Analysis of the Elephant Immunoglobulin Genes Figure 1. The elephant IgH locus. The elephant IgH locus is localized to scaffold 57. The length is approximately 2,974 kb from the most 59 VH segment (VH2-112p) to the most 39c segment. Filled bars: potentially functional VH genes; open bars: VH pseudogenes: open bars and V-p; thin bars: truncated VH segments and V-t; DH: diversity genes; JH: joining genes; m: IgM coding gene; d: IgD remnant; and c: IgG coding gene. VH and DH genes are numbered based on the families to which they belong and their positions in the locus. The number before the dash (VHn or DHn) indicates the family, while the number after dash represents the genomic position. JH genes are numbered based on the order of their locations in the locus. Double slashes indicate gaps .10 kb. Alpha and epsilon genes are not displayed on the map, but this does not imply their absence in the elephant IgH locus. doi:10.1371/journal.pone.0016889.g001 with those of human, mouse, and pig (Fig. 3). The six elephant Sc regions are similar, but share little sequence similarity with human and mouse Sc (Fig. 4 and data not shown). VH gene segments. A total of 112 VH segments were identified in the elephant IgH locus. 51 of them appear to be potentially functional, because they have leader exons, normal open reading frames (ORF), downstream RSSs, and V gene domain (framework regions (FRs) and complementarity determining regions (CDRs)). The remaining 61 segments contain either in-frame stop codons or frameshifts, and are thus designated as pseudogenes. In addition, there are 17 partial segments of about 200 bp in length, which are regarded as truncated VH sequences. There are gaps above 10 kb in the elephant genome among the VH gene segments (Fig. 1), suggesting that there might be more VH segments. To examine the relationships among the elephant germline VH segments, pseudogenes as well as functional genes were used to construct a phylogenetic tree (Fig. 5). The seven identified VH gene families (1, 2, 3, 4, 5, 6, and 7) were confirmed to be homologous with the corresponding human VH gene families. The elephant VH4 family contains the most members (72 VH segments), which could be further divided into three groups (Fig. 5). We chose representative VH sequences from elephant and other mammals, covering almost all VH families identified, to construct phylogenetic trees (Fig. 6). The elephant VH genes clearly fall into the three previously known VH clans. DH gene segments. In the elephant IgH locus, 87 DH segments were identified and are presented in Figure S4 (S4- 1,S4-10). It should be noted that there might be more DH segments because of the existence of sequence gaps. Except for DH76, which has a 10 bp spacer, all the DH segments are flanked by characteristic heptamers and nonamers separated by 12-bp spacers. The potential coding regions of DH segments are 10–37 bp in length (Figure S4, S4-1,S4-10). It has been suggested that coding regions of DH segments of humans can be described by the characteristics of their amino acids [57]. Inspection showed that a great number of polar/hydrophobic amino acids or stop codons occur widely in elephant DH coding regions (data not shown). In humans and mice, the germline DH segments can be classified into families based on the extent of sequence similarity [58,59]. Analysis of nucleotide similarity in the coding regions and flanking RSSs indicated that the 87 elephant DH segments could be divided into seven families. Members within the same family share at least 70% nucleotide identity (data not shown), while some members in a family have completely identical sequences (these are shadowed in Fig. 7). We present the sequence alignment of the seven families in PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e16889