Analysis of the equine lymphocyte antigen system by Southern blot hybridization

Fourteen Standardbred horses homozygous for one of six equine lymphocyte antigen (ELA) specificities (A1, A3, A4, A5, A6, or A10) were analyzed by Southern blot hybridization using DNA probes derived from the mouse major histocompatibility complex (MHC). Total genomic DNA from peripheral lymphocytes was digested with the restriction enzymes Hind III, Pvu II, or Eco RI. Twenty-three to thirty-three bands were generated for individual horses with the class I cDNA probe. The resulting band patterns revealed 12-14 nonpolymorphic fragments, which is consistent with the highly conserved Qa/Tla genes seen in other species. The remaining 10-19 bands displayed significant polymorphism; no two animals had identical band patterns when studied with all three enzymes. The polymorphism was markedly decreased between animals of the same ELA serotypes. Unique bands were identified in both A1 horses and all four A6 animals. Pvu II digestions of lymphocyte DNA were hybridized with mouse MHC class II probes. A cDNA probe for the E alpha gene revealed only a single nonpolymorphic band. In contrast, a cDNA probe for the H-2 A alpha locus displayed three to five strong bands in each animal with polymorphism that was most pronounced between horses of different ELA serotypes. Genomic DNA probes for A beta and E beta genes both revealed multiple polymorphic bands. However, cross-hybridization between these two probes prevented distinction between A beta and E beta equivalent loci. The reduced polymorphism evident within ELA specificities is consistent with the concept that the equine lymphocyte antigen system includes two families of closely linked MHC genes.