A complete DNA sequence map of the ovine major histocompatibility complex

Abstract

Background: The ovine Major Histocompatibility Complex (MHC) harbors clusters of genes involved in overall resistance/susceptibility of an animal to infectious pathogens. However, only a limited number of ovine MHC genes have been identified and no adequate sequence information is available, as compared to those of swine and bovine. We previously constructed a BAC clone-based physical map that covers entire class I, class II and class III region of ovine MHC. Here we describe the assembling of a complete DNA sequence map for the ovine MHC by shotgun sequencing of 26 overlapping BAC clones.

Results: DNA shotgun sequencing generated approximately 8-fold genome equivalent data that were successfully assembled into a finished sequence map of the ovine MHC. The sequence map spans approximately 2,434,000 nucleotides in length, covering almost all of the MHC loci currently known in the sheep and cattle. Gene annotation resulted in the identification of 177 protein-coding genes/ORFs, among which 145 were not previously reported in the sheep, and 10 were ovine species specific, absent in cattle or other mammals. A comparative sequence analyses among human, sheep and cattle revealed a high conservation in the MHC structure and loci order except for the class II, which were divided into IIa and IIb subregions in the sheep and cattle, separated by a large piece of non-MHC autosome of approximately 18.5 Mb. In addition, a total of 18 non-protein-coding microRNAs were predicted in the ovine MHC region for the first time.

Conclusion: An ovine MHC DNA sequence map was successfully assembled by shotgun sequencing of 26 overlapping BAC clone. This makes the sheep the second ruminant species for which the complete MHC sequence information is available for evolution and functional studies, following that of the bovine. The results of the comparative analysis support a hypothesis that an inversion of the ancestral chromosome containing the MHC has shaped the MHC structures of ruminants, as we currently observed in the sheep and cattle. Identification of relative large numbers of microRNAs in the ovine MHC region helps to provide evidence that microRNAs are actively involved in the regulation of MHC gene expression and function.

Figure 1

A feature map of Ovine MHC sequence. The map spans 2,434,000 nucleotide bases in length, containing 177 protein-coding genes/ORFs and 18 miRNA coding genes. Each locus is represented by an arrow or arrow head, and annotated according to type, orientation, and location within the MHC. The tiling path of the sequenced BACs and the MHC structure are shown on the top. Micro RNA (18 shown): Identified gene that has high sequence homology with conserved gene encoding the functional mircoRNA in other species, noted following the given name of that species. Predicted (36 shown): Gene that either has high sequence similarity to that of the predicted gene in other species, or has a predicted ORF but no high sequence homologies with ESTs of Ovine or other species. Novel (10 shown): Ovine-specific gene identified with a defined open reading frame (ORF) that has not been found in any other species to date. The novel genes are annotated with OaN1 to OaN8 (Oa for Ovis aries; N for novel; another two with no cloning data, no annotation), from left to the right of the map. Known (131 in total): The functional genes previously annotated in Bovine, Ovine or other species.

Figure 2

Gene order comparisons for the selected class II loci from HLA, OLA, and BoLA. Genetic loci in class II region were compared by aligning HLA, OLA and BoLA at telemere→centromere orientation. The orthologous loci were linked by solid lines. Solid and open box represent the selected class II loci and non-MHC loci, respectively. Shaded carmine boxes indicate regions of conservation among species. Red ellipses indicate the potential breaking points.Tel: Telomere, Cen: Centromere.

Figure 3

A comparative alignment of Btnl loci among known MHC/loci of different species. Solid and open box represent the known coding gene and predicted gene, respectively, with an arrow head of box indicating the orientation of gene transcription. A numeric number in a solid box indicates the gene family number of Btnl families. MHC of swine was interrupted by the Centromere as shown