A shotgun subclone library was constructed from sheared fragments of CHORI-241 BAC 100I10. Nine hundred sub-clones were bi-directionally sequenced, resulting in 513,390 bases with quality scores > 15, providing 3.95X coverage. The individual chromatogram files were analyzed by Phred, Phrap and Consed [33-37] and individual contigs were scaffolded on the human genome sequence using BLAST. The scaffold was further validated by the addition of multiple sequences from TraceDB [38] retrieved via BLAST searches using full length equine OAS1 mRNA [GenBank: AY321355]. The scaffold contained four genomic contigs spanning a substantial part of the equine OAS1 gene, including 4.5 kb of promoter sequence upstream of exon 1 and 1.6 kb of sequence downstream of exon 6, and was submitted to GenBank under accession number DQ536887. The genomic assembly also included sequence for the downstream equine OAS3 gene as well as an upstream gene orthologous to human RPH3A (data not shown). This assembly completely overlaps two whole genome shotgun sequences, AAWR01028567 (55,475 bp) and AAWR01028568 (31,407 bp), that were recently submitted to GenBank from the Broad Institute.

The genomic sequence assembly identified two microsatellites, one located within the promoter and the other downstream of exon 6. The promoter GT-microsatellite is located 575 bp upstream of the ATG translation initiation site. A shorter GT-microsatellite is in the same relative position in the human OAS1 promoter and the flanking regions were well conserved between the two sequences (Figure 1). This microsatellite may affect the functions of flanking regulatory elements. Sequencing the OAS1 promoter regions of 13 horses established that this promoter microsatellite is polymorphic in length. The second polymorphic microsatellite was a GT-dinucleotide repeat located 43 bp downstream of exon 6 within the 3' UTR. It has previously been reported that a 3' UTR microsatellite can alter the level of synthesis of a mRNA. [39].

The assembled OAS1 scaffold was aligned to the full length, 1.6 kb cDNA equine transcript [GenBank: AY321355] to delineate individual exons and flanking intron sequences from the genomic contigs. Genomic primers were designed within flanking intron sequences as well as for the proximal promoter (Table 1).

Sequence data obtained from the screening population and from CHORI BAC 100I10 were analyzed using Phred, Phrap and Consed programs [33-37]. Both visual analysis of the chromatogram data to identify heterozygotes and computer analysis using the Consed visualization tool identified 33 single nucleotide substitutions within the proximal promoter and exons of OAS1 (Table 2). Of these, 11 were within coding regions, 9 within non-coding regions and the remaining 13 within the proximal promoter upstream of exon 1. Four of the 9 non-coding polymorphisms were located within the 5' and 3' untranslated regions (UTR). Of the 11 coding polymorphisms, 4 were synonymous and 7 were non-synonymous. Five of the 7 non-synonymous SNPs resulted in substitutions of amino acids with different properties. Interestingly, the amino acids encoded by the major alleles of 4 of the 7 non-synonymous mutations were identical to the corresponding amino acids in the human OAS1 protein [UniProtKB: P00973]. The genotypes of each individual were used to identify potential haplotypes within equine OAS1 using PHASE v2.1 software [40,41]. Only those SNPs verified within multiple individuals were used for the haplotype analysis (minor allele frequency = 0.08). The best reconstruction produced 15 haplotypes from the 33 diallelic SNPs (Table 3). The polymorphic microsatellites were not included in the analysis.

A limited number of mammalian RNASEL mRNA sequences were previously deposited to GenBank and some of these sequences were predicted from whole genome annotations. However, this GenBank information was not sufficient to identify evolutionarily conserved regions in mammalian RNASEL sequences that could be used to design PCR primers to amplify equine RNASEL fragments. The predicted sequences of cattle [GenBank: XM_597290] and dog [GenBank: XM_547430] RNASEL ORFs were amplified from commercial cDNA (BioChain, Hayward, CA), directly sequenced and extended to full-length cDNA sequences by DNA walking. The full-length cattle and dog RNASEL sequences were submitted to GenBank under accession numbers DQ497162 and DQ497163, respectively. These two sequences as well as the human full-length RNASEL sequence NM_021133 were aligned and degenerate primers were designed from conserved regions (Table 4) and used to amplify the middle portions of equine RNASEL cDNA. This partial sequence was extended to the full-length sequence by DNA walking and submitted to GenBank under accession number DQ497159.

