Polymorphism of bovine MHC (BoLA) class I molecules has been characterised mainly by serology. The use of alloantisera has
led to the identification of 50 different serological specficities, most of which behave as alleles of a single highly polymorphic
locus. However, molecular biological and biochemical studies suggest that there are at least two class I genes expressed from any
one haplotype. The actual number of expressed MHC class I genes in cattle is as yet unknown. The aim of this study was to
isolate different functional class I genes from a heterozygous animal, class I typed as BoLA- A10/A11. In order to investigate
the expression and function of individual class I products, transfection and characterisation of a series of bovine class 1 genomic
and cDNA clones isolated from this animal were performed.
From a group of fifteen bovine class I genomic clones, five different clones (1.3, 4.2, 15.2, 17.3x and 19.1), characterised
by their exon 2 sequences and by restriction analysis of an amplified 3.2kb class I gene fragment, were transfected into mouse
L cells. Of the five clones transfected, only 19.1 and 4.2 showed expression at the L-cell surface by using a murine monoclonal
antibody specific for a non-polymorphic determinant on bovine MHC class I (IL-A88) in flow cytometry. The phage clone 19.1
which had been previously shown to express a bovine class I molecule with All specificity, served as a control in these
transfection experiments. The transfectants obtained from both expressing clones were characterised serologically, biochemically
and by cellular assays and both were found to encode All serological specificity. Interestingly, the products of the 19.1 and 4.2
transfected class I genes were indistinguishable by isoelectric focusing, despite having clearly different nucleotide sequences.
The class I gene from the 19.1 phage clone was subcloned into pBR322 to make pBoLA-19 which was used to determine its
sequence. This plasmid transfected at a very high frequency with more than 90% of HAT resistant colonies positive for class I
expression in the primary transfection. From the sequencing of the 19.1 gene and these transfection results it was clear that
pBoLA-19 contains all the upstream regulatory and promoter sequences necessary for class I expression. The lack of expression
from the three phage clones 1.3, 15.2 and 17.3x suggested that these clones might contain truncated class I genes or pseudogenes.
In order to test their ability to be expressed, these clones were reconstructed by replacing the coding region of pBoLA-19 with
the corresponding gene fragment from three non-expressing clones. For this the pBoLA-19 construct was modified into a class
I gene expression vector (pBoLA-21). Plasmid pBoLA-21 carries the 5' and 3'-ends of 19.1 class I genomic clone, flanking a
unique Eag I site which was used for subcloning PCR-amplified class I gene fragments. The exon 2 to 3'-untranslated region
fragments were generated using primers based on sequences conserved in exon 2 and in the 3'-untranslated regions of the published
bovine class I sequences. Both the 19.1 and 4.2 clones were reconstructed with this approach and expressed on transfection. The
three non-expressing phage clones did not express even after reconstruction, suggesting that they are pseudogenes.
The potential of the pBoLA-21 vector for cDNA cloning and expression was tested by subcloning the bovine class I cDNA clone
pBoLA-1, which is truncated at both the ends. The transfected cell line from pBoLA-1 showed detectable levels of class I
molecules using IL-A88. Since both of the expressible class I genomic clones isolated from this animal encoded A11 specificity,
a cDNA approach was adopted for the cloning of sequences representing possible A10 genes from this haplotype. A 1.2kb cDNA
was PCR-amplified using a selective primer based in the 3'ut region of the class I gene, and the PCR product was subcloned into
pBoLA-21 to test its expression. The transfectant obtained was recognised by IL-A88 and IL-A34 (an AlO-specific mAb)
indicating that the transfected cell line expressed a component of the A10 haplotype.
To correlate the serological specificities of the individual expressed products with nucleotide sequences, all three expressing
genes were sequenced. The new class I sequences from pBoLA-19, pBoLA-4 and pBoLA-10 were compared with each other and
the other published sequences in order to establish whether these sequences represented products of single locus or genes
representing different loci. It appeared from the sequence analysis that all three sequences isolated from this animal are likely
to represent different loci. The approach used in this project will facilitate the functional analysis of individual locus products,
and will allow the number of expressed class I loci to be resolved.
