Jang-Seu Ki

Cloning of a river pufferfish (Takifugu obscurus) metallothionein cDNA and study of its induction profile in cadmium-exposed fish.

We report here the full-length cDNA sequence of metallothionein (MT) gene from an anadromous river pufferfish, Takifugu obscurus (order: Tetradotiformes; family: Tetradontidae). Phylogenetic relationship analysis revealed that the identified MT has high sequence similarity with many Perciformes fish species. The tissue distribution and concentration- and time-dependent expression of MT mRNA were studied in fish exposed to cadmium. Liver showed the highest level of MT gene expression followed by other tissues (brain, gill and kidney) in response to cadmium exposure. Muscle showed a weak expression response of MT gene. Time-course study revealed highest early phase (at 6h) expression in the brain and late persistence of induction in the intestine. MT mRNA expression showed a concentration-dependent expression in all the tissues. However, induction in brain and liver occurred at much lower concentrations as compared to other tissues. Our results demonstrate that MT in T. obscurus is induced by cadmium exposure which indicates that it plays a functionally conserved function of metal detoxification.

Gonadotropin-releasing hormone receptor (GnRHR) gene expression is differently modulated in gender types of the hermaphroditic fish Kryptolebias marmoratus by endocrine disrupting chemicals

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the regulation of reproduction in vertebrates through interaction with a specific receptor. The GnRH-stimulated gonadotropin synthesis and release are regulated by the GnRH receptors (GnRHRs). In this study, we have identified a GnRH receptor (GnRHR) gene from the hermaphroditic fish Kryptolebias marmoratus. K. marmoratus GnRHR showed typical vertebrate GnRHR domains and motifs, and its cDNA contained 1634 bp including an open reading frame (ORF) of 1263 bp encoding a putative protein of 420 amino acids. To analyze expression patterns of GnRHR gene in various tissues and developmental stages of K. marmoratus, we carried out quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). The K. marmoratus GnRHR gene expression was detected in all the tissues of adult fish with highest level in brain and gonad. The expression of K. marmoratus GnRHR mRNA increased from stage 1 (2 day post fertilization, dpf) to stage 4 (12 dpf) but steeply decreased at hatching stage (stage 5). Expression of K. marmoratus GnRHR after exposure to endocrine-disrupting chemicals such bisphenol A (BPA, 600 microg/L for 96 h) and 4-tert-octylphenol (OP, 300 microg/L for 96 h) in hermaphrodites as well as secondary males was highly up-regulated in almost all the tissues. Another EDC, 4-nonylphenol (NP, 300 microg/L for 96 h) showed no consistent response. 17beta-estrodiol (E2, 100 ng/L for 96 h), a known natural estrogen, suppressed expression of GnRHR in most of the tissues from hermaphrodites as well as secondary males. Tamoxifen (TMX, 10 microg/L), an estrogen antagonist, on the other hand, caused upregulation of GnRHR expression in the liver of hermaphrodites and the gonad and liver of secondary males. This is the first report of a GnRHR gene from K. marmoratus and modulation of its expression by EDCs. This study provides an insight into the molecular mechanism of endocrinological functions of this unique fish.

cDNA cloning and expression of a xenobiotic metabolizing cytochrome P4501A (CYP1A) from the yellow catfish, Pelteobagrus fulvidraco (Siluriformes)

We cloned the full‐length cDNA sequence of xenobiotic metabolizing cytochrome P4501A (CYP1A) gene from the yellow catfish (Pelteobagrus fulvidraco, Siluriformes, Bagridae). P. fulvidraco is a commercially important fish generally distributed in Southeast Asian countries and Korea. This is the first report of any xenobiotic metabolizing gene from this fish species. Tissue distribution of CYP1A was studied by real‐time PCR. Induction pattern of CYP1A was studied by exposing fish to β‐naphthoflavone (BNF). Liver showed the highest level of expression in control as well as BNF‐treated fish. However, high levels of expression were also recorded in brain, intestine, and kidney and the least in the muscles. Information on CYP genes in fish from order Siluriformes is limited as compared with other fish orders. This study provides an insight into the xenobiotic metabolizing system of P. fulvidraco and offers baseline information for further research related to biomarker use of CYP1A and detoxification strategies of this commercially important fish species.

Comprehensive comparisons of three pennate diatoms, Diatoma tenuae, Fragilaria vaucheriae, and Navicula pelliculosa, isolated from summer Arctic reservoirs (Svalbard 79°N), by fine-scale morphology and nuclear 18S ribosomal DNA

Here we report morphological and molecular characteristics of dominant freshwater diatoms in summer Arctic reservoirs of Svalbard (Norway), using four culture isolates, when we collected the samples in the field on 15 August 2005. Analyses of morphology and BLAST searches with 18S rDNA sequences identified them to Diatoma tenue (HYNP006, HYNP013), Navicula pelliculosa (HYNP021), and Fragilaria vaucheriae (HYNP022), respectively. Comparative studies of morphology revealed that the body shapes of the three polar diatoms were nearly identical to the known morphology of each species; however, they were considerably shorter in body length than previously described identical species from other locations. The 18S rDNA sequences of the diatoms were nearly identical to the same species from temperate and other regions. Phylogenetic analysis showed that the polar diatoms each formed a clade with their identical species and genera according to their taxonomic positions. This suggests that the polar diatoms may possess little or no genetic or morphological variation compared to more temperate strains.

