Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products.
Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures.
Chung
C
C
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99165-7040, USA. chungw@vetmed.wsu.edu
Leib
S R
SR
Fraser
D G
DG
Ellis
S A
SA
McGuire
T C
TC
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006239:Haplotypes / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006736:Horses / Q000235:genetics*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
D015395:Histocompatibility Antigens Class I