• Journal of Life Science
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• v.24 no.4
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• pp.343-351
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• 2014

Phosphoinositide 3-kinase (PI3K) plays a central role in cell signaling and leads to cell proliferation, survival, motility, exocytosis, and cytoskeletal rearrangements, as well as specialized cell responses, superoxide production, and cardiac myocyte growth. PI3K is divided into three classes; type I PI3K is preferentially expressed in leukocytes and activated by ${\beta}{\gamma}$ subunits of heterotrimeric G-proteins. In this study, the cDNAs encoding the $PI3K{\gamma}$ gene were isolated from a brain cDNA library constructed using the flounder (Paralichthys olivaceus). The sequence of the isolated $PI3K{\gamma}$ was 1341 bp, encoding 447 amino acids. The nucleotide sequence of the $PI3K{\gamma}$ gene was analyzed with that of other species, including Oreochromis niloticus and Danio rerio, and it turned out to be well conserved during evolution. The $PI3K{\gamma}$ gene was subcloned into the expression vector pET-44a(+), and expressed in the E. coli BL21 (DE3) codon plus cell. The resulting protein was expressed as a fusion protein of approximately 49 kDa containing a C-terminal six-histidine extension that was derived from the expression vector. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to $PI3K{\gamma}$. The binding of wortmannin to $PI3K{\gamma}$, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. The results obtained from this study will provide a wider base of knowledge on the primary structure and characterization of the $PI3K{\gamma}$ at the molecular level.

• Journal of Animal Science and Technology
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• v.49 no.6
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• pp.711-718
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• 2007

Most cattle breeds have a coat color pattern that is characteristic for the breed. Korean cattle(Hanwoo) has a coat color ranging from yellowish brown to dark brown including a red coat color. Variation in the Hanwoo coat color is likely to be the effects of modified genes segregating within the Hanwoo breed. MC1R encoded by the Extension(E) locus was almost fixed with recessive red e allele in the Hanwoo, but other gene(s) might be affecting the variation of the Hanwoo coat color into yellowish to red brown. We have analyzed a segregation of coat color in the F2 families generated from two Hanwoo bulls(yellowish brown) mated to six F1 dams(black) derived from Hanwoo and Holstein crosses. Segregation of coat color in the offspring found a ratio of 1(yellowish brown) : 1(black) and this ratio indicates that a single gene may play a major role for the Hanwoo coat color. We further investigated SNPs in MC1R, ASIP and TYRP1 loci to determine genetic cause of the Hanwoo coat color. Several polymorphisms within ASIP intron 2 and TYRP1 exons were found but not conserved within the Hanwoo population. However, the segregation of the MC1R e allele was completely associated with the Hanwoo coat color. Based on this information, it is clear that the MC1R e allele is mainly responsible for the yellowish red Hanwoo coat color. Further study is warrant to identify possible genetic interaction between MC1R e allele and other coat color related gene(s) for the variation of Hanwoo coat color from yellowish brown to dark brown. (Key words : Hanwoo, Coat color, SNP, MC1R, ASIP, TYRP1)

• Journal of Life Science
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• v.20 no.3
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• pp.365-373
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• 2010

To monitor newly emerged influenza virus variants and to investigate the prevalence pattern, our laboratory performed isolation of the viruses from surveillance sentinel hospitals. In the present study, we analysed influenza A/H1N1, A/H3N2, B viruses isolated in Busan during the 2006/07 and 2007/08 seasons by sequence analysis of the hemagglutinin (HA1 subunit) and neuraminidase (NA) genes. The isolates studied here were selected by the stratified random sample method from a total of 277 isolates, in which 15 were A/H1N1, 16 were A/H3N2 and 29 were B. Based on the phylogenetic tree, the HA1 gene showed that A/H1N1 isolates had a 96.7% to 97.7% homology with the A/Brisbane/59/2007, A/H3N2 isolates had a 98.4% to 99.7% homology with the A/Brisbane/10/2007, and B isolates had a 96.5% to 99.7% homology with the B/Florida/4/2006(Yamagata lineage), which are all the vaccine strains for the Northern Hemisphere in 2008~2009 season. In the case of the NA gene, A/H1N1 isolates had 97.8% to 98.5% homologies, A/H3N2 isolates had 98.9% to 99.4% homologies, and B isolates had 98.9% to 100% homologies with each vaccine strain in the 2008~2009 season, respectively. Characterization of the hemagglutinin gene revealed that amino acids at the receptor-binding site and N-linked glycosylation site were highly conserved. These results provide useful information for the control of influenza viruses in Busan and for a better understanding of vaccine strain selection.

• Journal of Plant Biotechnology
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• v.32 no.3
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• pp.167-173
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• 2005

Three different cDNAS for cinnamate 4-hydroxylase (C4H) which are involved in the second step of the general phenylpropanoid pathway were isolated and designated as pc4h1 (1,755 bp), pc4h2 (1,655 bp), and pc4h3 (1,316 bp), respectively. The nucleotide sequence analysis revealed that both pc4h1 and pc4h2 clones encode polypeptides of 505 amino acids frame but pc4h3 clone was truncated at the 5'-end of coding region. The alignment of the deduced amino acid sequences showed that PC4H1 and PC4H2 are highly homologous (95.8% identical) with each other and contain three conserved domains which are typical in cytochrome P450 monooxygenase: proline-rich region, threonine-containing binding pocket for the oxygen molecule, and heme binding region. In addition, result of the phylogenic tree analysis revealed that both pepper C4Hs belong to Class 1. pc4h2 transcription was strongly induced in wounded fruit (400%) and root (200%) relative to its very low basal level but not in leaf or stem tissue. In case of pc4h1, the basal level of transcription was higher than pc4h2 but induction by wounding was lower in fruit and root while leaf and stem tissues did not respond to wounding. The basal level of pc4h3 transcripts was not, if any, detectable and response to wounding was not observed.

