Sequence characterization of the horse OAS1 gene in CHORI-241 BAC 100I10 enabled a partial genomic sequence assembly [GenBank: DQ536887] and comparison among multiple equine individuals. We identified 2 polymorphic microsatellites and 33 single nucleotide polymorphisms from a group of 13 individuals and CHORI-241 BAC 100I10 (Table 2). In an attempt to identify potential structural and/or functional consequences of the coding non-synonymous SNPs, each was analyzed using PolyPhen software [46–48]. Each polymorphic variant identified in equine OAS1 was predicted to cause benign effects at their respective residue position. However, the single mutation resulting in an Arg209Cys substitution may significantly change OAS1 enzymatic activity. Arg209 in the equine OAS1 protein corresponds to Arg544 in the human OAS2 protein, which is located in the donor binding domain. Substitution of Arg544 with either Ala or Tyr significantly decreased enzymatic activity of the OAS2 protein . In addition, the equine OAS1 promoter SNP at position 4531 is located in an interferon stimulating response element . Inactivation of this regulatory element by a single nucleotide substitution may alter expression of the equine OAS1 gene.
RNASEL enzymatic activity was previously reported in reptiles, birds, and mammals . However, no RNASEL genes have been found for amphibians or fishes to date. Interestingly, the same classes of vertebrates also do not have OAS genes.
The horse RNASEL gene was FISH mapped to chromosomal location ECA5p17-p16. Orthologous genes are located on primate chromosome 1 (human, chimpanzee and rhesus macaque), cattle chromosome 16, dog chromosome 7, mouse chromosome 1, rat chromosome 13 and chicken chromosome 8 . Using comparative chromosome painting (Zoo-FISH), similarities between human chromosome 1 and horse chromosome 5 , mouse chromosome 1, rat chromosome 13 , dog chromosome 7 [55, 56] and cattle chromosome 16  were previously established. Our results further confirm the conservation of RNASEL-containing syntenic chromosomal segments in horses.
Thirty one SNPs were identified for equine RNASEL (Table 6). Interestingly, all but three of the 20 coding SNPs identified are located within exon 2. The RNase L protein contains 9 N-terminal ankyrin repeats responsible for binding 2-5A molecules that are essential for activation . Exon 2 of the human RNASEL gene encodes the entire ankyrin repeat region (amino acid 24 to 329). The high frequency of non-synonymous polymorphisms within exon 2 suggests that a single SNP or haplotype could ablate 2-5A binding and/or other RNase L interactions. As well, the SNP identified within the promoter upstream of the first exon is located within a potential cAMP-response element binding site. Mutations within this promoter element have been shown to affect gene expression [58–60]. PolyPhen analysis was also conducted on the non-synonymous coding SNPs identified within equine RNASEL. All but 4 of the RNase L SNPs were predicted to have benign effects. However, the SNP at residue 287 was predicted to change hydrophobicity at a buried site within the RNase L protein and the effect of this on protein function is unknown. The predictions provided by PolyPhen analysis are based on functional effects identified using human nsSNPs and may differ for the horse RNase L. Four SNPs within the ankyrin repeat region in exon 2 (residues 414, 463, 467 and 487) were predicted to have a negative effect on function. These data support our hypothesis that a single SNP or haplotype could affect 2-5A binding within the equine RNase L ankyrin repeats.
A number of SNPs were detected within the 3'UTR region of the equine RNASEL gene. Of the eight SNPs found within this region, six result in transitions. The 3'UTR regions of mRNAs contain regulatory regions capable of protein and microRNA binding that control mRNA stability, translation and localization. A simple analysis of octamer motifs containing equine 3' UTR SNPs identified SNP 10247 as being within a human miRNA target site . If this target site is conserved in horses, this SNP could significantly affect the synthesis of RNase L. However, this particular octamer motif was not found in human or rodent RNASEL 3'UTRs. Furthermore, cross-species sequence comparison using mVISTA[62, 63] also revealed no significant longer range conservation in this region between species (data not shown).
Genotype analysis using PHASE v2.1 [40, 41] identified 15 and 10 haplotypes among equine OAS1 and RNASEL genes, respectively, and suggested the existence of haplotype blocks spanning most of each gene (Table 3). Even if efforts to show an association between viral-induced disease susceptibility and OAS1 and/or RNASEL SNPs are successful, it may prove difficult to unambiguously identify a single causal SNP because of potential linkage disequilibrium at these loci. As determined from our screening population, a single haplotype occurred more frequently than any other, with a frequency of 0.19 and 0.23 in OAS1 and RNASEL, respectively (Table 3).
The frequency of SNP identification in this study in two equine genes was high considering the previously estimated equine SNP frequency of 1 per 1500 bp . In dogs, the estimated SNP rate is ~1 per 1600 bp (based on entire genome re-sequencing), but a higher frequency of ~1 per 900 bp was estimated between breeds . Re-sequencing of specific genes in several breeds of the domestic dog identified polymorphisms at frequencies comparable to our estimates, with 1 SNP per ~250–330 bp [S. Canterbury, personal communication]. Furthermore, re-sequencing within an Elk (Cervus elaphus nelsoni) putative promoter region, which is highly conserved between mule deer, cow and sheep, detected an average SNP frequency of 1 per 69 bp [unpublished data].
The microsatellite identified within the promoter region in this study may also alter expression of the equine OAS1 gene. The alleles observed to date indicate that dinucleotide repeat lengths of 9 and 18 may represent the major alleles at this locus. The over-representation of these alleles may be due to the fact that they correspond to one complete rotation of the DNA helix. If this microsatellite separates cis-regulatory elements, alterations in its length could affect the binding of transcriptional regulators to these elements and significantly alter gene expression [66–71]. In support of this hypothesis, there is a high degree of conservation between human and horse OAS1 promoters in the regions flanking the microsatellite (Figure 1). As well, recent micro-array data provide evidence of an inverse relationship between gene expression and dinucleotide microsatellite length, supporting the significantly higher frequency with which we identified the (GT)9 allele within the individuals screened