MHC class II pseudogene and genomic signature of a 32-kb cosmid in the house finch (Carpodacus mexicanus)

Abstract

Large-scale sequencing studies in vertebrates have thus far focused primarily on the genomes of a few model organisms. Birds are of interest to genomics because of their much smaller and highly streamlined genomes compared to mammals. However, large-scale genetic work has been confined almost exclusively to the chicken; we know little about general aspects of genomes in nongame birds. This study examines the organization of a genomic region containing an Mhc class II B gene in a representative of another important lineage of the avian tree, the songbirds (Passeriformes). We used a shotgun sequencing approach to determine the sequence of a 32-kb cosmid insert containing a strongly hybridizing Mhc fragment from house finches (Carpodacus mexicanus). There were a total of three genes found on the cosmid clone, about the gene density expected for the mammalian Mhc: a class II Mhc beta-chain gene (Came-DAB1), a serine-threonine kinase, and a zinc finger motif. Frameshift mutations in both the second and third exons of Came-DAB1 and the unalignability of the gene after the third exon suggest that it is a nonfunctional pseudogene. In addition, the identifiable introns of Came-DAB1 are more than twice as large as those of chickens. Nucleotide diversity in the peptide-binding region of Came-DAB1 (Pi = 0.03) was much lower than polymorphic chicken and other functional Mhc genes but higher than the expected diversity for a neutral locus in birds, perhaps because of hitchhiking on a selected Mhc locus close by. The serine-threonine kinase gene is likely functional, whereas the zinc finger motif is likely nonfunctional. A paucity of long simple-sequence repeats and retroelements is consistent with emerging rules of chicken genomics, and a pictorial analysis of the "genomic signature" of this sequence, the first of its kind for birds, bears strong similarity to mammalian signatures, suggesting common higher-order structures in these homeothermic genomes. The house finch sequence is among a very few of its kind from nonmodel vertebrates and provides insight into the evolution of the avian Mhc and of avian genomes generally.

Figure 1

Organization of cosmid HFcos10A containing simple sequence repeats (A), predicted exons found by the program GeneMark (B), three genes identified using various analysis packages (see text for details) (C), and the GC content plotted with a moving window size of 500 bp and offset length of 50 bp using the program PercentGC (D). Direction of arrows in B indicates the transcriptional orientation of each gene. Dashed line in C is the average GC content over the entire cosmid.

Figure 2

Mhc class II B structure in house finches (Came–DAB1), red-winged blackbirds (Agph–DAB1; Edwards et al. 1998), and chickens (B-LBII; Zoorob et al. 1990). The asterisks represent frameshift mutations indicating the nonfunctional nature of Came–DAB1. The shaded exon represents the polymorphic second exon encoding the peptide binding region. Intron and exon sizes are to scale. Dashes signify that the gene continues downstream.

Figure 3

Alignment of Came–DAB1 exon 2 (A) and exon 3 (B) to a highly polymorphic chicken gene (B-LBI; Zoorob et al. 1993) and a house finch cDNA sequence (Edwards et al. 1995a). Deletions are indicated in the sequences by a − mark.

Figure 4

List of variable sites as inferred from HAPINFER (Clark 1990) for exon 2 sequences. (Top) Unresolved sequences with reconstructed genotypes in parentheses; (bottom) the reconstructed alleles. HAPINFER was unable to resolve phase two of the individuals (see text for details). Column numbers refer to the site where the polymorphisms occur and are consistent with the alignment from Fig. 3.

Figure 5

Comparison of the number of nonsynonymous (d_n) and silent (d_s) substitutions per site within house finches for exon 2 in Came–DAB1 (n=9), a functional chicken gene (n=12), and a “nonclassical” class II chicken gene (n=3). These comparisons were made with a number of chicken sequences downloaded from GenBank (Zoorob et al. 1993) and the nine inferred haplotypes from house finches obtained through PCR amplification, direct sequencing, and analysis using HAPINFER as described in text. The d_n and d_s values were calculated by the Jukes-Cantor method of Nei and Gojobori (1986) using the MEGA software package (Kumar et al. 1993).

Figure 6

Phylogenetic analysis of exon 2 (A) and exon 3 (B) sequences from different avian species (Lost=Bengalese finch, Vincek et al. 1995; Apco=scrub jay, Came=house finch, Agph=red-winged blackbird, Edwards et al. 1995a; HFcos10A=house finch cosmid 10A, HF=unresolved exon 3 sequences; Phco=ring-necked pheasant, Witzell et al. 1999; BLB=chicken, Zoorob et al. 1993). The trees shown are neighbor-joining trees (Saitou and Nei 1987) using a Tamura-Nei (1993) distance. The numbers above the branches are bootstrap scores with 1000 replicates. The trees are rooted with the pheasant and chicken sequences used collectively as an outgroup.

Figure 7

Genomic signature of house finch DNA for two-, five-, and eight-letter words based on the 32-kb cosmid sequence. Dark pixels indicate high-frequency words and light pixels indicate low-frequency words. The 16 (4²) words in the leftmost panel are indicated for convenience. The five-letter signature contains 4⁵ words (pixels) and the eight-letter signature 4⁸. The fact that the orientation of the four nucleotides is the same at all scales in the image, i.e., within any given quadrant of any size, contributes to the fractal nature of the image.