as pseudomonas putida K6, Pseudomonas acidovorans K82, Achromobacter gr. D. V. K24, Achromobacter xylosocidans K4, Moraxella sp. K21 and Moraxella sp. K22. All of them degraded 1000 ppm aniline completely within 30 to 36 hours. Most of these strains are resistant to antibiotics more than one, but Moraxella sp. has not any antibiotic marker tested. Most strains except for P. acidovorans K82 were shown to have resistance to the heavy metal ions such as Ni, Cu, Li, Ba, Co, etc. but not to Hg to which only P. putida K6 was resistant. M. sp. K21 was capable of degrading aniline to a maximum concentration of 2500 ppm without any repression. The incubation of the cell in limited pH ranges (4-8) had no great effect on aniline degradation. The addition of bactopeptone to the minimal media promoted the speed of aniline degradation, but the addition of glucose rather repressed the rate of aniline degradation. Through enzyme assay, A. gr. D. V. K 24 was shown to degrade aniline through artho-pathway and formed .betha.-ketoadipate as intermediate metabolite.