Figure 1.
VIT Gene Evolution in Tetrapods
The topology and divergence times of the tree are based on previous studies [19,24,25,41]. Latin crosses indicate VIT inactivation events in eutherians and monotremes. Inactivation estimates (including approximated 95% prediction intervals) based on opossum VIT sequences are indicated by colored bars at the top (see also Figure 3). Duplications (“x2”) are indicated. VITanc is the likely ancestor of both the amphibian vtgA1/vtgA2 and VIT2/VIT3 genes in birds. Functional VIT genes in extant species are indicated in red. The inactivation time of VIT1* on the amphibian branch could not be estimated because of its absence in Xenopus tropicalis.
Figure 2.
Genome Alignment (Dot Plot Representing SIM Alignments) of Human/Chicken Syntenic Regions VIT 1-VIT3 Regions
The chain with the best cumulative score is shown. Alignment of flanking genes confirms the synteny of the aligned regions. The combined alignments of VIT1 coding sequences showed significantly higher alignment scores than the genomic background (introns and intergenic regions) in the chain, as assessed by a Mann-Whitney U test (p < 0.05). Thus, we can statistically exclude that detected VIT1 remnants from humans represent spurious sequence matches. The coding sequence matches for VIT2/3 may be too short to provide statistical significance or partially spurious.
Figure 3.
Genome Alignment (Dot Plot Representing SIM Alignments) of Opossum/Chicken Syntenic Regions VIT1-VIT3 Regions
The chain with the best cumulative score is shown. Alignment of flanking genes confirms the synteny of the aligned regions. The subsets of alignments corresponding to VIT exons of the best chain for all three regions have significantly higher scores than genomic background hits in the chain (p < 0.05, Mann-Whitney U test). This shows that VIT1-VIT3 exon matches in opossum represent nonrandom hits and thus correspond to real coding sequence matches.
Figure 4.
Overview of Intact and Disabled VIT Genes from Opossum and Platypus
Thin red lines indicate VIT sequences that could be retrieved. The sequences are aligned to the known 35 (VIT1/VIT2) and predicted 36 (VIT3) coding exons from the chicken VIT genes. Indels (blue), stop codons (red), and neutral indels (gray) are shown (the cumulative indel count is provided in Table 1). We note that the platypus VIT gene was aligned to the chicken VIT2 gene to illustrate the absence of sequence disablements (the platypus VIT sequence could not be unambiguously identified as VIT2 or VIT3, see text for details).
Table 1.
Disablement Counts of VIT Genes
Figure 5.
Illustration of Shared Indels in American and Australian Marsupials
The total number of non-neutral indels (i.e., those that are not a multiple of 3) shared among marsupials, wallabies (tammar and swamp wallaby), and specific to opossum are shown in the table. They were obtained based on the complete alignment shown in Figure S3. The alignment shown is based on a merge of individual pairwise alignments of the marsupial VIT sequences to that of chicken (to preserve the original genomic VIT alignments obtained using SIM).
Figure 6.
Inactivation Simulations for VIT Genes in Monodelphis domestica (opossum) and Ornithorhynchus anatinus (platypus)
The fraction of simulations (0–400 Mya) that correspond to the observed stop codon and indel counts for the respective pseudogene are shown (see Materials and Methods and Figure S4 for details on the simulation procedure). The most probable inactivation time is indicated at the mode of the distribution. Shaded areas indicate inactivation times that can be excluded because of the following: (i) shared sequence disablements (VIT1-VIT3, providing lower bounds), (ii) non-overlapping distributions (the clear inactivation of VIT1 in platypus rules out an inactivation of this gene in the common mammalian ancestor), and/or (iii) functionality of all VIT genes (VIT1-VIT3) in chicken. Dotted lines correspond to major lineage splits as indicated in the phylogenetic tree.
Figure 7.
Selection of the Intact Platypus VIT Gene
Two tests were conducted to test for purifying selection of the intact VIT sequence from platypus.
(A) To test whether the VIT platypus (foreground) branch shows a significantly different dN/dS compared to the functional VIT lineages from birds and amphibians (background), we used codeml as implemented in PAML and compared a one-ratio model (“null” model, which assumes an equal dN/dS ratio for all the branches in the phylogeny) to a two-ratio model (“alt”, alternative model), where an additional dN/dS value is allowed on the platypus VIT2 lineage. The two models were compared using a likelihood ratio test [40], and they were found to not provide significantly different fits to the data (“ns”, p = 0.96).
(B) To test whether dN/dS on the lineage leading to the extant platypus VIT2 sequence is significantly different from 1, we compared the likelihood of the two-ratio model (alternative model), where dN/dS on this lineage is estimated from the data, to that of a model where dN/dS was fixed to 0.5 (null model).
Figure 8.
Alignment of Syntenic SCPP Regions between Human and Platypus Using SIM
Horizontal bars show localizations of known casein genes or exons in humans. Vertical bars indicate various features (specified in the figure) of the putative platypus casein sequences. Putative casein locus sequences were predicted using GenScan, and putative transcripts overlapping significant alignments with SIM were analyzed for serine abundance (for putative α/β-caseins) and casein signatures (PS00306). SIM alignments of the human κ-casein locus were compared to Genewise HMM predictions (with Pfam HMM PF00997, κ-casein).