ABSTRACT
Hepcidin antimicrobial peptides (HAMPs) are key molecules of the innate immune system against bacterial infections and in iron metabolism. In this study we report the molecular cloning and genomic characterization of four HAMP genes (referred to as HAMP1, HAMP2, HAMP3 and HAMP4) in the redbanded seabream (Pagrus auriga). All these genes possessed the eight characteristic cysteine residues involved in protein folding. No canonical sequence for convertase-mediated processing of the HAMP3 propeptide was identified. At the genomic level, all four HAMP genes consisted of two introns and three exons. Phylogenetic analysis revealed that HAMPs could group in two main clusters with HAMP2, HAMP3 and HAMP4 belonging to the more complex and diversified HAMP2-like group of acanthopterygians. Quantitation of mRNA levels in adult tissues showed that HAMP1 was ubiquitously expressed, HAMP2 mainly in kidney, spleen and intestine, whereas HAMP3 and HAMP4 in liver. During development, HAMP2 and HAMP3 were expressed at a high level in embryos. Moreover, the expression levels of the four HAMP genes increased between 5 and 15 days after hatching when larvae started external feeding. Induction experiments with lipopolysaccharide revealed significant changes in gene expression of the four HAMP genes in kidney, liver and spleen. However, expression profiles differed in magnitude and time course response. HAMP1 mRNAs increased rapidly in kidney at 1 h p.i. whereas HAMP2 did later at 24 h. Moreover, HAMP4 transcripts increased more than 5000-fold in liver whereas HAMP2 mRNAs dropped significantly in spleen at 3 h p.i. All these data suggest that HAMPs are involved in the response against bacterial infections although additional functions in iron regulation and embryogenesis in fish should be considered.
Subject(s)
Antimicrobial Cationic Peptides/genetics , Gene Expression Regulation , Genome/genetics , Sea Bream/genetics , Sea Bream/metabolism , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/chemistry , Cloning, Molecular , Gene Expression Profiling , Gene Expression Regulation/drug effects , Gene Expression Regulation, Developmental , Hepcidins , Kidney/immunology , Larva/growth & development , Larva/metabolism , Lipopolysaccharides/pharmacology , Molecular Sequence Data , Phylogeny , Sequence AlignmentABSTRACT
Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25- and 23-fold at 12 and 24h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion.
Subject(s)
Flatfishes/genetics , Flatfishes/metabolism , Gene Expression Regulation, Enzymologic , Muramidase/classification , Muramidase/genetics , Phylogeny , Amino Acid Sequence , Animals , Fish Diseases/enzymology , Fish Diseases/microbiology , Gene Expression Regulation, Enzymologic/drug effects , Gram-Negative Bacterial Infections/enzymology , Gram-Negative Bacterial Infections/veterinary , Lipopolysaccharides/pharmacology , Molecular Sequence Data , Photobacterium/immunology , Sequence AlignmentABSTRACT
The complete mitochondrial genome sequence of the blackspot seabream, Pagellus bogaraveo, was obtained using the long PCR/cloning method. The total length of the mitogenome was 16,941 bp, and had a gene content (13 protein-coding, two ribosomal RNAs, and 22 transfer RNAs) and organization similar to those observed in most other vertebrates. Nevertheless, two main features in the WANCY region revealed as unique in P. bogaraveo mitogenome. First, the O(L) was disrupted by the insertion of a 66 bp long element that was determined to be a tRNA(Cys) pseudogene; second, the existence of high levels of length heteroplasmy, both intra-and inter-individuals, as a result of sequence duplications and deletions. The tandem duplication and random loss (TDRL) model as well as recombination are proposed to account for the length heteroplasmy and gene rearrangements in the P. bogaraveo WANCY region.
