Evidence of genotypic variation, MHC class I copy number variation and recombination within the chicken major histocompatibility complex-y system

The major histocompatibility complex (MHC) is a gene region found in all jawed vertebrates. These highly polymorphic, tightly linked genes are responsible for immune regulation by presenting self and non-self antigens to the immune system. The MHC is the most polymorphic vertebrate genome region because its genes face considerable selective pressure since survival depends on the capacity to respond to bacterial, viral and parasitic diseases. The MHC organization in different species has clearly evolved in separate and unique ways since the time that birds and mammals diverged from a common ancestor ∼310 million years ago. The size and organization of chicken MHC genes is somewhat different from the MHC regions found in mice and humans.;The history of the chicken MHC discovery began with a highly polymorphic blood group system reported in 1950 by Briles, McGibbon and Irwin. This serologically-defined system, originally designated as the chicken B alloantigen system, was subsequently identified as the MHC locus through tissue graft compatibility experiments. The region renamed MHC-B was mapped to chicken microchromosome 16. Briles and colleagues (1993) discovered another group of MHC genes named MHC-Y. These genes also map to chromosome 16 but are separated from MHC-B by a currently unmapped region. Chicken MHC-B haplotypes strongly determine susceptibility or resistance to certain infectious diseases with additional evidence that same may be true for MHC-Y.;Due to its more recent discovery and absence of a mammalian equivalent, little is known about the MHC-Y. For this reason my project focused on this second MHC gene cluster. MHC-B was first identified serologically through hemagglutination assays, and for years it was routinely typed in this manner. The MHC-Y gene cluster remained undetected, since MHC-Y molecules are either expressed at lower levels or do not appear on the chicken red blood cell surface. . The MHC-Y system was first discovered through DNA hybridization, hence it was originally called the Rfp-Y system, but then later changed. Sequencing revealed MHC class I-like genes in MHC-Y. Early Southern hybridizations provided evidence for MHC-Y polymorphism. The MHC-Y class I (YF) architecture is similar to that of the classical MHC-B class I (BF) molecule. However, instead of binding peptides, the YF molecule possesses a narrow, hydrophobic binding groove that binds non-peptidic ligands.;The first project objective was to gain insight into the MHC-Y genotypic diversity. I examined nine different layer stocks of Leghorn origin including a Leghorn line maintained at Northern Illinois University, a University of Arkansas experimental line, and seven lines from an international company breeding egg laying chickens. I examined their MHC-Y haplotypes using Southern blots. Multiple DNA samples representing all nine lines were digested with Bgl I and hybridized with a MHC-Y class I region specific probe (163/164f). The blots revealed extreme MHC-Y polymorphism among the haplotypes present in the nine lines. Some haplotypes are very simple with only two or four fragments, whereas others have as many as 12 hybridizing fragments. These data suggest that MHC-Y haplotypes may contain a range of class I genes.;Next, to obtain a more direct measure of sequence variability, I examined MHC-Y class I locus exons 3 and 4 using denaturing gradient gel electrophoresis (DGGE). Another goal of employing DGGE was to develop a more convenient method for determining MHC-Y haplotypes to replace expensive, time consuming, and radioactive Southern hybridizations. These assays also provided additional data supporting the likelihood of different numbers of MHC-Y class I genes across multiple haplotypes.;To further investigate Y class I gene copy number variation between different haplotypes, I analyzed the NIU haplotypes in this study using quantitative PCR (qPCR). This third technique confirmed that significant copy number differences occur in certain MHC-Y haplotypes. Among the MHC-Y haplotypes examined from the NIU line, haplotype Y3 seems to have a higher YF loci copy number compared with all others.;Given the wide variability in gene numbers revealed in this study, I wanted to determine whether genetic recombination, crossing over, in the MHC-Y region might be contributing to haplotypic variation. YF molecules seem to have a high recombination frequency between its multiple copies, perhaps on the order of 1% (1 map unit) or higher. This may be due to many similar genes present within the MHC-Y region. Proximity of a 41 base pair repeat known as PO41 might also contribute to instability of the region.