• Fisheries and Aquatic Sciences
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• v.26 no.3
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• pp.204-215
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• 2023

The thermolabile haemolysin (tlh) of Vibrio parahaemolyticus (Vptlh) from V. parahaemolyticus is a multiple-function enzyme, initially describes as a haemolytic factor activated by lecithin and phospholipase A2 enzymatic activity (Shinoda, 1991; Vazquez-Morado, 2021; Yanagase et al., 1970). Until now, the tlh structure has hypothesized including N-terminal and C-terminal domain, but what domain of the Vptlh structure does the haemolytic activity has not been refined yet. In this study, a 450-bp VpTLH nucleotide sequence of the entire Vptlh gene encoded the C-terminal domain cloned firstly to examine its responsibility in the activity of the Vptlh. The C-terminal domain fused with a 6-His-tag named the His-tag-VpC-terminal domain was expressed successfully in soluble form in the BL21 (DE3) PlysS cell. Remarkably, both expression and purification results confirmed a high agreement in the molecular weight of the His-tag-VpC-terminal domain was 47 kDa. This work showed the His-tag-VpC-terminal domain lysed the erythrocyte membranes in the blood agar and the phosphate buffered saline (0.9%) media without adding the lecithin substrate of the phospholipase enzyme. Haemolysis occurred at all tested diluted concentrations of His-tag-VpC-terminal domain (p < 0.05), providing evidence for the independent haemolytic activity of the His-tag-VpC-terminal domain. The content of 100 ㎍ of the His-tag-VpC-terminal domain brought the highest haemolytic activity of 80% compared to that in the three remaining contents. Significantly, the His-tag-VpC-terminal domain demonstrated not to involve the phospholipase activity in Luria-Bertani agar supplemented with 1% (vol/vol) egg yolk emulsion. All results proved the vital responsibility of the His-tag-VpC-terminal domain in causing the haemolytic activity without the required activation by the phospholipase enzyme. Raw extracts of Phellinus igniarus and Phellinus pipi at 10^-1 mg/mL inhibited the haemolytic activity of the His-tag-VpC-terminal domain from 67.7% to 87.42%, respectively. Hence applying the His-tag-VpC-terminal domain as a simple biological material to evaluate quickly potential derivatives against the Vptlh in vivo conditions will accessible and more advantageous than using the whole of the Vptlh.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.7
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• pp.917-922
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• 2008

A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif (ADAMTS1) plays a critical role in follicular rupture and represents a major advance in the proteolytic events that control ovulation. In this study, a 9,026-bp DNA sequence containing the full coding region, all 8 introns and part of the 5'and 3' untranslated region of the porcine ADAMTS1 gene was obtained. Analysis of the ADAMTS1 gene using the porcine radiation hybrid panel indicated that pig ADAMTS1 is closely linkage with microsatellite marker S0215, located on SSC13q49. The open reading frame of its cDNA covered 2,844 bp and encoded 947 amino acids. The coding region of porcine ADAMTS1 as determined by sequence alignments shared 85% and 81% identity with human and mouse cDNAs, respectively. The deduced protein contained 947 amino acids showing 85% sequence similarity both to the human and mouse proteins, respectively. Comparative sequencing of three pig breeds revealed one single nucleotide polymorphism (SNP) within exon 7 of which a G-C substitution at position 6006 changes a codon for arginine into a codon for proline. The substitution was situated within a PvuII recognition site and developed as a PCR-RFLP marker for further use in population variation investigations and association analysis with litter size. Allele frequencies of this SNP were investigated in seven pig breeds/lines. An association analysis in a new Qingping female line suggested that different ADAMTS1 genotypes have significant differences in litter size (p<0.01).

• Fisheries and Aquatic Sciences
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• v.17 no.3
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• pp.325-337
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• 2014

To assess the functional structure of prepro-melanin-concentrating hormone (pMCH), we isolated and cloned pMCH (of-pMCH) mRNA from the brain of the olive flounder, Paralichthys olivaceus, and compared its amino acid sequence with those from other animals. In addition, to examine whether activation of the brain of-pMCH gene is influenced by background color, density, and feeding, we compared pMCH mRNA activities against different background colors (bright and dark) and at different densities (100% PCA and 200% PCA). To examine whether the pMCH gene is related with malpigmentation of blind-side skin and appetite, we compared pMCH gene expression between ordinary and hypermelanic flounders, and between feeding and fasting flounders. The of-pMCH cDNA was 405 bp in the open reading frame [ORF] and encoded a protein of 135 amino acids; MCH was 51 bp in length and encoded a protein of 17 amino acids. An obvious single band of the expected size was obtained from the brain and pituitary by RT-PCR. In addition, of-pMCH gene activity was significantly higher in the bright background only at low density (< 100% PCA) making the ocular skin of fish whitening, and in ordinary fish. However, the gene activity was significantly decreased in dark background, at high density (>200% PCA), and in hypermelano fish. These results suggest that skin whitening camouflage of the flounder is induced by high MCH gene activity, and the density disturbs the function of background color in the physiological color change. Moreover, our data suggest that a low level of MCH gene activity may be related to malpigmentation of the blind-side skin. In feeding, although pMCH gene activity was significantly increased by feeding in the white background, the pMCH gene activity in the dark background was not influenced by feeding, indicating that the MCH gene activity increased by feeding can be offset by dark background color, or is unaffected by appetite. In conclusion, this study showed that MCH gene expression is related to ocular-skin whitening camouflage and blind-skin hypermelanosis, and is influenced by background color and density.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.78.2-79
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• 2003

