Diversity Analysis of the Immunoglobulin M Heavy Chain Gene in Nile Tilapia, Oreochromis niloticus (Linnaeus)

A full-length cDNA encoding the immunoglobulin (IgM) heavy chain gene of Nile tilapia was successfully cloned using the 5’ and 3’ RACE techniques. The complete cDNA of the Nile tilapia IgM heavy chain gene is 1,921 bp in length and has an open reading frame (ORF) of 1,740 bp, which corresponds to 580 amino acid residues. The deduced amino acid sequence of the Nile tilapia IgM heavy chain includes a typical secretory IgM heavy chain designated "On-sIgM" and a variable region that is connected to 4 constant regions to form the LH-VH-Cµ1-Cµ2-Cµ3-Cµ4 pattern. Comparisons of the nucleotide and amino acid sequences of On-sIgM with IgM heavy chains of other organisms showed the highest similarity scores of 62.6 and 55.4%, respectively, to the orange-spotted grouper (Epinephelus coioides). Structural analysis of 126 cDNAs encoding variable domains of the IgM heavy chain revealed that at least 9 VH families, 6 DH segments and 4 JH families were utilized using several mechanisms to generate the repertoire of antigen-binding domains. Variation analysis of the variable domains indicated that the amino acid sequences of the framework regions (FRs) were less variable than those of the complementarity determining regions (CDRs), among which the most variable was CDR3. Tissue expression profile analysis using quantitative real-time RT-PCR of healthy Nile tilapia showed that the IgM heavy chain gene was ubiquitously expressed in all 13 tested tissues, but the highest expression level was observed in the head kidney, followed by the spleen, intestine and peripheral blood leukocytes (PBLs). Furthermore, Southern blot analysis of the constant region of the IgM heavy chain gene of 3 different fishes indicated that Nile tilapia genomes may contain 2 copies of the IgM gene.Keywords: Nile Tilapia, IgM Heavy Chain, Variable Region, Diversity, Secreted Form, Southern Blo

Characterization of the Major Histocompatibility Complex Class II Genes in Miiuy Croaker

Major histocompatibility complex (MHC) has a central role in the adaptive immune system by presenting foreign peptide to the T-cell receptor. In order to study the molecular function and genomic characteristic of class II genes in teleost, the full lengths of MHC class IIA and IIB cDNA and genomic sequence were cloned from miiuy croaker (Miichthys miiuy). As in other teleost, four exons and three introns were identified in miiuy croaker class IIA gene; but the difference is that six exons and five introns were identified in the miiuy croaker class IIB gene. The deduced amino acid sequence of class IIA and class IIB had 26.3–85.7% and 11.0–88.8% identity with those of mammal and teleost, respectively. Real-time quantitative RT-PCR demonstrated that the MHC class IIA and IIB were ubiquitously expressed in ten normal tissues; expression levels of MHC genes were found first upregulated and then downregulated, and finally by a recovery to normal level throughout the pathogenic bacteria infection process. In addition, we report on the underlying mechanism that maintains sequences diversity among many fish species. A series of site-model tests implemented in the CODEML program revealed that positive Darwinian selection is likely the cause of the molecular evolution in the fish MHC class II genes