Cloning and characterisation of MHC class 1 genes in cattle

Abstract

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.