Pepper defensin ( CADEFl) clone was isolated from cDNA library constructed from pepper leaves infected with avirulent strain Bv5-4a of Xanthomonu campestris pv. vesicatoria. The deduced amino acid sequence of CADEFl is 82-64％ identical to that of other plant defensins. Putative protein encoded by CADEFl gene consists of 78 amino acids and 8 conserved cysteine residues to form four structure-stabilizing disulfide bridges. Transcription of the CADEF1 gene was earlier and stronger induced by X campestris pv. vesicatoria infection in the incompatible than in the compatible interaction. CADEF1 mRNA was constitutively expressed in stem, root and green fruit of pepper. Transcripts of CADEFl gene drastically accumulated in pepper leaf tissues treated With Salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), hydrogen Peroxide (H$_2$O$_2$), benzothiadiazole (BTH) and DL-${\beta}$-amino-n-butyric acid (BABA). In situ hybridization results revealed that CADEF1 mRNA was localized in the phloem areas of vascular bundles in leaf tissues treated with exogenous SA, MeJA and ABA. Strong accumulation of CADEF1 mRNA occurred in pepper leaves in response to wounding, high salinity and drought stress. These results suggest that bacterial pathogen infection, abiotic elicitors and some environmental stresses may play a significant role in signal transduction pathway for CADEF1 gene expression.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.81.2-82
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• 2003

Plants have evolved along with pathogens, and they have developed sophisticated defense systems against specific microorganisms to survive. G-protons are considered one of the upstream signaling components working as a key for the defense signal transduction pathway. For activation and inactivation of G-protein, GTP-biding proteins are involved. GTP -binding proteins are found in all organisms. Small GTP-binding proteins, having masses of 21 to 30kD, belong to a superfamily, often named the Ras supefamily because the founding members are encoded by human Ras genes initially discovered as cellular homologs of the viral ras oncogene. Members of this supefamily share several common structural features, including several guanine nucleotide binding domains and an effector binding domain. However, exhibiting a remarkable diversity in both structure and function. They are important molecular switches that cycle between the GDP-bound inactive form into the GTP-bound active form through GDP/GTP replacement. In addition, most GTP-binding proteins cycle between membrane-bound and cytosolic forms. such as the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture secondary wall formation, meristem signaling, and defense against pathogens. But their molecular mechanisms and functions are not well known. We isolated a RacB homolog from rice to study its role of defense against pathogens. We introduced the constitutively active and the dominant negative forms of the GTP-hinging protein OsRacB into the wild type rice. The dominant negative foms are using two forms (full-sequence and specific RNA interference with RacB). Employing southern, and protein analysis, we examine to different things between the wild type and the transformed plant. And analyzing biolistic bombardment of onion epidermal cell with GFP-RacB fusion protein revealed association with the nucle.

• 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.

• BMB Reports
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• v.40 no.3
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• pp.426-431
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• 2007

To investigate the possible mechanisms of high-altitude native animals in adapting to high altitude, we cloned hemoglobin alpha-chain (alpha-chain Hb) gene from Pantholops hodgsonii, an animal species that indigenously lives at elevations of 3700-5500 m on the Qinghai-Tibetan plateau. Using reverse transcription polymerase chain reaction (RT-PCR) technique, the alpha-chain Hb gene was amplified from total RNA in the liver of the Pantholops hodgsonii. TA cloning technique was used and the PCR product was cloned into pGEM-T vector. The DNA sequence of the gene was highly homologous with sheep (99.1%), goat (98.6%), cattle (95.6%) and human (86.5%). The alpha-chain Hb gene encoded a 142-amino acid protein that could be identified with the homology of alpha-chain Hb protein in sheep (98%), goat (96%), cattle (91%) and human (87%). However, 18 alternations were detected when compared with the alpha-chain Hb gene in human, and 2 in sheep. Moreover, the alterations of a117 GluAsp and $\alpha$132 AsnSer in important regions were noted in human and sheep, respectively. Phylogenetic analysis suggested that the structure of alpha-chain Hb was highly similar to that in sheep. This study provided essential information for elucidating the possible roles of hemoglobin in adapting to extremely high altitude in Pantholops hodgsonii.