Several additional mammalian RNASEL sequences were also determined and subsequently used to perform a phylogenetic analysis. The GenBank feline Whole Genome Sequence (WGS) database was searched with the canine RNASEL sequence [GenBank: DQ497163]. Four genomic contigs, AANG01026257, AANG01026302, AANG01630549 and AANG01026248, were detected. These contigs contain the first, second and third, as well as the fifth and sixth feline RNASEL coding exons, respectively. No contigs containing the fourth coding exon of the feline RNASEL gene were found in GenBank. Two primers were designed based on the 3'- end AANG01026302 sequence and the 5'-end AANG01630549 sequence (Table 4) and used to amplify and sequence this region from a commercial cat genomic DNA (Novagen, Madison, Wisconsin). The sequence of this exon was submitted to GenBank under accession number EF062998. Using this sequence as well as the other exon sequences derived from GenBank (see above), the predicted full-length mRNA sequence of the feline RNASEL gene was assembled.

The TIGR porcine database [42] was searched using the cattle sequence [GenBank: DQ497162] and five partial RNASEL sequences were found. The TC212507 and TC212872 sequences correspond to the 5'-end of porcine RNASEL mRNA, while the TC218317, TC237301, and TC236970 sequences represent the 3'-end. An additional 5'-end cDNA sequence, 20060611S-038813, was detected in the Pig EST Data Explorer [43]. A pair of primers were designed based on the partial sequence (Table 4) and used to amplify pooled cDNA (kindly provided by Dr. Jonathan E. Beever, University of Illinois at Urbana-Champaign). The middle portions of the porcine RNASEL cDNA were directly sequenced. The partial sequence was then extended to the full-length sequence by DNA walking and submitted to GenBank under accession number DQ497160.

The GenBank Guinea Pig whole genome sequence database was searched using both mouse [GenBank: NM_011882] and rat [GenBank: NM_182673] full-length RNASEL sequences. Two Guinea pig sequences, AAKN01052053 and AAKN01424676, showed significant similarity to the 5' and 3' regions of the rodent RNASEL sequences, respectively. These two sequences were used to design primers (Table 4) to amplify commercial cDNA (BioChain, Hayward, CA) and directly sequence the middle portions of Guinea pig RNASEL cDNA. This partial sequence was extended to the full-length sequence by DNA walking, and submitted to GenBank under accession number DQ497161.

Cattle, dog, horse and pig RNASEL sequences were used to search the GenBank elephant genome trace archive using the discontiguous Mega BLAST program. The same sequences were also used to search the GenBank elephant whole genome sequence database using the BLASTN program. The sequences for all potential exons of the elephant RNASEL gene were identified. Based on these sequences, five primer pairs (Table 4) were designed to amplify genomic DNA (kindly provided by Drs. Alfred L. Roca and Stephen J. O'Brien, National Cancer Institute) and directly sequence each of the elephant RNASEL exons. The resulting sequence was submitted to GenBank under accession number DQ497164.

The RNASEL ORF sequence of the laboratory opossum (Monodelphis domestica) was predicted by searching the UCSC genome browser [44] using the BLAT program. No sequence traces similar to RNASEL were detected in frog (Xenopus tropicalis) or several fish species (Danio rerio, Takifugu rubripes and Tetraodon nigroviridis).

Only sequences of human and mouse RNASEL genes were previously reported [26]. Sequences of orthologous rat (GenBank: AY262823) and chicken (GenBank: AM0492248) genes were recently submitted to GenBank but have not been reported in any publications. In addition, annotations of chimpanzee, orangutan and rhesus macaque genomes using a GNOMON method resulted in predicted RNASEL sequences in these three species. Primate, rodent and avian RNASEL sequences were downloaded from GenBank and aligned to orthologous sequences described above to build a phylogenetic tree (Figure 2). Rodents showed the highest rate of nucleotide substitutions, while primates showed the lowest rate of evolution. Evolution rates were found to be fairly uniform in the three different RNase L domains: ankyrin repeats, serine/threonine protein kinase domain, and ribonuclease domain. The percent identity between the RNASEL ORFs of horse and the other species compared is shown in Table 5.

