Molecular Characterization Of Major Histocompatibility Complex (MHC) Genes In The Chinese Alligator

Major histocompatibility complex(MHC)is a gene dense region found in all jawed vertebrates,which plays essential roles in antigen presentation to T cells in animal adaptive immune response.The MHC gene locus contains a high percentage of immune genes,especially genes encoding antigen-presentating proteins,which are generally highly polymorphic.As the closest relatives of birds,crocodiles represent a key evolutionary position linking amphibians,reptiles,birds and mammals.Therefore,studies on the crocodilian MHC genes can be expected to provide significant clues to understanding of the evolutionary pattern of MHC genes in jawed vertebrates.In this thesis,we have carried out a thorough analysis of MHC genes in the Chinese alligator,a so-called species of "living fossil".From the Chinese alligator genomic BAC(bacterial artificial chromosome)library previously constructed in our laboratory,we isolated 14 BAC clones using a PCR screening approach,and these 14 clones were fully sequenced.In combination with genome walking and BLAST searches against the released Alligator sinensis whole genome shotgun sequence(WGS)assembly,we obtained a 3.1 Mb MHC gene-containing genomic sequence,which was predicted to contain 216 genes.Among them,seventy-three are MHC or MHC related genes,which include 15 clssical class ? ? genes(5 psudogenes),1 classical class ? a gene,23 classical class ? p genes(19 psudogenes),1 nonclassical class ? DMA psudogene,2 nonclassical class ? DMB genes,23 class ? genes and antigen processing genes LMP2 and LMP7,5 TAP transporter genes(3 psudogenes)and 1 TAPBP gene.Additionally,MHC framework genes such as TRIM39 are also found within the genomic region.These data indicate that many MHC genes in the Chinese alligator are polygenic,especially the class ? ? and class ? ? genes,which are present in the locus as multiple copies,and many of them appearing to be pseudogenes.The size of MHC gene locus in the Chinese alligator is close to human(?3.6 Mb),but much longer than the chicken MHC locus(?92-739 Kb).The Chinese alligator MHC genes are organized in four subregions in an order as follows:extended class ?-class ?-class ?-MHC class ?.Whereas LMP and TAP genes involved in the antigen processing are found to be located in the class ? region,tapasin encoding gene was identified in the extended class ? region.Syntenic analysis of the MHC loci in tetrapods suggests that the Chinese alligator has a gene organization simialr to those in amphibians,but appearing to be an intermediate between the bird and and mammalian MHC gene loci.Phylogenetic analyses of MHC class ? genes indicate that there are three subtypes of class ? genes in the Chinese alligator.Subtype ? is closely related to the bird genes,whereas subtype ? is related to mammalian genes,and subtype ? related to the ZLA in zebrafish.Phylogenetic analyses of DMB genes in jawed vertebrates also reveal that there are two types of DMB genes:one present in cartilaginous fish,amphibians,reptiles and monotremes,the other one present in bony fish,amphibians,reptiles,birds and mammals.Our data also show that there are two subtypes of ? ? genes in testudines and crocodilians,one of which is evolutionarily ancient,closely related to the genes in bony fish,and the other one is closely related to the genes in amphibians,birds and mammals.All these analyses indicate that the MHC genes in the Chinese alligator could evolutionarily link the MHC genes in amphibians,birds and mammals.In summary,we have,in this thesis,conducted a thorough analysis of MHC genes in the Chinese alligator,based on which a systematic evolutionary analysis of MHC genes was conducted.These data provide significant clues to understanding of the evolutionary pattern of MHC genes in vertebrates.