Characterization of MHC class I and beta(2)-microglobulin sequences in Atlantic cod reveals an unusually high number of expressed class I genes

Abstract

Degenerate polymerase chain reaction (PCR) primers based on conserved residues from alignments of species with already characterized major histocompatibility complex (MHC)-encoded sequences were used in the search for class I and beta(2)-microglobulin (b(2)m) genes in Atlantic cod (Gadus morhua L. ). After PCR amplification and subsequent sequencing a putative class I sequence was identified, from which a probe was designed and used to screen a spleen cDNA library from one single individual. The full-length clone obtained was sequenced and shown to be a classical Mhc class I-encoded sequence. It revealed the characteristic alpha1-, alpha2-, and alpha3-domains and transmembrane and cytoplasmic region, with several conserved amino acids. A PCR amplification from the alpha2-domain to the CY-region was performed on the same library, using a proof-reading enzyme. At least 11 unique additional sequences were isolated. Moreover, sequencing of the additional cDNA clones resulted in a total of 17 different Mhc class I sequences in this individual. A Southern hybridization of DNA from four different individuals using an alpha3-specific probe confirmed this large number of genes. Interestingly, based on differences mainly in their transmembrane region, the sequences obtained could be divided into two distinct groups. Within the groups no support could be obtained for any further subdivision. Southern experiments using an alpha1-specific probe gave almost the same restriction fragment length polymorphism with a high number of hybridizing bands, suggesting a low divergence in this part of the gene. Sequencing of PCR clones obtained with a proof-reading enzyme confirmed this at the nucleotide level. PCR amplification to isolate and characterize the b(2)m gene resulted in a sequence which was used to screen a thymus cDNA library. Two different alleles were obtained and these showed the characteristic features of known teleostean beta(2)m sequences. A Southern hybridization with genomic DNA from four different individuals suggested the presence of one b(2)m locus in Atlantic cod.