• ALGAE
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• v.35 no.2
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• pp.177-187
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• 2020

Cyanobacteria have been widely reported to produce a variety of UV-absorbing mycosporine-like amino acids (MAAs). Herein, we reported production of the unusual MAA, mycosporine-glycine-alanine (MGA) in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using a newly developed UHPLC-DAD-MS/HRMS (ultra-high performance liquid chromatography-diode array detection-high resolution tandem mass spectrometry) method. MGA had previously been first identified in a red-algae, but S. torques-reginae strain ITEP-024 is the first cyanobacteria to be reported as an MGA producer. Herein, the chemical structure of MGA is fully elucidated from one-dimensional / two-dimensional nuclear magnetic resonance and HRMS data analyses. MAAs are unusually produced constitutively in S. torques-reginae ITEP-024, and this production was further enhanced following UV-irradiance. It has been proposed that MAA biosynthesis proceeds in cyanobacteria from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate. Annotation of a gene cluster encoded in the genome sequence of S. torques-reginae ITEP-024 supports these gene products could catalyse the biosynthesis of MAAs. However, addition of glyphosate to cultures of S. torques-reginae ITEP-024 abolished constitutive and ultra-violet radiation induced production of MGA, shinorine and porphyra-334. This finding supports involvement of the shikimic acid pathway in the biosynthesis of MAAs by this species.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.35-39
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• 2001

The Integration Host factor (IHF) of Escherichia coli is a small, basic protein that is required for a variety of functions including site-specific recombination, transposition, gene regulation, plasmid replication, and DNA packaging. It ,is composed of two subunits that are encoded by the ihfA ($\alpha$-subunit) and ihjB ($\beta$-subunit) genes. IHF binding sites are composed of three elements called the WATCAR, TTG, and poly (dAT) elements. We have characterized IHF binding to the H site of bacteriophage λ. We have isolated suppressors that bind to altered H' sites using a challenge phage selection. Two different suppressors were isolated that changed the adjacent $\alpha$P64 and $\alpha$K65 residues. The suppressors recognized both the wild-type site and a site with a change in the WATCAR element. Three suppressors were isolated at $\beta$-E44. These suppressors bound the wild-type and a mutant site with a T：A to A：T change (H44A) in the middle of the TIR element. Site-directed mutagenesis was used to make several additional changes at $\beta$E44. The wild-type and $\beta$E44D mutant could not bind the wild-type site but were able to bind the H44A mutant site. Other mutants with neutral, polar, or a positive charge at $\beta$E44 were able to repress both the wild-type and H44A sites. Examination of the IHF crystal structure suggests that the ability of the wild-type and $\beta$E44D proteins to discriminate between the T：A and A：T basepairs is due to indirect interactions. The $\beta$-E44 residue does not contact the DNA directly. It imposes binding specificity indirectly by interactions with residues that contact the DNA. Details of the proposed interactions are discussed.

• Entomological Research
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• v.48 no.5
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• pp.416-428
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• 2018

To gain an insight into the function of heat shock proteins (HSPs) in insects during thermal stress, three HSP cDNAs were identified in the transcriptome of adult Heortia vitessoides, one of the most destructive defoliating pests in Aquilaria sinensis (Loureiro) Sprenger forests. The open reading frames of HvHsp60, HvHsp70, and HvHsp90 were 1,719, 2,070, and 2,151 bp in length, respectively, and encoded proteins with molecular weights of 61.05, 75.02, and 82.23 kDa, respectively. Sequence analysis revealed that all three HSPs were highly conserved in structure. Regarding the stage-specific expression profiles, HvHsp60, HvHsp70, and HvHsp90 mRNAs were detected in all developmental stages. Regarding the tissue-specific expression profiles, the expression levels of the three HSP genes were different in various larval and adult tissues. Moreover, the expression patterns of heat-stressed larvae, pupae, and adults indicated that HvHsp60, HvHsp70, and HvHsp90 were heat-inducible. In particular, HvHsp60 transcripts increased dramatically in larvae and pupae that were heat-stressed at $40^{\circ}C$ and were upregulated in adults that were heat-stressed at $35^{\circ}C$ and $40^{\circ}C$. The expression of HvHsp70 significantly increased in all of the three different developmental stages at $35^{\circ}C$, $40^{\circ}C$, and $45^{\circ}C$. The expression of HvHsp90 obviously increased at $30^{\circ}C$, $35^{\circ}C$, and $40^{\circ}C$ in larvae and could be induced at $35^{\circ}C$ in pupae and adults. The results suggest that HSP60, HSP70, and HSP90 play a major role in protecting H. vitessoides against high-temperature stress.