The horse CHORI-241 BAC library was searched with a probe derived from the partial equine RNASEL cDNA fragment. Twelve positive clones were identified and two of them, 108P15 and 189I19, were FISH mapped to assign the RNASEL gene to the horse chromosomal location ECA5p17-p16 (Figure 3).

Partial sequence for the equine RNASEL gene was obtained by sequencing PCR fragments of BAC 159N12. The mRNA sequence [GenBank: DQ497159] was used as a reference for determining intron/exon junctions. Sufficient genomic sequence was obtained to build a scaffold as described for the equine OAS1 gene. The scaffold was verified using sequences from TraceDB [38] and submitted to GenBank under accession number EF070193. This scaffold completely overlaps the whole genome shotgun sequence AAWR01030439 (193510 bp) that was recently submitted to GenBank from the Broad Institute. Comparison of genomic and mRNA sequences revealed six coding and one 5'-terminal non-coding exon in the equine RNASEL gene. This exonic composition is similar to that of a number of other mammalian RNASEL genes. However, two and three 5'-terminal non-coding exons were found in the chicken and mouse RNASEL genes, respectively. The coding vertebrate RNASEL exons were designated A through F. Comparison of the genomic and mRNA sequences of vertebrate RNASEL genes revealed significant length variation in both the 5'- (1402–1510 bp) and 3'-terminal (130–187 bp) coding exons (Table 5).

After identification of the equine RNASEL introns, exon-specific genomic primers were designed (Table 1). Exon-specific sequencing of DNA from the screening population identified 31 SNPs within the RNASEL gene (Table 6). Of the 10 non-coding polymorphisms, one was within the second intron and the others were located in the 5' and 3' UTRs. Seventeen of the 31 SNPs were located within the ankyrin repeat-encoding exon 2, 13 of which are non-synonymous, with 10 resulting in substitutions of amino acids with different properties. Three non-synonymous polymorphisms were identified within exons 3 and 5. The remaining exons, including the non-coding exon 1 were invariant among these horses. The amino acids encoded by the major allele of 11 of the 16 non-synonymous mutations were identical to the corresponding human RNase L amino acid [UniProtKB: Q05823]. Using MOTIF Search [45] to identify putative transcription factor binding motifs in the TRANSFAC database, the promoter SNP was found to be located within a potential cAMP-response element binding site (Score: 90) upstream of the first exon. Haplotypes were assembled in the same manner as for the equine OAS1 gene. The best reconstruction from Phase analysis produced 10 haplotypes among the 31 verified diallelic SNPs with minor allele frequencies = 0.08 (Table 3). As with OAS1, only good quality, unambiguous resequencing data were used for the haplotype analysis.

Identifying single nucleotide polymorphisms by sequencing DNA from multiple individuals enhances the possibility of artifacts either from PCR or sequencing error. The 64 SNPs identified from the equine OAS1 and RNASEL genes were considered valid if each allele was identified in at least two individuals. Eight additional SNPs were identified but could not be verified in more than one individual (minor allele frequency < 0.08). Within the 3,864 and 5,406 base pairs re-sequenced during the SNP identification for OAS1 and RNASEL, respectively, equine OAS1 contained an average of one polymorphism per 117 bases, while equine RNASEL averaged one polymorphism per 174 bases.

