shotgun, can be obtained from the Ensembl database (http:// www.ensembl.org). LoxAfr3, an assembly of the genome of African Elephant, has been sequenced to 76 coverage (loxAfr3, 76 coverage, July 2009). The elephant immunoglobulin gene sequences were retrieved from the UCSC genome browser (http://genome.ucsc.edu/). Identification of the elephant Ig genes Human immunoglobulin gene sequences were used as queries to search the elephant genome scaffolds that contained immuno- globulin genes. A conventional TBLASTN approach was used to identify constant region genes of the elephant immunoglobulins. FUZZNUC, an online software (http://embossgui.sourceforge. net/demo/fuzznuc.html) was used to find adjacent recombination signal sequences (RSSs) for identification of variable, diversity, and joining gene segments. Five or more mismatched bases were allowed to cover all genes. The locations of the annotated elephant gene sequences on the elephant genome are shown in Table S1 (S1-1,S1-5). Sequence alignments Editing and comparison of sequences were carried out using the DNAstar program. Alignment of multiple sequences was per- formed using the Clustal W algorithm, then aligned with Clustal X software, and exported by BioEdit software with view conservation by plotting identities to a standard as a dot. Dot matrix analysis A dot matrix analysis (window size 30 bp and mismatch limit 9 bp) was used for comparing two sequences to identify a possible alignment of characters between the sequences. Phylogenetic analysis Phylogenetic studies were carried out using MrBayes3.1 and viewed with the TreeView package. All the trees were obtained with 1 million generations for the chains, a sample frequency of a 100, and a burn in of 2,500 (ngen = 1000000; Samplefreq = 100; burnin = 2,500). The site by site rate variation was set to a gamma distribution (rates = gamma) for all the Bayesian trees and a General Time-Reversible (GTR) (nst = 6) model of substitution was chosen. The sequences from other species used in phyloge- netic analyses are presented in Table S2 (S2-1,S2-2). Definition of the VH/VL gene families In mammals, germline VH and VL gene segments can be grouped into families based on their nucleotide sequence similarity [49]. The established criteria are that the same family members share more than 80% nucleotide similarity, those with less than 70% similarity are put into different families, and those possessing between 70% and 80% similarity are inspected on a case-by-case basis [50]. In our analysis, we placed VH and VL segments having similarity greater than 70% into the same family. Results Elephant immunoglobulin heavy chain genes IgH locus. The public elephant genome assembly used in this study was loxAfr3, which is an assembly of the genome of the African Elephant (Loxodonta africana), sequenced to 76 coverage. The high genome coverage of this assembly confers a high reliability on the gene analysis. BLAST searching localized the elephant IgH locus to genomic scaffold 57. It spans approximately 2,974 kb from the most 59 VH segment (VH2-112p) to the most 39 Analysis of the Elephant Immunoglobulin Genes c gene (Fig. 1). A single m and eight c genes were identified in this scaffold. Neither e nor a genes could be found, most likely due to sequence gaps. Constant region genes. Like other mammalian species, the elephant m gene contains four CH and two transmembrane exons. A sequence comparison of m genes among thirteen vertebrate species demonstrated that the critical amino acids for immunoglobulin folding, Cysteine (C) and Tryptophan (W) [51], were highly conserved in elephants (Figure S1). In addition, the elephant IgM constant region showed the highest amino acid sequence identity to human (63.8%), and the least to echidna (50.8%). Most mammals also express a d gene, which is always situated immediately downstream of the m, and the distance between m and d usually does not exceed 7 kb. A BLAST search against the elephant whole genome using both DNA and amino acid sequences of the d genes of other mammalian species showed no intact d gene. However, approximately 10 kb downstream of the elephant m (no sequence gaps for 90 kb downstream), we identified a short fragment encoding a polypeptide (Figure S2) homologous to the IgD CH3 domain of other mammals. This was done by a thorough examination of amino acid sequences encoded by the DNA sequences between m and c1 (based on translation of all reading frames of both sense and anti-sense sequences). An alignment of the elephant IgD remnant and the IgD CH3 domains of several mammalian species is presented in Figure S2. This indicates that the gene has been highly mutated and pseudogen- ized in the elephant. In addition to the eight c genes (c1 to c8) in scaffold 57 (Fig. 1), an additional c gene (tentatively named as c9) was identified in scaffold 495 (data not shown), which spans 77 kb. Scaffold 495 is not assembled together with scaffold 57; therefore, c9 could potentially be either an additional subclass encoding gene or an allelic variant. The identification of multiple IgG subclass- encoding genes is in accordance with a previous report, which indicated that there were at least five subclasses of IgG in African elephant sera [20]. Sequence analysis showed no additional Ig genes in genomic scaffold 495, except for the c9 gene. The greatest variation among mammalian IgG subclasses is usually concen- trated in their hinge regions [52–54]. However, no elephant IgG cDNA sequences have been sequenced, it is very difficult to accurately assess the hinge regions of the elephant IgG heavy chains. The hinge region is usually encoded on a separate exon that could not be identified in the elephant due to the low level of conservation and the absence of cDNA sequences. An amino acid alignment of the nine elephant IgG subclasses is presented in Fig. 2. The first exons (CH1) of c1 and c2 are both missing because of gaps. The CH3 exon of c3 is pseudogenized because of a premature stop codon (marked with a star in Fig. 2), and a frame- shift mutation (marked with shadowing in Fig. 2) caused by nucleotide (adenine) insertions at positions 148 and 158, respectively. To clarify the relationship among c chains from mammalian species, a phylogenetic tree of IgG CH2 and CH3 exons was constructed and is shown in Figure S3. The elephant c genes form a distinct cluster. This is consistent with previous analysis, which showed that the divergence of IgG subclasses occurred after speciation [52]. Dot matrix analysis of the elephant IgH locus showed there are switch regions upstream of the m gene and six c genes (c1, c4, c5, c6, c7, and c8), as in humans and mice [55,56]. The switch regions of c2, c3, and c9 could not be identified, most likely due to sequence gaps. Structurally, the switch regions, as in other species, are all composed of pentameric repeats (GGGCT and GAGCT). The elephant Sm region shows substantial nucleotide similarity PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e16889