Show simple item record

dc.contributor.authorMiltiadou, Despoinen
dc.date.accessioned2018-05-14T10:14:41Z
dc.date.available2018-05-14T10:14:41Z
dc.date.issued2006en
dc.identifier.urihttp://hdl.handle.net/1842/29891
dc.description.abstracten
dc.description.abstractThe 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.en
dc.publisherThe University of Edinburghen
dc.relation.ispartofAnnexe Thesis Digitisation Project 2018 Block 18en
dc.relation.isreferencedbyAlready catalogueden
dc.titleCharacterization of the ovine Major Histocompatibility Complex (MHC) class I genesen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


Files in this item

This item appears in the following Collection(s)

Show simple item record