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