Haplotype characterization of transcribed ovine major histocompatibility complex (MHC) class I genes

Abstract

The ovine major histocompatibility complex (MHC) remains poorly characterized compared with those of other livestock species. Molecular genetic analysis of the bovine MHC has revealed considerable haplotype and allelic diversity that earlier serological analysis had not detected. To develop cellular and molecular tools to support development of vaccines against intracellular pathogens of sheep, we have undertaken a molecular genetic analysis of four distinct ovine MHC haplotypes carried by two heterozygous Blackface rams. We have identified 12 novel class I transcripts and used a class I sequence-specific genotyping system to assign each of these transcripts to individual haplotypes. Using a combination of phylogenetic analysis, haplotype and transcript expression data, we identified at least four distinct polymorphic class I MHC loci, three of which appear together in a number of combinations in individual haplotypes. The haplotypes were further characterized at the highly polymorphic Ovar-DRB1 locus, allowing selection of the progeny of the two founder rams for the establishment of an MHC-defined resource population.

Constraints on haplotype structure and variable gene frequencies suggest a functional hierarchy within cattle MHC class I

“…In recent years, it has become clear that there is a wide range of variation in different species, ranging from the most simple (chicken) to higher primates with two or three class I genes and fixed haplotypes, through to a range of species including lower primates, ruminants, horses and birds that have very variable gene numbers and multiple haplotype structures (Kaufman et al 1995;Holmes and Ellis 1999;Miltiadou et al 2005;Otting et al 2005;Balakrishnan et al 2010). In many cases, no investigations have been conducted to assess or predict functional distinctions between genes or haplotype structures.…”

Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells

“…Therefore, it was unsurprising that most alleles identified in the Swaledale/Leicester cross sheep used in this study were novel and diverse. However, it is of interest that alleles 2G, H, I, J from the responding sheep 4247 have a unique polymorphic cluster corresponding to changes Phe 113 and Met 114 of the reference sequence N*00301 ; Met 114 was predicted to bind peptides [33]. Allele 2K also possessed this unique cluster, although it is one of three alleles identified in an unresponsive sheep.…”

“…MHC class I genotyping was carried out as previously described [33] with some modifications. Total RNA was extracted with the Qiagen RNeasy Mini kit from cryopreserved lymphocytes of T .…”

“…Contaminating DNA was removed with the Turbo DNA-free™ kit (Applied Biosystems) before cDNA synthesis, which was carried out using the AMV LongAmp® Taq RT-PCR kit (New England Biolabs). Partial MHC class I sequences were amplified from cDNA samples using class I generic primers 416 (5' CGGCTACGTGGACGACAYG 3') and Cr (5' ATGGGTCACATGTGYCTTTG 3'), which bind within exons 1 and 3 [33], generating a 500 bp product. The cycling conditions for PCR were 94°C for 4 min, 30 cycles 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with Promega Go Taq DNA polymerase.…”

Cattle MHC nomenclature: is it possible to assign sequences to discrete class I genes?

“…sheep (Miltiadou et al 2005), assignment of sequences to defined classical MHC class I loci has proved problematic (Holmes et al 2003). Class I sequences are therefore currently prefixed 'N' to indicate 'not assigned', and are numbered in a single series.…”