• Fisheries and Aquatic Sciences
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• 제12권2호
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• pp.98-103
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• 2009

The potential utility of the rockbream (Oplegnathus fasciatus) $\beta$-actin 5'-upstream sequence as a regulatory element for DNA vaccination was evaluated based on in vitro and in vivo heterologous expression assays. In the in vitro transfection experiment, the efficacy of the rockbream $\beta$-actin promoter to drive the expression of a downstream lacZ gene was significantly higher (more than fourfold) than that of the human cytomegalovirus (hCMV) promoter in two fish cell lines (grunt Haemulon plumierii fin and bluegill Lepomis macrochirus fry cell lines). In contrast, the functional activity of the rockbream $\beta$-actin promoter was hardly detectable in a mammalian mouse embryonic fibroblast cell line. Rockbream skeletal muscles injected in vivo with a GFP reporter construct driven by the $\beta$-actin promoter displayed the significantly higher expression of a GFP protein (more than threefold) than did those injected with hCMV promoter driven construct. Data from this study suggest that the homologous rockbream $\beta$-actin promoter could be used as a potential regulator for DNA vaccination in this species.

• Fisheries and Aquatic Sciences
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• 제12권2호
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• pp.90-97
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• 2009

The cytoskeletal $\beta$-actin gene and its 5'-upstream region were isolated and characterized in the rockbream (Oplegnathus fasciatus). Complementary DNA of the rockbream $\beta$-actin represented a 1,125 bp of an open reading frame encoding 375 amino acids, and the rockbream $\beta$-actin cDNA and deduced amino acid sequences were highly homologous to those of other vertebrate orthologs. At the genomic level, the $\beta$-actin gene also exhibited an organization typical of vertebrate cytoskeletal actin genes (2,159 bp composed of five translated exons interrupted by four introns) with a conserved GT/AG exon-intron splicing rule. The putative non-translated exon predicted in the rockbream $\beta$-actin gene was much more homologous with those of teleostean $\beta$-actin genes than those of mammals. The 5'-upstream regulatory region isolated by genome walking displayed conserved and essential elements such as TATA, CArG and CAAT boxes in its proximal part, while several other immune- or stress-related motifs such as those for NF-kappa B, USF, HNF, AP-1 and C/EBP were in the distal part. Semi-quantitative RT-PCR assay results demonstrated that the rockbream $\beta$-actin transcripts were ubiquitously but different-tially expressed across the tissues of juveniles.

• 한국수산과학회지
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• 제44권2호
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• pp.126-140
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• 2011

Genetic determinants of two metallothionein isoforms (MT-A and MT-B) were isolated and characterized from the perciform species, rockbream (Oplegnathus fasciatus). Rockbream MT-A and MT-B shared a high degree of homology at amino acid levels with representative orthologs from other perciform species, especially with respect to the conserved cysteine residues. At the genomic level, both MT-A and MT-B genes represent a tripartite structure typical of vertebrate MT genes. However, rockbream MT-B showed unusually large introns (1.2 kb and 0.8 kb for intron I and II, respectively), a phenomenon that has rarely been seen in other vertebrate MT genes. MT-A and MT-B transcripts were ubiquitously detected in a wide array of tissues, wherein brain and eye showed the highest basal expression levels, and the fin exhibited the lowest expression of both isoforms. The basal expression of MT-A in most tissues was significantly higher (ranging from 4- to 10-fold) than that of MT-B. Upon heavy metal exposures to Cd, Cu or Zn at 25 ppb for 48 h, MT-A and MT-B transcripts in the liver were significantly activated by Cd and moderately by Zn. On the other hand, exposure to Cu did not result in alterations of MT-A, nor in the significant suppression of MT-B. Following bacterial challenges with Escherichia coli, Edwardsiella tarda or Streptococcus iniae, MT isoforms in the liver, kidney and spleen were highly modulated and exhibited a pattern that was dependent on the bacterial species, tissues and isoforms. These results suggest that the two MT isoforms could be taken into account as potential indicators of metal toxicity and immune perturbations of this aquaculture-relevant species.

