Immunogenetics (2005) 57: 499–509 DOI 10.1007/s00251-005-0008-y ORIGINAL PAPER Despoina Miltiadou . Keith T. Ballingall . Shirley A. Ellis . George C. Russell . Declan J. McKeever Haplotype characterization of transcribed ovine major histocompatibility complex (MHC) class I genes Received: 14 January 2005 / Accepted: 23 May 2005 / Published online: 19 July 2005 # Springer-Verlag 2005 AbstractThe ovinemajorhistocompatibility complex (MHC) remains poorly characterized compared with those of other livestock species. Molecular genetic analysis of the bovine MHC has revealed considerable haplotype and al- lelic diversity that earlier serological analysis had not de- tected. To develop cellular and molecular tools to support development of vaccines against intracellular pathogens of sheep, we have undertaken a molecular genetic analysis of four distinct ovine MHC haplotypes carried by two het- erozygous Blackface rams. We have identified 12 novel class I transcripts and used a class I sequence-specific ge- notyping system to assign each of these transcripts to individual haplotypes. Using a combination of phyloge- netic analysis, haplotype and transcript expression data, we identified at leastfour distinct polymorphic class I MHC loci,three of which appear together in a number of com- binations in individual haplotypes. The haplotypes were further characterized at the highly polymorphic Ovar-DRB1 locus, allowing selection of the progeny of the two founder rams for the establishment of an MHC-defined resource population. KeywordsOvine . Class I . Major histocompatibility complex (MHC) . Haplotype . Single-strand conformational polymorphism (SSCP) . Sequence-specific primer-polymerase chain reaction (SSP-PCR) Introduction The highly polymorphic major histocompatibility comple (MHC) class I genes encode glycoproteins expressed at cellsurface in association with peptides derived from in- tracellular pathogens or from selfproteins. Infection is detected by CD8 + T lymphocytes which recognise the combination of self MHC and foreign peptide (reviewed by Harty et al. 2000).The majority of polymorphic sites within classical MHC class I genes encode amino acids th line the peptide binding groove (Bjorkman et al. 1987). repertoire of peptides presented to T cells in an individu dependent on the complement of class I molecules inhe ited on each haplotype. This diversity in the repertoire of peptides presented to T cells can influence the quality o immune response to infection. As a consequence, poly- morphic MHC loci are often used as genetic markers in studies of disease resistance or susceptibility and in cas where variations are observed in responses to infection et al. 1991; Messaoudi et al. 2002) or vaccination (Eckel 2000). Three polymorphic classical class I loci, HLA-A, -B and -C, are present on all human MHC haplotypes (reviewed Trowsdale 1995). Such consistency appears to be the ex- ception rather than the norm for other species. For exam the number of class I genes varies in different inbred str of mouse (reviewed by Trowsdale 1995) and variable num- bers of MHC class I genes have been reported in differen serologically defined swine haplotypes (Ruohonen-Lehto et al. 1998). Similarly, although two polymorphic genes ubiquitously expressed in equids, at least four putative n classical genes may also be expressed at low levels (Elli etal. 1995;Holmes and Ellis 1999). In cattle, there is ev- idence for at least six classical MHC class I loci (reviewe by Ellis 2004), which occur in a number of combinations on different haplotypes (Ellis et al. 1999). Haplotype variation may also be a feature of ovine cla I genes. A number of serological studies have suggested two expressed loci (Cullen et al. 1982; Garrido et al. 199 Jugo and Vicario 2001; Jugo et al. 2002; Stear and Spoon 1981),whereas others propose three (Grossberger et al. Nucleotide sequence data reported here are available in the DDBJ/ EMBL/GenBank databases under the accession numbers: AJ874673, AJ874674, AJ874675, AJ874676, AJ874677, AJ874678, AJ874679, AJ874680, AJ874681, AJ874682, AJ874683, AJ874684. D. Miltiadou (*) . K. T. Ballingall . G. C. Russell . D. J. McKeever (*) Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, EH26 0PZ, UK e-mail: Declan.Mckeever@mri.sari.ac.uk Tel.: +44-131-4455111 Fax: +44-131-4456235 S. A. Ellis Institute for Animal Health, Compton, RG20 7NN, UK