• Fisheries and Aquatic Sciences
• /
• v.12 no.4
• /
• pp.286-292
• /
• 2009

We have constructed a cDNA library using brain samples of olive flounder Paralichthys olivaceus. Here, we described the study on gene identification by screening 356 clones from the brain cDNA library of olive flounder. Here, we screened 356 clones from the library to identify genes. Of these, 176 (49.5%) were identified as orthologs of known genes from olive flounder and other organisms. Among the 176 EST clones, 33 (18.7%) represented 11 unique genes that are identical to expressed sequence tags (ESTs) reported for olive flounder, and 120 (68.2%) represented 102 unique genes known from other organisms. The percentage of unknown genes (50.5%) is higher than in other olive flounder cDNA libraries (Lee et al., 2003, 2006, 2007), reflecting the high complexity of brain tissue. Further studies of expression characterization and developmental behavior related to these genes should provide useful insight into the physiological functions of the brain in olive flounder.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.4
• /
• pp.838-843
• /
• 2005

Brain-derived neurotrophic factor (BDNF) is a small secretory protein and a member of the nerve growth factor (NGF) gene family. We cloned the flounder BDNF gene from a flounder brain cDNA library. The nucleotide sequence of the cloned gene showed an open reading frame (ORF) consisting of 810 bp, corresponding to 269 amino acid residues. The tissue distribution of flounder BDNF was determined by reverse transcription-polymerase chain reaction (RT-PCR) in brain, embryo, and muscle tissues. To express fBDNF using a eukaryotic expression system, we constructed the vector mpCTV-BDNF containing the fBDNF gene and transformed this vector into Chlorella ellipsoidea. Stable integration of introduced DNA was confirmed by PCR analysis of genomic DNA, and mRNA expression in C. ellipsoidae was confirmed by RT-PCR analysis.

• Fisheries and Aquatic Sciences
• /
• v.4 no.1
• /
• pp.25-31
• /
• 2001

4-Aminobutyrate aminotransferase plays an essential role in the 4-aminobutyric acid shunt, converting 4-aminobutyrate to succinic semi aldehyde. We isolated and sequenced' a fish cDNA fragment that encodes 4-aminobutyrate aminotransferase. A brain cDNA library from flounder (Paralichthys olivaceus) was constructed using the ZAP- III XR vector and screened for the fish 4-aminobutyrate aminotransferase gene using a probe derived from the conserved sequences of known mammalian 4-aminobutyrate aminotransferases. A partial cDNA for 4-aminobutyrate aminotransferase was cloned and found to be 700 bp in length corresponding to 66 amino acids. Nucleotide sequence of the clone was aligned with NCBI (National Center for Biotechnology Information) DNA sequence data base. The result showed high sequence identity with previously reported mammalian 4-aminobutyrate aminotransferases. The trans­criptional level of flounder 4-aminobutyrate aminotransferase was detected with the presence of mRNA at different flounder tissues by reverse transcription-polymerase chain reaction (RT-PCR). The expression of flounder 4-aminobutyrate aminotransferase was also tested and detected from the flounder tissues of the brain, liver, kidney and pancreas.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.36 no.5
• /
• pp.442-448
• /
• 2003

Melanin concentrating hormone (MCH) regulating color change of fish skin was identified from brain cDNA library of Olive flounder (Paralichthys olivaceus) during the analysis of Expressed Sequence Tags (ESTs). Olive flounder MCH gene consisted of 598 nucleotides encoding 150 amino acids. Olive flounder MCH protein revealed to contain signal peptide of 19 amino acid residues, pro-MCH of 131 amino acids being processed to biologically active and mature form of hormone with 25 amino acid residues at the carboxyl terminus. A comparative structural analysis revealed that Olive flounder MCH precursor had low sequence identity with other fish species and mammalian counterparts, while the amino acid sequences of mature hormone had a relatively high identity and more conserved. RT-PCR analysis revealed that olive flounder MCH precersor gene was expressed spectically only in the brain and not in other tissues.

• Fisheries and Aquatic Sciences
• /
• v.6 no.4
• /
• pp.225-227
• /
• 2003

Acetylcholinesterase (AChE) activity is a potential biomarker for phenanthrene exposure in aquatic organisms. Olive flounder (Paralichthys olivaceus) were exposed to three different concentrations (0.5, 1.0 and 2.0, uM) of phenanthrene for four weeks. AChE activities in the brain, heart and eyes were documented. Inhibition of AChE activity was found significant in flounder treated with a concentration greater than $1.0 {\mu}M$ of phenanthrene. This indicates that a chronic exposure to phenanthrene induces damage in various organs (brain, heart and eyes) and changes of AChE activities might be a useful biomarker to assess the impacts induced by polycyclic aromatic hydrocarbon (PAH). Evidence from this study confirms that the measurement of AChE in the brain and eyes of flounder is a valuable tool that along with other biomarkers can maximize an ecotoxicologists' confidence in assessing the impacts of oil and PAH pollution in the aquatic environment.

• 한국생물공학회:학술대회논문집
• /
• 2003.04a
• /
• pp.736-738
• /
• 2003

Ornithine decarboxylase (ODC) is the key enzyme in the synthetic pathway of polyamines. This enzyme is a homodimeric and a pyridoxal 5-phosphate (PLP) dependent enzyme. We have isolated, a cDNA clone encoding ODC from brain cDNA library constructed from flounder (Paralichthys olivaceus). The ODC cDNA contained a complete ORF consisting of 460 amino acids and one stop codon with comparison to nucleotide sequences of the flounder, zebrafish and rat ODC genes, the ODC genes were highly conserved. The transcription of ODC was analyzed with reverse transcription-polymerase chain reaction (RT-PCR) species in brain, kidney, liver, and embryo.