Preparation of horse cDNA was described previously [32]. Partial RNASEL sequences were extended using a DNA Walking SpeedUp Kit (Seegene USA, Del Mar, CA) according to the manufacturer's protocol. Four high-density filters for segment 1 of the CHORI-241 equine genomic BAC library were purchased from the Children's Hospital Oakland Research Institute (CHORI), Oakland, CA. These filters were screened using a P³²-labeled equine RNASEL cDNA probe according to the supplier's protocol. Two positive equine BAC clones were purchased from CHORI. Each of these BAC clones was grown individually in 500 mL of LB media. BAC DNA was isolated using the NucleoBond BAC Maxi Kit (BD Biosciences Clontech, Palo Alto, CA) and used as the template for direct partial sequencing with a BigDye terminator v1.1 Cycle Sequencing Kit on an ABI 3100 Genetic Analyzer according to the manufacturer's recommendations. DNA from equine BAC clones 108P15 and 189I19 was FISH mapped as described previously [67]. International cytogenetic nomenclature of the domestic horse [68] was used to identify individual horse chromosomes.

The njtree program was used to construct a phylogenetic tree as described previously [51] and tree topology was inferred by the Neighbor-Joining algorithm. The bootstrap algorithm with 1000 replications was used to estimate the confidence of each node. The njtree program is available upon request.

BAC clone 100I10 was isolated from segment 1 of the CHORI-241 equine BAC library at Texas A&M University and confirmed by PCR as containing OAS1. The colony-isolated clone was cultured and BAC DNA was isolated by standard alkaline/lysis miniprep using Millipore Solutions and treated with Plasmid-Safe ATP-dependent DNAse (Epicentre, Madison, WI). BAC DNA was fragmented using a HydroShear^® DNA Shearing Device (GeneMachine, San Carlos, CA) at Speed Code 8 for an estimated fragment size of 2.5 kb. The fragmented product was analyzed by agarose gel electrophoresis stained with ethidium bromide and gel extracted using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Extractions were eluted in water according to the manufacturer's protocol. Purified fragments were cloned into vector pCR^® 4Blunt-TOPO^® using the TOPO^® Shotgun Subcloning Kit (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. Ligation reactions were incubated 30 minutes at room temperature and electroporated into E. coli. Colonies were screened for lack of β-galactosidase activity and selected for ampicillin resistance on LB-agarose plates containing 50 μg/mL ampicillin. White colonies were cultured and screened for appropriate insert size by PCR using vector-sequence M13 primer sites flanking the cloned insert, prior to sequencing.

Individual OAS1 inserts were amplified directly from individual colonies by PCR using vector-sequence M13 primer sites flanking the cloned insert. Amplification products were purified by centrifugation with the PSI-Clone PCR 96 kit (Princeton Separations, Adelphia, NJ) according to manufacturer's protocol. Purified products were sequenced in separate reactions with each M13 primer using a cycle sequence of 96C, 10 sec; 50C, 5 sec; 60C, 4 min with BigDye^® Terminator Mix v1.1 (Applied Biosystems, Foster City, CA). Sequencing reactions were analyzed using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Primers were designed to amplify the immediate promoter and exons of OAS1 and RNASEL genes from 13 individual horses by PCR (Table 1). Sequencing was carried out in the same manner as used for the library subclones. Sequences obtained were compared between individuals to identify SNPs within the amplified regions.

Sequences were assembled and analyzed using Phrap assembly software [33,34,37] and viewed with the Consed visualization tool [35-37]. Contig and singleton reads were assembled by scaffolding onto the human genome using BLASTN [69-71].

Additional sequences were added to the assembly data and re-analyzed with Phrap and BLAST until the consensus sequence spanned the genes from the promoter to the 3' UTR. The genomic equine consensus sequence was confirmed using data from the Equine Genome Sequencing Project (2x) [38] and intron/exon boundaries were assigned by local alignment to the full-length equine OAS1 [GenBank: AY321355] and RNASEL [GenBank: DQ497159] cDNAs. The equine genomic sequences of OAS1 and RNASEL were submitted to GenBank and assigned the accession numbers DQ536887 and EF070193, respectively.

Blood samples were collected at the Texas A&M University Equestrian Center in accordance with ethical standards. The sampled set used for screening consisted of 13 horses, including 10 geldings/stallions and 3 mares, ranging in age from 21 months to 20 years. Breeds represented include American Quarter Horse (9), Arabian (1), American Paint Horse (1), Appaloosa (1) and Thoroughbred (1).