• Journal of Plant Biotechnology
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• v.5 no.3
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• pp.143-148
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• 2003

Bax, a mammalian pro-apoptotic member of the Bcl-2 family induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MSP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to playa novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.

• Microbiology and Biotechnology Letters
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• v.39 no.1
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• pp.93-96
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• 2011

It has been shown that a nucleotide substitution at position 770 in Escherichia coli 16S rRNA, which is implicated in forming the evolutionary conserved B2c intersubunit bridge, has a detrimental effect on ribosome function. In order to isolate second-site revertants that complement ribosomes containing C770G, we performed a random mutagenesis of the 16S rRNA gene and selected clones that could produce more CAT protein translated by specialized ribosome. One of the clones contained two nucleotide substitutions at positions 569 and 904 (C569G and U904C) and these mutations partially complemented the loss of protein-synthesis ability caused by C770G. Further studies using the isolated revertant will provide information about which part of 16S rRNA is interacting with C770 and the consequence of the structure formed by these interactions in the process of protein synthesis.

• Journal of Life Science
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• v.26 no.4
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• pp.406-413
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• 2016

Abalones are gastropod mollusks belonging to the genus Haliotis. Pacific abalones are regarded as a very important marine gastropod mollusk in Korea, Japan, China, and also in food industries around the world. In Korea, 6 species of abalone have been reported to occur along the coasts: Haliotis discus hannai, Haliotis discus discus, Haliotis madaka, Haliotis gigantea, Haliotis diversicolor supertexta, and Haliotis diversicolor diversicolor. This study was performed to discriminate the genetic variances by the partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) genes and random amplified polymorphic DNA (RAPD) analysis against four species of Pacific abalone (H. discus hannai, H. discus, H. madaka, H. gigantea). COI gene is reasonably well conserved and has been sequenced in various invertebrate taxa. The RAPD analysis technique is a relatively simple and low cost method that allows differentiation of taxa without the need to know their genomes. In this study, we investigated the genetic diversity, phylogenetic relationships within each species. The COI and RAPD analysis were able to distinguish between H. gigantea and the other three species. However, these analysis methods were inadequate to distinguish between H. discus and H. madaka. These results are believed to be able to provide a basis data for future hybrid breeding research by defining the genetically closely related four species of abalone, which is to develop new hybrid abalone for export using hybrid breeding.

• Molecules and Cells
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• v.40 no.4
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• pp.299-306
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• 2017

The transcriptional activator AphB has been implicated in acid resistance and pathogenesis in the food borne pathogens Vibrio vulnificus and Vibrio cholerae. To date, the full-length AphB crystal structure of V. cholerae has been determined and characterized by a tetrameric assembly of AphB consisting of a DNA binding domain and a regulatory domain (RD). Although acidic pH and low oxygen tension might be involved in the activation of AphB, it remains unknown which ligand or stimulus activates AphB at the molecular level. In this study, we determine the crystal structure of the AphB RD from V. vulnificus under aerobic conditions without modification at the conserved cysteine residue of the RD, even in the presence of the oxidizing agent cumene hydroperoxide. A cysteine to serine amino acid residue mutant RD protein further confirmed that the cysteine residue is not involved in sensing oxidative stress in vitro. Interestingly, an unidentified small molecule was observed in the inter-subdomain cavity in the RD when the crystal was incubated with cumene hydroperoxide molecules, suggesting a new ligand-binding site. In addition, we confirmed the role of AphB in acid tolerance by observing an aphB-dependent increase in cadC transcript level when V. vulnificus was exposed to acidic pH. Our study contributes to the understanding of the AphB molecular mechanism in the process of recognizing the host environment.

• Research in Plant Disease
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• v.9 no.3
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• pp.107-115
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• 2003

Chinese yam (Dioscorea opposita cv. Jang-Ma) plants showing necrotic mosaic symptom were collected from their growing fields in Andong, Euisong, Gunwi and Daegu, Korea. Direct negative stainning method by electron microscope showed filamentous particles of about 660 nm in length. Immunosorbent electron microscopy (ISEM) revealed filamentous particles of 660nm decorated with antiserum of Chinese yam necrotic mosaic virus (ChYNMV). The virues purified partially were used to isolate viral RNA as template for RT-PCR to amplify about 1.2 kbp of 3'-terminal region (coat protein, 3'-UTR) with ChYNMV specific and oligo-dT primers. Amino acids sequences of amplified CP genes revealed that the viruses shared 97.9% similarity with ChYNMV (AB044386) wh ich was known as the member of Macluravirus. So the viruses from Chinese yam (D. opposita cv. Jang-Ma) plants were identified as ChYNMV. Comparing the CP amion acid sequences of ChYNMV strains with other macluraviruses such as Cardamon mosaic virus (CdMV), Narcissus latent virus (NLV) and Maclura mosaic virus (MacMV) revealed that N-terminal was the most varialbe region and conserved regions were present within the genus Macluravirus.

• BMB Reports
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• v.40 no.4
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• pp.459-466
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• 2007

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7$\cdot}$Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.