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Cloning and analysis of expression of Mx cDNA in Japanese flounder, Paralichthys olivaceus
1Laboratory of Genetics and Biochemistry, Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan, Minato-ku, Tokyo, Japan.
Abstract:
Myxovirus resistance (Mx) is an interferon (IFN)-induced protein that specifically prevents growth, in vivo and in vitro, of certain classes of virus. We isolated the Mx cDNA from a leukocyte cDNA library obtained from a Japanese flounder infected with Hirame rhabdovirus (HRV). The Mx cDNA was found to contain 2385 bp coding for 620 amino acids. The amino acid sequence had approximately 51 and 78% identities to the sequences of human and rainbow trout Mx cDNAs, respectively. We propose that the amino acid sequence encoded by this cDNA clone contains a tripartite guanosine-5'-triphosphate (GTP)-binding motif, GXXXSGKS/T, DXXG and T/NKXD, and a signature of the dynamin family, LPRG(S/K)GIVTR, which are conserved in all IFN-induced Mx proteins of vertebrates. In addition, the C-terminal region of the Japanese flounder Mx protein, like those of other Mx proteins, contains a highly conserved leucine zipper motif. Japanese flounder infected with the HRV showed increased expression of Mx mRNA in the leukocytes starting at 48 h and peaking at 72 h. An RT-PCR (reverse transcription-polymerase chain-reaction) analysis of Mx mRNA from a healthy fish showed that the Mx mRNA was predominantly expressed in the kidney, spleen, intestine, brain, peritoneal cavity fluid and gill, and only minimally expressed in leukocytes, liver, muscle and mucus.
Insights
Researchers identified a Japanese flounder myxovirus resistance (Mx) gene crucial for antiviral defense. This interferon-induced protein shows conserved motifs, suggesting a vital role in fish immunity against viral infections.
Area of Science:
- * Molecular biology and immunology
- * Fish virology and genetics
Background:
- * Myxovirus resistance (Mx) proteins are interferon (IFN)-induced, providing antiviral defense in vertebrates.
- * Understanding Mx protein function in fish is crucial for aquaculture and disease management.
Purpose of the Study:
- * To isolate and characterize the myxovirus resistance (Mx) cDNA from Japanese flounder (Paralichthys olivaceus).
- * To investigate the expression patterns of Mx mRNA in Japanese flounder following Hirame rhabdovirus (HRV) infection.
Main Methods:
- * Isolation of Mx cDNA from a leukocyte cDNA library of HRV-infected Japanese flounder.
- * Sequence analysis to identify conserved motifs (GTP-binding, dynamin family, leucine zipper).
- * Reverse transcription-polymerase chain-reaction (RT-PCR) to analyze Mx mRNA expression levels and tissue distribution.
Main Results:
- * A 2385 bp cDNA encoding a 620 amino acid Mx protein was isolated, showing 51% and 78% identity to human and rainbow trout Mx, respectively.
- * Conserved tripartite GTP-binding motif, dynamin family signature, and leucine zipper motif were identified.
- * Mx mRNA expression in leukocytes increased significantly 48-72 hours post-HRV infection. Predominant expression found in kidney, spleen, intestine, brain, peritoneal fluid, and gills.
Conclusions:
- * The identified Japanese flounder Mx protein shares conserved functional domains with other vertebrate Mx proteins, indicating a similar antiviral role.
- * Mx gene expression is inducible by viral infection and exhibits specific tissue distribution patterns in Japanese flounder, highlighting its importance in innate immunity.
