Abstract
The ovine major histocompatibility complex (MHC) remains poorly characterized compared
with those of other livestock species. To develop cellular and molecular tools to support
development of vaccines against intracellular pathogens of sheep, a molecular genetic analysis
of four distinct ovine MHC haplotypes carried by two heterozygous Blackface rams (501 and
504) was conducted. A total of 17 novel sequences was identified, 11 of which were obtained
full length. Initially, using a combination of reverse transcriptase - polymerase chain reaction
(RT-PCR), Single Stranded Conformational Polymorphism (SSCP) and sequence analysis, six
distinct SSCP patterns were identified from ram 501 and eight from ram 504 each
corresponding to a different sequence (chapter 3). SSCP patterns and their corresponding
sequences D5, D3, C7, E2, F8 and F12 accounted for 86% of 360 clones analysed from both
rams, suggesting that they encode the mainly expressed classical class I molecules, while F7
and G12 represented only 0.56%, indicating they are putative low expressed class I like genes or
pseudogenes. Genotyping of 17 offspring from both rams using a novel sequence specific PCR
method, allowed the segregation of sequences within the four haplotypes (chapter 4). An
additional sequence C7b was identified during genotyping, indicating that at least 6 class I
genes are transcribed in a single haplotype. F7 and G12 were shared between all four
haplotypes. Full length transcripts for ten of the sequences identified in chapter 3 were obtained
by RT-PCR, all having a characteristic MHC gene structure (chapter 5). During full length
amplification two additional sequences, N1 and N2, were identified with characteristics
consistent with non classical class I loci. Phylogenetic analysis using the identified transcripts
and published ovine, bovine and other ruminant class I sequences belonging to the Bovidae
family indicated that there are at least six ovine MHC class I loci (chapter 6). Sequences N1 and
N2 are closer to the non classical bovine MHC class I sequence HD15 than to the remaining
ovine class I
transcripts. Seven out of eight full length transcripts subcloned into a mammalian
expression vector expressed detectable class I cell surface glycoproteins in COS-7 cells (chapter
7). The combination of phylogenetic analysis, haplotype, transcription and expression data
suggest that there are at least four distinct polymorphic ovine MHC class 1 loci, three of which
appear to be expressed in a number of combinations in individual haplotypes, a couple of non
polymorphic poorly transcribed class I like sequences and at least one additional diverged non
classical class I locus (chapter 8). Similarities and differences of the ovine MHC class I region
with that of other species are discussed. Using the data generated here, an MHC defined sheep
flock, which includes animals homozygous for each of the four MHC haplotypes is currently
under development. The MHC defined resource population, along with the transfected cell lines
expressing each of the full length ovine MHC class I sequences, comprise tools for
immunization and disease association experiments studying the protective immunity to
intracellular pathogens of sheep.
