The bovine MHC class I loci have largely been characterised using
immunological techniques. This has identified in excess of thirty
alleles all corresponding to one locus, BoLA-A . Detailed analysis of
bovine MHC class I genes was undertaken using recombinant DNA
technology in an effort to expand on this knowledge.
A bovine liver cDNA library was constructed in Xgt10 and screened
with a human class I MHC DNA probe. Two bovine class I cDNA clones
were isolated. The largest clone, pBoLA-1, size 1263bp, was
incomplete lacking a transcription initiation codon and a
polyadenylation signal. pBol_A-1 on translation into amino acid
sequence could encode a mature protein of 339 amino acids.
Comparison of the amino acid sequence of pBoLA-1 to two other
bovine cDNA clones revealed that pBoLA-1 may be allelic to one of
them. Analysis of these bovine clones facilitated the identification of
putative species specific residues. Extensive comparison of pBoLA-1
protein to class I proteins of other species revealed that the protein
was more similar to class la proteins than to class lb proteins. The
second cDNA clone, pBoLA-2, was approximately 600bp in size.
pBoLA-2 mapped to the 3' end of pBoLA-1, extending it to 1.7kb, the
approximate size of class I transcripts. The exact nature of the
sequence encoded by pBoLA-2 was not established, although Northern
blots suggested it may contain a repeated element.
Two bovine genomic clones (which were isolated from a bovine
genomic DNA library by Dr Jean-Luc Vilotte using the pBoLA-1 cDNA
clone as a probe) were chosen for further characterisation. The first,
phage 41, encoded two non-contiguous hybridising pieces of DNA.
Restriction enzyme digests of this phage were compared on a
Southern blot to similar digests of genomic DNA, the isolated phage
clone appeared to contain a sequence which was probably present. as 1
copy in the genome. The conclusion reached was that phage 41
carries two class 1 genes, seperated by ISRb. This close proximity of class 1 genes has also been observed in mouse. The second genomic
clone, phage 33, encoded a class I pseudogene. The gene lacked a
defined 5' end but shared 52-76.5% nucleotide similarity with
pBoLA-1 stretching over exons five to eight.
Lastly, pBoLA-1 was used to dissect the detailed multi-band
hybridisation pattern obtained when probing Southern blots. Use of 5'
and 3' probes allowed an estimate of at least 2-3 bands recognised in
common. Use of a 5' probe at high stringency revealed that pBoLA-1
only detected polymorphic bands, these by inference are class la
genes with nonpolymorphic bands corresponding to class lb genes. The
polymorphic bands recognised by pBoLA-1 were correlated to the
BoLA-A serological type of the animal. Bands which segregated with
BoLA-w6.2, -w8 an6-w10 were identified.
Knowledge of the class I genes of the bovine MHC was extended
from a single serologically defined locus to a large multi-gene family.
This multi-gene family consists of class la genes which include
pBoLA-1 and possibly class lb genes as defined by genomic clones and
Southern blotting analysis of bovine DNA.