• Fisheries and Aquatic Sciences
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• 제14권1호
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• pp.31-42
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• 2011

Three novel hepcidin isoforms were isolated and characterized from the perciform fish species Oplegnathus fasciatus. These hepcidin isoforms (designated rbhepc5, rbhepc6 and rbhepc7) were found to share a conserved, tripartite gene structure and a considerable sequence homology one another. A comparison of their mature peptide sequences with those of other perciform hepcidin orthologs indicated that these three hepcidin isoforms as well as four other isoforms previously identified in this species, appear to belong to the HAMP2 group of hepcidin genes. Analysis of the 5'-upstream sequences showed that the proximal non-coding regions of rbhepc5~7 do not possess canonical TATA signals; instead, they harbor several binding motifs for transcription factors involved in immune modulation. Reverse transcriptase-PCR analysis demonstrated that the rbhepc5~7 are expressed predominantly in the liver, and that the transcription of rbhepc5~7 is rapidly induced in the liver, but not in other tissues, by experimental challenge with any of three different bacterial species. However, transcription of rbhepc6 appeared to be negligible under both basal and stimulated conditions, as judged by the redundancy count of randomly chosen reverse transcriptase-PCR clones.

• 한국어병학회지
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• 제20권1호
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• pp.41-47
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• 2007

돌돔 치어 종묘 생산장에서 부화 후 약 20일 전후 대량 폐사 현상의 원인 조사를 위해 사육 수질의 영양염 및 일반 세균수와 사육 해수의 영양염 농도별 자어의 폐사율을 조사한 결과는 다음과 같다.돌돔 부화 경과 일수별 사육수의 pH는 1일령 8.21, 7일령 7.56으로 일령이 증가할수록 pH는 감소하였으며 암모니아는 1일령 0.49 ppm, 10일령 0.85 ppm으로 증가하였다.먹이생물이 사육수에 미치는 영향을 조사한 결과, 클로렐라 첨가하기 전보다 첨가 후 사육수의 COD는 3.3배 증가, 사육 해수에 로티퍼 첨가 후 COD는 약 1.2배 증가하였으며 인산염과 암모니아는 클로렐라 첨가 후 각각 1.7배, 2.3배 증가하여 해역Ⅱ등급 기준인 0.015 ppm, 0.1 ppm을 훨씬 초과하는 값이었다.돌돔 사육수 수질은 지수 사육시 COD는 3.85 ㎎/ℓ였으며 유수량 증가시 다소 양호해지는 경향이었고 지수 사육에 비해 유수 사육시 인산염, 암모니아 모두 양호한 경향이었으나 수질 해역Ⅱ등급을 훨씬 초과하는 값이었다.사육 수질의 정화를 위해 사용하는 PSB (광합성세균)의 1시간 처리 전후의 사육수 수질 변화는 사용 전에 비해 사용 후 COD 1.7배 증가, 인산염 2.7배 증가, 암모니아 1.4배의 증가를 보여 수질 개선효과는 없었다.사육수의 세균 조사 결과, 클로렐라 첨가 전 사육수의 총균 수는 8.7×102 cfu/㎖, 비브리오균수 2.7×102cfu/㎖, 클로렐라 첨가후 총균 수 1.4배, 비브리오균 수 1.6배 증가하였으며 부화경과 일수에 따라 사육수의 세균 수는 증가하였다.사육 해수의 암모니아 농도별 돌돔자어 (5일령)의 폐사율은 100 ppm에서 18시간, 50 ppm에서 20시간, 10 ppm 에서 28시간에 100%의 폐사를 나타내었으며 2 ppm에서 28시간에 40%, 1 ppm에서 28시간에 10%의 폐사를 가져왔다.

• 한국양식학회지
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• 제22권1호
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• pp.51-55
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• 2009

Transcriptional response of the second isoform of glyceraldehyde (GAPDH-2) to infectious challenges using various bacterial species and the rockbream iridovirus (RBIV) was examined in the spleen of the barred knifejaw (Oplegnathus fasciatus). Bacterial challenges of the juvenile barred knifejaws with Escherichia coli, Edwardsiella tarda, Vibrio anguillarum and Streptococcus iniae resulted in the significant elevation of the GAPDH-2 transcripts in the spleen up to 12-fold relative to that in the non-challenged controls (PBS-injected). In addition, the barred knifejaw fingerlings injected with RBIV stock also represented the highly upregulated mRNA expression of the GAPDH-2 up to more than 20-fold when compared to that of control fingerings. Results obtained from this study strongly suggest that the GAPDH-2 is no longer a housekeeping glycolytic protein and rather than that it might be associated with immune-relevant cellular activities. From this finding, the traditional dogma for the use of GAPDH as an invariant standard for gene expression assays should be carefully revised depending on the kinds of biological stimulations applied in this species.