Abstract

Major histocompatibility complex (MHC) proteins are encoded by a series of genes clustered in one region of the genome. These proteins function to present immunogenic peptides to T lymphocytes. The organization and structure of the MHC and its genes are major factors that allow the immune system to recognize such a vast array of antigens. For instance, the MHC is polygenic containing both class I and class II genes that encode molecules with different peptide-binding properties and specificity. MHC genes also are extremely polymorphic (i.e., the products of different alleles can be extremely varied, differing from one another by up to 20 amino acids). Class I MHC genes are involved in processing and presenting viral peptides and abnormal cellular byproducts to the immune system. Previous studies into the pinniped MHC have been restricted to one or two MHC genes, and have largely focused on genes within the class II region. In this study, we examined the expression of class I genes in two seal species and developed techniques by which the combination of MHC genes possessed by individuals (the MHC haplotype) can be defined and compared. This information will be useful in predicting the susceptibility of particular populations to viral and intracellular infections and, in captive or working populations, may help in designing breeding programs that maximize diversity of these immunologically important genes. Most structural differences observed between MHC alleles are localized to the portions of the molecule that bind the immunogenic peptides, namely the α1 and α2 domains. In this study we use an MHC-typing technique that combines PCR amplification, denaturing gradient gel electrophoresis (DGGE) and direct sequencing. This technique circumvents the need for cloning, and can be used to characterize and compare each domain of the MHC genes between individuals. A RACE (rapid amplification of cDNA ends) cDNA library was constructed from total mRNA extracted from peripheral blood mononuclear cells (PBMCs) of three Pacific harbor seals (Phoca vitulina) and three California sea lions (Zalophus californianus). Full length class I genes were amplified using degenerate class I-specific primers. Over 30 unique class I sequences were identified from each library of three animals. Furthermore, sequence information from the more conserved downstream regions of the class I genes showed three consistent motifs, which may be useful in interspecies phylogeny comparisons. Seal sequence-specific class I primers were designed to examine the α1 and α2 domains of the class I genes of an additional 15 animals of each species. These primers are currently being tested and, if successful, will facilitate rapid MHC genotyping of pinnipeds in the future.