Modulatory effect of environmental endocrine disruptors on N-ras oncogene expression in the hermaphroditic fish, Kryptolebias marmoratus

Kryptolebias marmoratus is the only known internally self-fertilizing vertebrate. It shows high susceptibility to many chemical carcinogens and has been proposed as a potential cancer model species alternative to mammals. Since use of this fish species is expected to rise in cancer research, regulation of oncogenes from K. marmoratus needs proper understanding. We cloned and deduced full-length sequence of cDNA of N-ras oncogene from K. marmoratus. Study of expression profile of N-ras by using quantitative real-time RT-PCR revealed that brain had the highest level of expression compared to other tissues. Some embryonic stages showed more N-ras expression than juveniles and adults. Exposure to two environmental endocrine disrupting chemicals (EDCs), bisphenol A (BPA) and 4-nonylphenyl (NP) caused up-regulation of N-ras in gonad, intestine and liver of hermaphrodite K. marmoratus. It is suggested that K. marmoratus may be a suitable model species for oncogene expression studies. The observed EDC-induced expression of N-ras supports the assumption that EDC exposure may predispose the host to the risk of environmental carcinogenesis.

Simultaneous detection of Aurelia and Chrysaora scyphozoan jellyfish on a DNA microarray

To demonstrate the effectiveness of microarrays for the detection of jellyfish, we developed a low density DNA chip based on the mitochondrial COI gene sequences of scyphozoans (jellyfish). We designed species-specific oligonucleotide probes by sequence comparisons between scyphozoans and other cnidarians such as hydrozoans and anthozoans. Each amine-labelled capture probe was arrayed onto a silylated slide. PCR products of the COI gene were hybridized to the DNA microarray that contained COI consensus sequences. We tested the ability of the DNA chip to discriminate between species from the genera Aurelia and Chrysaora based on samples of both species from the polyp and ephyra stages. The array produced unique hybridization patterns for each of the two tested jellyfish species. Furthermore, we were able to simultaneously detect individual jellyfish species from mixtures of these two different species in the laboratory and from environmental samples. These results show that the low density DNA chip that we designed can be used as a technical platform for parallel molecular detection of various jellyfish species.

Complete mitochondrial genome of the Arctic green sea urchin Strongylocentrotus droebachiensis (Strongylocentrotidae, Echinoidea)

The complete mitochondrial genome was obtained from the assembled genome data sequenced by next-generation sequencing technology from the Arctic green sea urchin Strongylocentrotus droebachiensis. The mitochondrial genome sequence was 15,710 bp in size, and the gene order and contents were identical with previously reported sea urchin mitochondrial genomes. Of 13 protein-coding genes (PCGs), 1 gene (Cytb) had an incomplete stop codon. The base composition of the mitogenome of Arctic S. droebachiensis showed high A+T (58.36%) and anti-G bias (14.86%) on the third position of PCGs.

Molecular relationships of gammaridean amphipods from Arctic sea ice

The information on the biology and ecology of the Arctic sea ice-associated amphipods (Apherusa glacialis, Gammarus wilkitzkii, Onisimus glacialis, and O. nanseni) has increased, but their molecular taxonomic information still remains undisclosed. In the present study, we investigated long-range DNA sequences spanning 18S to 28S rDNA of these four sea ice-associated amphipods and analyzed their genetic relationships with other amphipod taxa. Variations of rDNA within the individuals of the same species were not detected. Phylogenetic analyses showed that each ice amphipod was separated, forming clusters with other conspecifics. Pairwise comparisons led to similar phylogenetic results, showing that the molecular taxonomy of the ice amphipods was in accordance with morphological systematics. In addition, these findings suggest that all four amphipods have little genetic variation compared with their morphologically defined conspecifics from temperate regions. Based on DNA taxonomy, G. wilkitzkii was supported as a species in good standing, refuting a recent synonymization with Gammarus duebeni. Considerably low genetic divergences of O. glacialis and O. nanseni in 18S, ITS, and 28S rDNA suggest the presence of population distinctions within species.

Re-description of Chrysaora pacifica (Goette, 1886) (Cnidaria, Scyphozoa) from Korean Coastal Waters: Morphology and Molecular Comparisons

The nomenclature of the sea nettle jellyfish from Korea was initially described as Dactylometra quinquecirrha Agassiz, 1862. However its identity has been questioned on the basis of its local distribution and molecular data. Here, we examined morphology and DNA sequences of nuclear rDNA using specimens collected from southern Korean waters in August 2014. Based on morphological characteristics (bell size, umbrella pattern, number of tentacles and lappets) and distribution locality, we reassign the Korean D. quinquecirrha to Chrysaora pacifica (Goette, 1886), and provide a re-description accordingly. The molecular identity of C. pacifica was further confirmed by comparison of nuclear ribosomal DNA sequences.

Origin and roles of a novel copper-zinc superoxide dismutase (CuZnSOD) gene from the harmful dinoflagellate Prorocentrum minimum

Superoxide dismutase (SOD) acts as the first line of defence against reactive oxygen species (ROS) within cells. In this study, we characterized a novel SOD gene (PmCuZnSOD) from the dinoflagellate Prorocentrum minimum, and examined its structural features, putative origin and gene expression. The SOD cDNA is 895 bp in length, containing dinoflagellate splice-leader (dinoSL) sequence, 714-bp ORF (237 aa), and poly (A) tail. In addition, PmCuZnSOD is coded on the dinoflagellate nuclear genome without introns and in a non-tandem repeat manner; however, the encoded protein is probably localized in chloroplasts. Phylogenetic analysis indicated that it might be acquired from cyanobacteria via horizontal gene transfer (HGT) and then the gene possibly relocated from the chloroplast to the nuclear genome. Excess copper dramatically increased the PmCuZnSOD transcripts and SOD activity in cells, caused by ROS generation and decrease of photosynthetic efficiency in the treated cells. These suggest that CuZnSOD may function to defend against oxidative stress for the survival of the dinoflagellate.