Genes of the ovine major histocompatibility complex class II region

Abstract

The major histocompatibility complex (MHC) is a multi-gene family encoding proteins which play important roles in the immune response to antigenic challenge. Three distinct regions designated class 1, 11, and 111 have been defined in the MHC of mouse and man. This thesis focuses on the genes of the class 11 region of the sheep. The products of the class 11 alpha (A) and beta (B) genes are heterodimeric glycoproteins whose physiological function is to present exogenous peptides to helper T cells. The recognition of MHC class 11/peptide complexes by the T cell receptor signals the release of a cytokine cascade resulting in T and B cell proliferation, macrophage activation and B cell differentiation with the production of increased amounts of pathogen-specific antibody.Much is known about the detailed structure and function of the MHC of man and mouse. However, when this project began little was known about the detailed structure of the MHC of the ungulates, the economically important group of animals which contains cattle, pigs, horses and sheep. As part of a study investigating fundamental cellular immunology in the sheep, this thesis describes the characterisation and expression of the genes of the sheep MHC class 11 region.Cosmid libraries prepared from DNA from three unrelated sheep were screened with probes from the DP, DQ and DR sub-regions of the human and mouse MHC class 11 regions. Cosmids were used because they facilitate the cloning of relatively large genomic inserts. Restriction maps of the cosmids have been produced showing that some of the clones overlapped. The MHC A and B genes within the clones have been sequenced and assigned to a specific sub-type. Functional genes have been identified by the reaction of their products with anti-sheep class 11 monoclonal antibodies following DNA-mediated transfection into the mouse L cell, a fibroblast cell line which does not express endogenous mouse class 11 genes. Transcription of some of the genes has been demonstrated by Northern blots and reverse transcription polymerase chain reaction.A restriction map of the sheep class 11 DQ sub-region has been constructed and shown to contain two distinct DQA loci with associated DQB genes. The DQ1 A/B gene pair was expressed in the mouse L cell. The sheep class 11 DQ1 product at the cell surface reacted with a sub-set of the available anti-sheep class 11 monoclonal antibodies. The DQ2 genes were transcribed and some evidence for their cell surface expression was obtained, although this was not formally proved.A previous study demonstrated the expression of a putative sheep DRA gene when co-transfected with a sequenced DRB gene. The sequence of the sheep DRA gene is described here together with sequence data from a number of DRB genes or pseudogenes which show that, depending on haplotype, the sheep DR sub-region may contain up to five DRB genes.The cloning and sequencing of ruminant orthologues of the HLA-DNA and -DOB genes is described for the first time. Although evidence was obtained for the transcription of the sheep DNA gene, its DOB gene partner is transcriptionally silent.A class 11 locus designated DY which is not found in mouse or man is described from the sheep MHC. Cloning and sequencing has shown that it contains a class 11 A/B gene pair like that of the expressed DQ1 locus, however, it appears to be transcriptionally silent.