Immunoglobulin Genomics in the Guinea Pig Figure 8. Southern blotting analysis of guinea pig genomic DNA. Southern blotting analysis of the guinea pig heavy chain variable region genes. Genomic DNA was digested with BamH I (B), EcoR I (E), Hind III (H) and Xba I (X), and hybridized with probes for each of three guinea pig families. doi:10.1371/journal.pone.0039298.g008 Analysis of the Guinea Pig Constant Region Genes To acquire the cDNA sequence for four heavy chain isotypes (m, c, e, and a), 39RACE were performed on splenic RNA using specific primers. Using this strategy we successfully identified genes encoding the constant region exons for IgM, IgG, IgE, and IgA (Figure S4). As predicted from the analysis of the guinea pig Ig genomic genes, the domain structure for the four isotypes was typical of that for other mammalian species (Figure S5). Within the guinea pig genome, CH3 and parts of the CH4 segments of the m gene were determined to be missing because of the existence of gaps (Figure 1). By using the 39 RACE method, the complete secreted IgM, including four exons, was successfully cloned (Figure S4–S5). Most mammals also express the d gene, with the exception of the rabbit [24] and opossum [25]. The area predicted to contain an IgD C region, and in particular the 39 region of the IgM exons between IgM and IgG, as well as the whole cavPor3 assembly, was thoroughly searched in two different orientations for coding sequences that might correspond to a putative IgD. These searches detected d trace fragments that are homologous with mammalian IgD (Figure 3) and showed an opposite transcription direction to the upstream m gene. Based on sequence alignment, the three- fragment d trace was found to belong to the CH2 and CH3 domains (Figure 3). Although two IgG isotypes (IgG1 and IgG2) were previously identified in domestic guinea pig serum [26], we only identified one IgG gene in the guinea pig genome perhaps due to sequence gaps. Our sequence alignment further showed that the recognized IgG in guinea pig genome shared the highest similarity with IgG1 (six amino acid difference) (Figure S6). Interestingly, we were just able to clone the IgG2 mRNA transcript by 39 RACE. To address this question, a further Southern blotting experiment using the CH1 exon (high similarity between IgG1 and IgG2) as a probe, which showed that there were more than one c genes in the guinea pig genome (Figure S7). Taken together, these data suggested that the guinea pig had two c genes in its genome but only one was preferentially expressed. A phylogenetic tree constructed with CH2 and CH3 exons of IgG from different mammalian species revealed that the guinea pig IgG genes were clustered with rodents IgG genes (Figure 4). IgG2 and IgA also exhibit a hinge region, which is thought to have evolved by condensation of the CH2 exon in an ancestral isotype such as IgY in birds [25,27]. The hinge region of IgA is encoded by the 59 end of the CH2 exon, as observed in other eutherian mammals [28,29,30] (Figure S4–S5). In mammals, especially in humans and mice, a pentameric tandem repeat sequence is found upstream of the heavy chain constant region, which acts as a switch or S region. S regions have previously been mapped and sequenced, and are relevant in Ig class switch recombination. Such characteristic tandem repeats were also found within the upstream Cm, Ce and Ca gene sequences of the guinea pig. Three putative S regions span 2.2 kb to 3 kb, and exhibit similar repeats (GAGCT and GGGCT) to those observed in humans and mice. However, the characteristic sequence of the switch regions could not be identified within the c gene, most likely due to sequence gaps. Dot matrix analysis of the guinea pig S region revealed substantial nucleotide similarity with those of humans and mice (Figure 5). Analysis of the Guinea Pig VH Gene Segments A total of 507 VH segments were identified in scaffolds 54 and 75 (Figure 1, Figure 2). Ninety-four of these appeared to be potentially functional because they contained leader exons (L), uninterrupted open reading frames (ORF), downstream RSS, and a V gene domain (framework regions and complementarity determining regions). The remaining 413 segments that contain either in-frame stop codons or are partial sequences were designated as pseudogenes (Figure 1, Figure 2 and Table S3). Given that gaps existed within the assembly, it is possible that as yet unidentified VH genes are also present in the guinea pig genome. Phylogenetic analysis and multiple sequence alignments, including all functional guinea pig germline VH gene segments, revealed the VH gene families 1, 2 and 3, which were comprised of 22, 17 and 55 members, respectively (Figure 6, Figure 7). The PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39298