Genomic Organization And Repertoire Generation Of The IgL Genes In Pekin Ducks

Because of economic importance in agriculture and its immunological characters, the duck, as a representative species of water fowls, has gradually attracted attentions of many immunologists. Immunoglobulins (Igs) are key molecular elements of the adaptive immune system. In the IgL gene loci of nearly all bird species examined to date, there is only a single functional variable gene segment that can recombine with joining (J) gene segments, introducing very limited sequence diversities. Thus, to generate sufficient diversity, the bird IgL genes rely mainly on gene conversion (GCV) using pseudogenes as sequence donors to modify the single rearranged variable gene. Some other species such as rabbits and artiodactyls (pigs, cattle and sheep) can also use gene conversion to generate their Ig diversities. However, during expression of the human and mouse Ig genes, gene conversion events are nearly not found. In this thesis, we have performed a thorough analysis of the Pekin duck IgL genes, providing a complete germline structure of the IgL locus. Several molecular mechanisms such as VJ recombination, somatic hypermutation and gene conversion, that are tightly involved in generation of Ig diversity, are also analyzed. We aim to provide a more complete understanding of IgL genes and their diversity-generating mechanisms in ducks.We have screened a Pekin duck bacterial artificial chromosome (BAC) genomic library and sequenced a BAC clone containing the entire duck IgL gene locus, and the total length of the BAC insert is about 210 kb. A detailed analysis of the BAC sequence, together with our RACE and Southern blotting data, allowed us to provide a complete genomic structure of the Pekin duck IgL gene locus. Like chickens and some other birds, only a single pair of Jλand Cλ was identified. While 88 Vλ gene segments were identified in the IgL locus,79 of them appear to be pseudogenes, and the remaining 9 were functional genes. The 88 Vλ, genes can be classified into two subgroups, and the 9 functional Vx genes are highly homologous and classified into the same subgroup. Phylogenetic analysis showes that the 9 duck functional Vx genes clearly cluster with functional Vx genes of other birds, suggesting that the 9 functional Vx genes may have been derived from a single gene through duplications. Specific PCR of the nine functional VxJx segments and sequencing show that, all the 9 functional Vx genes are able to rearrange with the Jx, which could increase the duck IgL VλJλrecombinational diversities compared with chickens and other birds, although the recombinational diversities are still limited if compared with humans and mice. The Pekin duck CDRL3 length and amino acid usage were calculated and compared with those of mice, humans and chickens. The duck CDRL3 repertoire favored longer sequences, and had a higher cysteine content compared to mouse/human/chicken. We also found that gene conversion played a critical role in generation of duck IgL diversity (GCV frequency at 25.3% of Vxl, Vx9 and Vλ19 sequences in the 21-day-old duck, with a mean GCV tract length of 45 ± 41 bp; and 36.5% in the 140-day-old duck, with a mean GCV tract length of 86 ± 46 bp). Notably, GCV events occur at all nine functional Vx genes in the duck, which further introduce more diversities into duck IgL genes. A few N and P nucleotide insertions were identified at the duck VxJxjoints, and somatic mutations downstream of the Jλ segment were also examined, both of which could introduce the duck IgL diversity.To further investigate the reasons why mice can not use gene conversion to introduce immunoglobulin diversities, we modified the BAC clone covering the entire Pekin duck IgL locus, which was further used to prepare transgenic mice. In the transgenic mice, all the exogenous duck Vλ genes could rearrange with the Jλgene segment. At the RNA level, the endogenous mouse Igλ and Igκ genes, and the exogenous duck Igλ, all expressed normally in the transgenic mice. Western blotting detection showed that, the exogenous duck Igλ, gene was also expressed at the protein level. Gene conversions were found to occur in the exogenous duck Vλ6, Vλ9, Vλ19 genes with a low level (2.69%). The values were higher than that in the endogenous mouse Ig genes (IgG,0.1%), but much lower than that in the Pekin ducks themselves. Based on the gene conversion analysis in the transgenic mice, we speculate that, Ig gene structure and in vivo microenvironment of the WT mice may together account for the low level of gene conversion of the mouse Ig genes.In summary, we for the first time provide a complete germline structure of the IgL gene locus, and a thorough analysis of IgL diversity-generating mechanisms in ducks. The data reported in this thesis are quite useful for us to understand the adaptive immunity in ducks.