A genome-wide screen identifies a single beta-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins

Abstract

Background: Defensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, alpha-, beta-, and theta-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial.

Results: Following a comprehensive screen, here we report that the chicken genome encodes a total of 13 different beta-defensins but with no other groups of defensins being discovered. These chicken beta-defensin genes, designated as Gallinacin 1-13, are clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7. The deduced peptides vary from 63 to 104 amino acid residues in length sharing the characteristic defensin motif. Based on the tissue expression pattern, 13 beta-defensin genes can be divided into two subgroups with Gallinacin 1-7 being predominantly expressed in bone marrow and the respiratory tract and the remaining genes being restricted to liver and the urogenital tract. Comparative analysis of the defensin clusters among chicken, mouse, and human suggested that vertebrate defensins have evolved from a single beta-defensin-like gene, which has undergone rapid duplication, diversification, and translocation in various vertebrate lineages during evolution.

Conclusions: We conclude that the chicken genome encodes only beta-defensin sequences and that all mammalian defensins are evolved from a common beta-defensin-like ancestor. The alpha-defensins arose from beta-defensins by gene duplication, which may have occurred after the divergence of mammals from other vertebrates, and theta-defensins have arisen from alpha-defensins specific to the primate lineage. Further analysis of these defensins in different vertebrate lineages will shed light on the mechanisms of host defense and evolution of innate immunity.

Figure 1

Multiple sequence alignment of chicken β-defensins. The intervening region between signal and mature peptide sequence is the short propiece. The conserved residues are shaded. Also shown is the length of each peptide. Notice the six-cysteine defensin motif is highly conserved. The six cysteines in the second tandem copy of the defensin motif in Gal11 are boxed.

Figure 2

Phylogenetic relationship of vertebrate β-defensins. The tree was constructed by the neighbor-joining method and the reliability of each branch was assessed by using 1000 bootstrap replications. Numbers on the branches indicate the percentage of 1000 bootstrap samples supporting the branch. Only branches supported by a bootstrap value of at least 50% are indicated. Chicken β-defensins are highlighted in yellow. Abbreviations: BNBD, bovine neutrophil β-defensin; LAP, lingual antimicrobial peptide; EBD, enteric β-defensin; TAP, tracheal antimicrobial peptide; PBD, porcine β-defensin; DEFB/Defb, β-defensin; Gal, Gallinacin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Genomic organization of the chicken β-defensin gene cluster. The horizontal lines at the bottom represent the three overlapping genomic clones that were used to assemble the continuous, gap-free contig. The position of each gene is represented by a solid vertical bar and the width of each bar is proportional to the size of each gene. The direction of transcription is indicated by the triangle above each gene. The genes with solid triangles are transcribed in the direction opposite to the ones with open triangles. Slanted lines refer to the sequences omitted. Note that the three fragments of AC110874 sequence have been re-ordered and the gaps have been filled following alignment with AC146292.

Figure 4

Chromosomal localization of the chicken β-defensin gene cluster by fluorescence in situ hybridization. The BAC clone TAM31-54I4, which harbors 11 Gal genes, was mapped to chicken chromosome 3q3.5-q3.7. Arrows indicate the hybridization signals.

Figure 5

Tissue expression patterns of 10 novel chicken β-defensins by RT-PCR. See Materials and Methods for details. The number of PCR cycles was optimized for each gene, and the specificity of each PCR product was confirmed by sequencing. The house-keeping gene, GAPDH, was used for normalization of the template input.

Figure 6

Comparative analysis of defensin clusters among the chicken, mouse, and human. The gene clusters were drawn proportionally according to their sizes. Each vertical line/bar represents the position of a gene, and the width of each line/bar is proportional to the size of each gene. Three highly conserved genes (CTSB, BE072524, and HARL2754) surrounding the defensin clusters in the chicken, mouse, and human were connected by solid lines. The position of the α-defensin locus (DEFA) was indicated as an open square. Note that the human θ-defensin pseudogene resides in the DEFA locus. The positions and orders of defensin genes in human and mouse were drawn based on the genome assemblies released in July 2003 and October 2003, respectively. Abbreviations: GGA, chicken chromosome; MMA, mouse chromosome; HSA, human chromosome; Tel, telomere; Cen, centromere.