• Journal of Life Science
• /
• v.7 no.1
• /
• pp.17-23
• /
• 1997

This study was designed as a part of efforts to investigate the biochemical pollutant markers for diagnosis of maine pollutions by changes in cholinesterase activity of the flounder (Paralichthys olivaceus)in Yellow Sea of Korea. Acetylcholinesterase (AChE) activities in brain and muscle of cultured flounders in Yellow Sea were remarkably lower (40-50% and 40-55%, respectively)than those of wild flounder in Pohang (control) of East Sea, but AChE activities in brain and muscle of wild flounders in Yellow Sea were significantly lower(15-40% and 25-35%, respectively)than those of wild flounder in Pohang of East Sea. Butyrylcholinesterase(BChE) activities in barin and muscle of cultured flounders in Yellow Sea were remarkably lower(70-75% and 65-75%, respectively) than those of wild flounder in Pohang of East Sea, but BChE activities in barin and muscle of wild flounders in Yellow Sea were significantly lower (15-40%and 25-35%, respectively)than those of wild flounder in Pohang of East Sea. Lactate dehydrogenase (LDH) activities in serum of cultured flounders in Yellow Sea were significantly 10-50% higher than those of wild flounder in Pohang of East Sea, but LDH activities in serum of wild flounders in Yellow Sea were significantly 20-25% higher than those of wild flounder in Pohang of East Sea. It suggests that AChE and BChE activities in brain and muscle of cultured and wild flounders of Yellow Sea may be used as the most effective mean in a biochemical markers for diagnosis of pollutant effects by organophosphorus pesticides.

• Fisheries and Aquatic Sciences
• /
• v.7 no.3
• /
• pp.171-173
• /
• 2004

Activity of acetylcholinesterase (AChE) is well known as a biomarker of exposure to organophosphate compounds in aquatic organisms. However, the effect of diethyl phthalate (DEP), a widely used plasticizer, on the chance of AChE activity is not yet known. Olive flounder (Paralichthys olivaceus) were exposed to DEP 300 and 1,000 mg DEP/kg b.w. through three times of intraperitoneal injection and effects were assessed in AChE activity of brain, muscle, heart and eyes of the exposed fish. AChE activity in various tissues of flounder was inhibited after exposure to DEP as a concentration-dependent manner, especially in brain, muscle and heart. Among tissues examined, heart is supposed to be a major part of body which is seriously damaged by DEP exposure. It indicates that DEP induces toxic effects in various organs (brain, muscle and heart), and changes of AChE activities. Such changed activities of AChE might be a useful biomarker to assess the impacts induced by phthalate esters including DEP.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.2
• /
• pp.321-329
• /
• 2005

Ornithine decarboxylase (ODC) antizyme is a key regulatory protein in the control of cellular polyamines. We have isolated two distinct ODC antizyme cDNA clones (AZS and AZL) from a flounder (Paralichthys olivaceus) brain cDNA library. Their sequences revealed that both clones required translational frameshifting for expression. Taking + 1 frameshifting into account, AZS and AZL products were 221 and 218 amino acid residues long, respectively, and shared $83.3\%$ amino acid sequence identity. Comparison of the structure and nucleotide sequence of the antizyme genes showed that the genes were highly conserved in flounder, zebrafish, mouse, and human. A phylogenetic tree was constructed, based on the antizyme amino acid sequences from various species. The presence of the two types of antizyme mRNA species in brain, kidney, liver, and embryo was confirmed by using the reverse transcription­polymerase chain reaction (RT-PCR) and Northern blot analysis. Recombinant proteins of flounder ODC antizymes, containing His-Nus-S tag at the amino-terminus, were overexpressed as His-AZL and His-AZS fusion proteins in Escherichia coli BL21 (DE3) pLys by using the pET­44a(+) expression vector.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.34 no.4
• /
• pp.370-377
• /
• 2001

Flounder brain cytosol contains protein inhibitors that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) which is dependent on phosphatidylinositol 4,5-bisphosphate ($PIP_2$) but insensitive to ADP-ribosylation factor (ARF), The PLD inhibitors have been enriched through several chromatographic steps and characterized with respect to size and mechanism of inhibition. Sequential chromatography of the brain cytosol yielded six inhibitor fractions, Two (IIA and IIB) of six inhibitor fractions showed the $PIP_2$-phosphatase activities. IIA was identified as synaptojanin, a nerve terminal protein that has known to be a member of the inositolpolyphosphate 5-phosphatase family, by immunoblot analysis. IIB showed an apparent molecular mass of 158 kDa by Superose 12 gel filtration chromatography and was immunologically distinct from synaptojanin. IIB hydrolyzed $PIP_2$, yielding only phosphatidylinositol phosphate (PIP) as product, suggesting that IIB hydrolyzes only one phosphate from either the 4- or 5-position of PI (4,5)$P_2$. These studies demonstrate that the existence of multiple $PIP_2$-phosphatases have been implicated in the negative regulation of $PIP_2$-dependent PLD activity within flounder brain.