Genomic Structural And Phylogenetic Analyses Of T Cell Receptor Genes In The Alligator Sinensis

Involved in cellular immunity,T lymphocytes play important roles in animal adaptive immune system,such as undertaking signal transduction,activating B cells,and killing target cells,thus regulating immune response.To fulfill these roles,T lymphocytes depend T cell receptors(TCR)to recognize antigen-MHC complex.Four conventional TCR types of ?,?,? and ? genes have been shown to be present in all jawed vertebrates,forming membrane-bound heterodimers comprising ?? and ??chains,respectively.There has also been other atypical TCR gene types found in lower mammals,birds,amphibians and cartilaginous fish(TCR?,VH?(V genes in TCR?/? locus homologous to IGHV),NAR-TCR).Although reptiles represent a key evolutionary position linking amphibians,birds and mammals,very few studies have been conducted on the TCR genes of this group of animals.Representing primitive reptiles,crocodilians are the closest relatives of birds,and are widely believed to have a strong immune system.In this study,we have determined the genomic structure and expressed repertoire of TCR genes in the Chinese alligator(Alligator sinensis),a reptilian species indigenous only to China.Firstly,we used the chicken TCR constant region sequences to conduct BLAST searches against the Chinese alligator whole genome shotgun sequence(WGS)assembly database.Based on the information obtained,PCR primers were designed and used to identify 17 BAC(bacterial artificial chromosome)clones from a Chinese alligator genomic BAC library,and 9 clones of them were fully sequenced.According to the sequencing data,the alligator TCR? locus was shown to span approximately 500 Kb of genomic DNA,whereas the TCRy locus spans 109 Kb.TCR?/? genes are distributed in two loci,where locus A spans about 1 Mb DNA,and locus B spanning approximately 400 Kb.Combining the transcriptional data obtained from the RACE(rapid amplification of cDNA ends)experiments with the Southern blotting results,we showed that the alligator TCR? locus was organized as V?(39)-J?1-?V?1-D?2-J?2(11)-C?2-V?(4),whereas the TCRy locus was organized as V?(18)-J?(9)-C?.The TCR?/? locus A is composed of V(104)-D?1(3)-J?1(3)-C?1-J?1(91)-C?1-VH?(2)(including 80 V?/? and 26 VH8 gene segments),and the locus B contains V(39)-D?2(3)-J?2(3)-C?2-V?-VH?-J?3-?C?3-VH?(3)-D?4-J?4-C?4-VH?(2)-D?5-J?5-C?5-J?2(63)-C?2(including 37 V?/? and 11 VH? gene segments).RACE results showed that the locus A and B were adjacent to each other,as they could share variable genes in expression.The expression data showed no evidence supporting somatic hypermutation occurred in the expressed TCR genes.However there are a plenty of N-and P-nucleotides in the CDR3 region.The TCR8 chains could use at least 50%of the V? segments and most of the functional VH? segments,as well as multiple D segments,which all together could increase their CDR3 diversity significantly.Additionally,quantitative PCR showed that the expression level of ?? TCR was similar to that of ??TCR in peripheral lymphoid tissues,while the expression level of ?? TCR was higher in mucosal tissues.The alligator TCR? and TCRy loci exhibit conserved syntenic structre with their genomic counterparts in other species.The alligator TCR?/? loci contain a large number of VH8 segments,which are co-expressed with C? segments.The VH? segments in the alligator TCR?/? loci can be divided into three groups:the first group(VH? ?)is highly similar with the VH8 segments in birds(VH Clan ?);the second group(VH??)is similar with the VH? and V?.segments in platypus and opossum(VH Clan ?);the third group(VH? ?)is most similar to crocodilian IGHV(immunoglobulin heavy chain variable region)(VH Clan ?,?,?),but the majority of VH? ? members are pseudogenes.Based on sequence alignments,phylogenetic analyses and syntenic genes analyses,a hypothetic evolutionary process of the amniotes TCR?/? loci was proposed.In this model,the common ancestor of reptiles,birds and mammals is thought to have one TCR?/? locus,which contains VH? ? and VH?? segments(VH?? may have occurred after the divergence of mammals and reptiles).Along with the evolution of mammals,the VH?? genes were retained in monotremes and marsupials,but lost in placental mammals.C? segment duplications should have occurred in the ancestor of reptiles and birds to create more C? segments(C?segments are divided into three groups:C??,C?? and C?? in reptiles).Whereas birds retain VH??and C??,and generated a new VH?-C? locus by genomic duplication,VH?? and VH?? were maintained in crocodilians with the newly appearance of VH??.Apparently,there should have been a duplication of the entire TCR?/? locus during the speciation of crocodilians.While the alligator TCR?/?locus B is more structurally conserved,the locus A should have been duplicated from the locus B.However,the expression level of genes in the TCR?/? locus A in lymphoid tissues was much higher than the genes in locus B.In summary,we have thoroughly analyzed the genomic structure,expressional patterns and phylogenetic characteristics of Chinese alligator TCR genes,and proposed a hypothetic evolutionary process of the amniotes TCR?/? loci,which provide important clues for the study of origin and evolution of VH? and TCR genes in tetrapods.