• The Korean Journal of Mycology
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• v.25 no.1 s.80
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• pp.1-5
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• 1997

The cDNA encoding ${\beta}-tubulin$ of Pleurotus sajor-caju was isolated using an internal gene segment probe amplified by polymerase chain reaction (PCR) of genomic DNA and by cDNA library screening. The cDNA was consisted of 1560 nucleotides(nt), including a 5'-untranslation region (UTR) of 27nt, an open reading frame (ORF) of 1341nt, and a 3'-UTR of 191nt. The ORF encoded a protein of 446 amino acids(aa), which shows over 80% homology with ${\beta}-tubulins$ of other filamentous fungi. Southern hybridization analysis showed that there were two isotypes of ${\beta}-tubulin$ genes in P. sajor-caju. Through sequence analysis we found that ${\beta}-tubulin$ had a unusual $Cys^{165}$ residue, which might be a significant factor for the insensitivity of fungi to fungicide benomyl.

• Journal of Applied Biological Chemistry
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• v.51 no.1
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• pp.1-6
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• 2008

Estrogens, a group of steroid compounds functioning as the primary female sex hormone, play an important role in the development and progression of breast cancer. In this study, using a novel annealing control primer-based GeneFishing PCR technology, five differentially expressed genes (DEGs), expressed using 10nM mitogenic estrogens, $17{\beta}$-estradiol (E2) and $16{\alpha}$-hydroxyestrone ($16{\alpha}$-OHE1), were selected from the estrogen receptor (ER)-positive MCF-7 human breast cancer cells. The DEGs, MRPL42, TUBA1B, SSBP1, KNCT2, and RUVBL1, were identified by comparison with the known genes via direct sequencing and sequence homology search in BLAST. Quantitative real-time PCR data showed that two DEGs, tubulin ${\alpha}1b$ and kinetochore associated 2, were greater than 2-fold upregulated by E2 or $16{\alpha}$-OHE1. Both genes could be new biomarkers for the treatment and prognosis of cancers, and further study may provide insights into the molecular mechanisms underlying development and progression of breast cancer.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2003.10a
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• pp.277-287
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• 2003

Acetolactate synthase (ALS, EC 4.1.3.18; also referred to as acetohydroxy acid synthase) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in microorganisms and plants. Recently X-ray structure of yeast ALS was available. Pair-wise alignment of yeast and tobacco ALS sequences revealed 63% sequence similarity. Using Deep View and automatic modeling on Swiss model server, we have generated reliable models of tobacco ALS based on yeast ALS template with a calculated pair-wise RMSD of 0.86 Angstrom. Functional roles of four residues located on the subunit interface (H142, El43, M350, and R376) were examined by site-directed mutagenesis. Seven mutants were generated and purified, of which three mutants (H142T, M350V, and R376F) were found to be inactivated under various assay conditions. The H142k mutant showed moderately altered kinetic properties. The E143A mutant increased 10-fold in K$_m$ value while other parameters remained unchanged. The M350C mutant was strongly resistant to three tested herbicides, while the R376k mutant can bind with herbicide carder at similar affinity to that of wild type enzyme, as determined by tryptophan quenching study. Except M350V mutant, all other mutants were ate to bind with cofactor FAD. Taken together, it is likely that residues H142 and E143 are located at the active site, while residues M350 and R376 are possibly located at the overlapping region of active site and herbicide binding site of the enzyme. Our data also allows us to hypothesize that the interaction between side chains of residues M350 and R376 are probably essential for the correct conformation of the active site. It remains to be elucidated that, whether the herbicide, upon binding with enzyme, inactivates the enzyme by causing change in the active site allosterically, which is unfavorable for catalytic activity.

• BMB Reports
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• v.38 no.1
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• pp.17-23
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• 2005

During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of $55^{\circ}C$. When tested using xylan from birchwood, it showed $K_m$ and $V_{max}$ values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by $CuSO_4$, EDTA, and by $FeSO_4$. The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-$\beta$-xylanase. The full-length gene encoding endo-1,4-$\beta$-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-$\^{a}$-xylanase B previously identified in A. niger.

