Abstract
We isolated and sequenced Fas ligand cDNA and its gene from Japanese flounder (JF), Paralichthys olivaceus. The JF-Fas ligand cDNA consisted of 1016 bp and encoded 230 amino acid residues. The identities of the deduced amino acid sequence of the JF-Fas ligand to human Fas ligand, Tumor necrosis factor-α and Lymphotoxin-α were 26.1%, 24.5% and 23.0%, respectively. A proline-rich domain (PRD) that is important for localization of the protein was found in the N-terminal region, and two cysteine residues, which form a disulfide bond, were conserved. The JF-Fas ligand gene has a length of 1.8 kb and consists of four exons and three introns. The length of the JF-Fas ligand second intron is shorter than that in the human and pig Fas ligand genes. However, the organization of the exons and intorons is similar to that of mammals. RT-PCR was conducted for 12 tissues, and expression of JF-Fas ligand mRNA was detected in the kidney, thymus, gills, stomach and spleen. The recombinant JF-Fas ligand prepared in an Escherichia coli protein expression system showed cytotoxic activity against Japanese flounder cell line HINAE and caused the fragmentation of genomic DNA. The cytotoxic activity was measured by MTT assay. These results indicate that fish possess a Fas ligand system.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 687-695 |
| Number of pages | 9 |
| Journal | Developmental and Comparative Immunology |
| Volume | 31 |
| Issue number | 7 |
| DOIs | |
| State | Published - Apr 4 2007 |
| Externally published | Yes |
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Keywords
- 3-(4
- 5-dimethylthiazol-2-yl)-2
- 5-diphenyl tetrasodium bromide (MTT) assay
- Apoptosis
- DNA ladder
- Fas ligand
- Innate immunity
- Japanese flounder
ASJC Scopus subject areas
- Immunology
- Developmental Biology
Cite this
Molecular cloning, characterization, expression and functional analysis of Japanese flounder Paralichthys olivaceus Fas ligand. / Kurobe, Tomofumi; Hirono, Ikuo; Kondo, Hidehiro; Saito-Taki, Tatsuo; Aoki, Takashi.
In: Developmental and Comparative Immunology, Vol. 31, No. 7, 04.04.2007, p. 687-695.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Molecular cloning, characterization, expression and functional analysis of Japanese flounder Paralichthys olivaceus Fas ligand
AU - Kurobe, Tomofumi
AU - Hirono, Ikuo
AU - Kondo, Hidehiro
AU - Saito-Taki, Tatsuo
AU - Aoki, Takashi
PY - 2007/4/4
Y1 - 2007/4/4
N2 - We isolated and sequenced Fas ligand cDNA and its gene from Japanese flounder (JF), Paralichthys olivaceus. The JF-Fas ligand cDNA consisted of 1016 bp and encoded 230 amino acid residues. The identities of the deduced amino acid sequence of the JF-Fas ligand to human Fas ligand, Tumor necrosis factor-α and Lymphotoxin-α were 26.1%, 24.5% and 23.0%, respectively. A proline-rich domain (PRD) that is important for localization of the protein was found in the N-terminal region, and two cysteine residues, which form a disulfide bond, were conserved. The JF-Fas ligand gene has a length of 1.8 kb and consists of four exons and three introns. The length of the JF-Fas ligand second intron is shorter than that in the human and pig Fas ligand genes. However, the organization of the exons and intorons is similar to that of mammals. RT-PCR was conducted for 12 tissues, and expression of JF-Fas ligand mRNA was detected in the kidney, thymus, gills, stomach and spleen. The recombinant JF-Fas ligand prepared in an Escherichia coli protein expression system showed cytotoxic activity against Japanese flounder cell line HINAE and caused the fragmentation of genomic DNA. The cytotoxic activity was measured by MTT assay. These results indicate that fish possess a Fas ligand system.
AB - We isolated and sequenced Fas ligand cDNA and its gene from Japanese flounder (JF), Paralichthys olivaceus. The JF-Fas ligand cDNA consisted of 1016 bp and encoded 230 amino acid residues. The identities of the deduced amino acid sequence of the JF-Fas ligand to human Fas ligand, Tumor necrosis factor-α and Lymphotoxin-α were 26.1%, 24.5% and 23.0%, respectively. A proline-rich domain (PRD) that is important for localization of the protein was found in the N-terminal region, and two cysteine residues, which form a disulfide bond, were conserved. The JF-Fas ligand gene has a length of 1.8 kb and consists of four exons and three introns. The length of the JF-Fas ligand second intron is shorter than that in the human and pig Fas ligand genes. However, the organization of the exons and intorons is similar to that of mammals. RT-PCR was conducted for 12 tissues, and expression of JF-Fas ligand mRNA was detected in the kidney, thymus, gills, stomach and spleen. The recombinant JF-Fas ligand prepared in an Escherichia coli protein expression system showed cytotoxic activity against Japanese flounder cell line HINAE and caused the fragmentation of genomic DNA. The cytotoxic activity was measured by MTT assay. These results indicate that fish possess a Fas ligand system.
KW - 3-(4
KW - 5-dimethylthiazol-2-yl)-2
KW - 5-diphenyl tetrasodium bromide (MTT) assay
KW - Apoptosis
KW - DNA ladder
KW - Fas ligand
KW - Innate immunity
KW - Japanese flounder
UR - http://www.scopus.com/inward/record.url?scp=33947713153&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33947713153&partnerID=8YFLogxK
U2 - 10.1016/j.dci.2006.08.006
DO - 10.1016/j.dci.2006.08.006
M3 - Article
VL - 31
SP - 687
EP - 695
JO - Developmental and Comparative Immunology
JF - Developmental and Comparative Immunology
SN - 0145-305X
IS - 7
ER -