Characterization of 23 CC chemokine genes and analysis of their expression in channel catfish (Ictalurus punctatus)
Introduction
Interest in comparisons of the innate immune mechanisms of a wide spectrum of vertebrate species has grown considerably over the last decade [1], [2], [3]. Progress in sequencing the genomes (Takifugu rubripes, Danio rerio, Tetraodon nigroviridis) and expressed sequence tags (Oncorhynchus mykiss, Ictalurus sp.) of several fish species has been especially important for gaining a better understanding of the evolution of the molecular components working against pathogen invasion. Fish are the earliest class of vertebrates with active components of both the innate and adaptive systems of immunity. Additionally, their tremendous diversity and ability to adapt to a wide range of environmental challenges makes them an ideal group for study of early innate immune components [4].
Chemokines are a large family of chemotactic cytokines in mammals and a crucial part of the innate immune response of higher vertebrates. There they play roles in immunosurveillance under homeostasis as well as stimulating the recruitment, activation and adhesion of cells to sites of infection or injury [5], [6], [7]. They are structurally related small peptides, with the majority containing four conserved cysteine residues. Based on the arrangement of these conserved cysteine residues [8], chemokines were divided into four subfamilies—CXC (α), CC (β), C, and CX3C. CC chemokines constitute the largest subfamily of chemokines with 28 CC chemokines identified from mammalian species [9]. The largest number of CC chemokines found in a single species is 24 from humans, missing orthologues to the murine CCL6, CCL9/CCL10 and CCL12. The majority of human, murine and chicken CC chemokine genes are organized in gene clusters within their genomes. The largest clusters are found on human chromosome 17, mouse chromosome 11 and chicken chromosome 19 [10], [11]. CC chemokines can be loosely characterized by their genomic location, with inflammatory CC chemokines constituting the large clusters, and a few homeostatic CC chemokines distributed individually among several chromosomes. Additionally, orthologies among species are relatively high between the non-clustered CC chemokines, but low when comparing the clustered CC chemokines of several species [11], [12].
With the exception of reports of a small number of genomic CC chemokine sequences from banded dogfish, Japanese flounder and rainbow trout [13], [14], [15], research on fish CC chemokines has been limited to sequencing of cDNAs. Most studies have been restricted in scope to several sequences. Only studies in trout [16] and, in our lab, catfish [12], [17], have attempted to survey the complete diversity of CC chemokines within a teleost species. Catfish has emerged lately as a model species for the study of the teleost immunity, with efforts being made to understand both adaptive and innate system components [18], [19], [20], [21], [22]. Our identification of 26 catfish CC chemokine cDNAs provided a foundation from which to conduct genomic sequencing and comparative analysis across species. The lack of even a draft genomic sequence in catfish made this task considerably more difficult than that faced by those with an assembled genome, because a large amount of sequencing is required as a first step. In this study, we used overgo and cDNA hybridizations to a catfish BAC library to isolate and sequence catfish CC chemokine genes. Here, we report their genomic structure, sequences and characteristics in relation to known CC chemokine genes in other vertebrate species. Despite the rapid identification of CC chemokines through EST analysis in catfish [12], [17], their expression patterns have never been analyzed. Here, we also report the expression patterns of the 26 genes and their associated transcripts in various tissues.
Section snippets
BAC library screening and BAC isolation
High-density filters of channel catfish BAC library were purchased from Children's Hospital of the Oakland Research Institute (CHORI, Oakland, CA), and screened using overgo hybridization probes [23], [24], [25]. Each set of filters contained 10× genome coverage of the channel catfish BAC clones from BAC library CHORI 212 (http://bacpac.chori.org/library.php?id=103). The catfish BAC library was screened using a two-step procedure. First, pooled overgos representing catfish CC chemokines were
Identification of the channel catfish CC chemokine genes
Despite the recent identification of a large number of CC chemokine cDNAs from channel catfish through the analysis of expressed sequence tags (ESTs), their encoding genes have never been identified. In order to identify their encoding genes, overgo probes were designed based on the cDNA sequences and hybridized to high-density BAC filters. Due to the large number of CC chemokine genes and their corresponding BACs, we first identified and confirmed at least one BAC for sequencing analysis. The
Discussion
In the present study, we have sequenced 23 channel catfish CC chemokine genes. This represents the first and largest set of fish chemokine genes that has been characterized at the genomic level. Over 56,000 bp of genomic sequences were sequenced. Genomic organization of the 23 CC chemokine genes was determined by comparing the generated genomic sequences with previously identified cDNA sequences. Microsatellites were identified from 16 catfish CC chemokine genes allowing them to be utilized for
Acknowledgements
This project was supported by a grant from USDA NRI Animal Genome Basic Genome Reagents and Tools Program (USDA/NRICGP 2003-35205-12827), and in part by E-Institute of Shanghai Municipal Education Commission, Project no. E03009. We thank Renee Beam, Karen Veverica, Esau Arana and Randell Goodman for their excellence in the production and maintenance of fish used in this study, and their assistance during challenge experiments.
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Both authors contributed equally.