• BMB Reports
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• v.31 no.4
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• pp.370-376
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• 1998

Src-Homology 2 (SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SID domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the $p56^{lck}$ SH2 domain. In our assay system, SID domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the biotinylated phosphotyrosine (pY)containing synthetic pep tides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated streptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2 domain-pY peptide pair; and (3) The binding of Lck SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction with reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of $p56^{lck}$ SH2 function.

• BMB Reports
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• v.39 no.1
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• pp.46-54
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• 2006

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of $11,180{\pm}50$ Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa $\alpha$ and 34 kDa $\beta$ subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at $55^{\circ}C$, pH 7.0. Maximum activity was observed at $70^{\circ}C$ and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with $K_m$ and $k_{cat}$ values of $163\;{\mu}M$ and $452\;min^{-1}$, respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.

• Parasites, Hosts and Diseases
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• v.56 no.2
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• pp.121-127
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• 2018

To survey the prevalence of Sarcocystis infections, 210 heart samples were collected from Korean native cattle (Bos taurus coreanae) at an abattoir in Daejeon Metropolitan City, Republic of Korea. Sarcocysts were detected form 31 specimens (14.8%) and identified as Sarcocystis cruzi via transmission electron microscopy. The wall of S. cruzi has flattened protrusions that did not contain fibrils or microfilaments. The protrusions arose irregularly from the base, contained a fine granular substance, lacked internal microfilaments, and measured approximately $0.21-1.25{\mu}m$ in length and $0.05-0.07{\mu}m$ in width. Sequence analysis revealed 99.5% homology to S. cruzi. This is the first report on the prevalence of S. cruzi in native cattle from the Republic of Korea.

• 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.5
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• pp.959-966
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• 2004

Twenty-three cultures of harmful algal bloom (HAB)-(causing dinoflagellates were isolated from the coastal waters of Korea. For each of the 14 morphospecies, the nuclearencoded small subunit (SSU) rDNA was analyzed to determine the phylogenetic relatedness of the species. Despite temporal and spatial isolation, 3-4 clonal cultures of Alexandrium catenella, Cochlodinium polykrikoides, and Gymnodinium catenatum had 100% identical SSU rDNA sequences. In contrast, heterogeneities in the SSU rDNA sequences were observed in Akashiwo sanguinea and Lingulodinium polyedrum strains. Extreme sequence polymorphism was shown within the SSU rRNA genes of an Al. tamarense clonal culture. A homology search in GenBank revealed that 11 dinoflagellate species were located in clusters corresponding to their morphological classification. The SSU rDNA sequences of C. polykrikoides, Gyrodinium instriatum, and Pheopolykrikos hartmannii, which were determined for the first time in this study, showed the following phylogenetic relationships: C. polykrikoides formed an independent branch separated from other dinoflagellates; Gyr. instriatum was placed in a monophyletic group with Gyr. dorsum and Gyr. uncatenum; and Ph. hartmanii, which forms a distinct two-celled pseudocolony, belonged to Gymnodinium sensu Hansen and Moestrup.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2003.05a
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• pp.187-187
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• 2003

Methylmercury (MeHg), one of the heavy metal compounds, can cause severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. In this study, two methods, cDNA Microarray and SSH, were performed to assess the expression profile against MeHg and to identify differentially expressed genes by MeHg in neuroblastoma cell line. TwinChip Human-8K (Digital Genomics) was used with total RNA from SH-SY5Y (human neuroblastoma cell line) treated with solvent (DMSO) and 6.25 uM (IC50) MeHg. And we performed forward and reverse SSH method on mRNA derived from SH-SY5Y treated with DMSO and MeHg (6.25 uM). Differentially expressed cDNA clones were sequenced and were screened by dot blot and ribonuclease protection assay to confirm that individual clones indeed represent differentially expressed genes. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences may provide an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as environmental pollutants.