Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products.
Abstract: 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.
Publication Date: 2003-12-17 PubMed ID: 14675391DOI: 10.1111/j.1365-2370.2003.00420.xGoogle Scholar: Lookup
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- Journal Article
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research article discusses the identification of novel classical MHC class I alleles in horses, using reverse transcription-PCR products. The researchers highlight that improved typing of this class is needed for better definition and to understand their variability. This information is crucial in understanding cytotoxic T lymphocyte responses in horses.
Research Methodology
- The researchers initiated the study by analyzing horse classical MHC class I genes. They did this using reverse transcription (RT)-PCR amplification of sequences. These sequences encode the polymorphic peptide binding region and the more conserved alpha 3, transmembrane, and cytoplasmic regions.
- The analysis involved the use of specific primer sets, including a horse classical MHC class I-specific reverse primer, and a forward primer conserved in all known horse MHC class I genes.
- The researchers sequenced a minimum of 25 clones that contained MHC class I sequences from 13 different horses. This process led to the identification of 25 novel sequences and three previously described ones.
Findings on MHC Class I Alleles
- Different horses or different RT-PCR identified nine alleles. Further, the researchers found 19 putative alleles in multiple clones from the same RT-PCR.
- The used primer pairs did not amplify putative non-classical MHC class I genes. All 462 clones contained only classical MHC class I and related pseudogenes.
- This method also helped identify classical MHC class I alleles shared between horses by descent and defined the differences in alleles in horses with different equine leukocyte antigen (ELA)-A haplotypes as per serology.
- However, researchers observed that horses sharing ELA-A haplotypes as defined by serotyping did not always share cDNA sequences. This observation suggests that there may be subhaplotypic variations within serologically defined ELA-A haplotypes.
- The 13 horses in the study displayed two to five classical MHC class I sequences, suggesting that multiple loci code for these genes.
Implications and Applications
- The researchers concluded that the method of sequencing clones from RT-PCR with classical MHC class I-specific primers proved beneficial for selecting haplotype-matched and mismatched horses for CTL studies.
- This method also provides sequence information necessary for developing simpler and more discriminating typing procedures.
Cite This Article
APA
Chung C, Leib SR, Fraser DG, Ellis SA, McGuire TC.
(2003).
Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products.
Eur J Immunogenet, 30(6), 387-396.
https://doi.org/10.1111/j.1365-2370.2003.00420.x Publication
Researcher Affiliations
- Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99165-7040, USA. chungw@vetmed.wsu.edu
MeSH Terms
- Alleles
- Amino Acid Sequence
- Animals
- Base Sequence
- Haplotypes / genetics
- Histocompatibility Antigens Class I / genetics
- Horses / genetics
- Molecular Sequence Data
- Protein Structure, Tertiary
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Sequence Analysis, DNA
Grant Funding
- AI-24291 / NIAID NIH HHS
- AI-47660 / NIAID NIH HHS
Citations
This article has been cited 12 times.- Vasoya D, Tzelos T, Benedictus L, Karagianni AE, Pirie S, Marr C, Oddsdóttir C, Fintl C, Connelley T. High-Resolution Genotyping of Expressed Equine MHC Reveals a Highly Complex MHC Structure. Genes (Basel) 2023 Jul 10;14(7).
- Rapacz-Leonard A, Dąbrowska M, Janowski T. Major histocompatibility complex I mediates immunological tolerance of the trophoblast during pregnancy and may mediate rejection during parturition. Mediators Inflamm 2014;2014:579279.
- Yao S, Qi J, Liu J, Chen R, Pan X, Li X, Gao F, Xia C. Expression, refolding and preliminary X-ray crystallographic analysis of equine MHC class I molecule complexed with an EIAV-Env CTL epitope. Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Jan 1;68(Pt 1):20-3.
- Ventura T, Aflalo ED, Weil S, Kashkush K, Sagi A. Isolation and characterization of a female-specific DNA marker in the giant freshwater prawn Macrobrachium rosenbergii. Heredity (Edinb) 2011 Oct;107(5):456-61.
- Ramsay JD, Leib SR, Orfe L, Call DR, Tallmadge RL, Fraser DG, Mealey RH. Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population. Immunogenetics 2010 Sep;62(9):633-9.
- Tallmadge RL, Campbell JA, Miller DC, Antczak DF. Analysis of MHC class I genes across horse MHC haplotypes. Immunogenetics 2010 Mar;62(3):159-72.
- Mealey RH, Leib SR, Littke MH, Wagner B, Horohov DW, McGuire TC. Viral load and clinical disease enhancement associated with a lentivirus cytotoxic T lymphocyte vaccine regimen. Vaccine 2009 Apr 21;27(18):2453-68.
- Mealey RH, Lee JH, Leib SR, Littke MH, McGuire TC. A single amino acid difference within the alpha-2 domain of two naturally occurring equine MHC class I molecules alters the recognition of Gag and Rev epitopes by equine infectious anemia virus-specific CTL. J Immunol 2006 Nov 15;177(10):7377-90.
- Tallmadge RL, Lear TL, Antczak DF. Genomic characterization of MHC class I genes of the horse. Immunogenetics 2005 Nov;57(10):763-74.
- Chung C, Mealey RH, McGuire TC. Evaluation of high functional avidity CTL to Gag epitope clusters in EIAV carrier horses. Virology 2005 Nov 25;342(2):228-39.
- Fraser DG, Leib SR, Zhang BS, Mealey RH, Brown WC, McGuire TC. Lymphocyte proliferation responses induced to broadly reactive Th peptides did not protect against equine infectious anemia virus challenge. Clin Diagn Lab Immunol 2005 Aug;12(8):983-93.
- Chung C, Mealey RH, McGuire TC. CTL from EIAV carrier horses with diverse MHC class I alleles recognize epitope clusters in Gag matrix and capsid proteins. Virology 2004 Sep 15;327(1):144-54.
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