Abstract
The major histocompatibility complex class I genes play crucial roles in the adaptive immune system of vertebrates against intracellular pathogens. To date, no class I genes from the giant panda (Ailuropoda melanoleuca) has been reported, even none from species of Ursidae. In this study, we successfully identified three class I genes from a giant panda bacterial artificial chromosome library and designated them as Aime-128, 152, and 1906, respectively. Pairwise sequence alignments revealed that (1) the Aime-1906 always possessed the lowest identities (52–86%) in different regions compared with the Aime-128 and 152 and (2) the Aime-128 also varied from the Aime-152 in the regions of 5′ untranslated region (UTR), 3′ UTR, and exon1, whose similarities were 83%, 87%, and 91%, respectively. Comparison of structure characteristics indicated that the Aime-128 possessed all conserved amino acids important to the function of antigen presentation while the Aime-152 and 1906 presented two and five mutated residues. Analysis of phylogenetic trees demonstrated that the Aime-128, 152, and 1906 were clustered into three different branches with 99% or 100% bootstrap values. As a result, these three kinds of evidence supported that the Aime-1906, 152, and 128 should be derived from different loci. Furthermore, in view of a prestop codon in the exon 7 and patterns of amino acid replacement within alleles, the Aime-1906 gene is predicted to be a nonclassical locus, which is most closely related to dog leukocyte antigen 79 in the phylogenetic tree constructed with various mammalian class I loci.
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Acknowledgements
This work was supported by grants from the National Basic Research Program of China (973 program; No.2007CB411600) and from the National Science Fund for Distinguished Young Scholars (No. 30325009). We thank Mr. Chris Wood for his help in English language.
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ESM 1
Primer sets used in PCR reactions for cDNA and gDNA are listed in the table below. The E23-S and E23-AS were used to screen the cDNA and genomic BAC library. The 3′-CDS and 5′ PCR primer were from SMART cDNA Library Construction Kit (Clontech). UP-TSP and DOWN-TSP primers were designed to obtain upstream and downstream sequences of the Aime-1906 gene. DW-ACP, DW-ACPN, and universal primer were from DNA walking SpeedUp™ Premix kit (Seegene). RD1 was designed to isolate the promoter sequence of Aime-152. The 128whole, 152whole, and 1906whole aimed to obtain the whole sequences of the three genes and 128-E23, 152-E123, and 1906-E23 were used for allelic diversity detection. Ta represents annealing temperature and E(min) is extension time (DOC 40 kb).
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Pan, HJ., Wan, QH. & Fang, SG. Molecular characterization of major histocompatibility complex class I genes from the giant panda (Ailuropoda melanoleuca). Immunogenetics 60, 185–193 (2008). https://doi.org/10.1007/s00251-008-0281-7
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DOI: https://doi.org/10.1007/s00251-008-0281-7