BACKGROUND
in tandemly repeated gene families, in which all members share a common function, there is a tendency for concerted evolution that is characterised by homogenisation of gene sequences  <cit> .
classical examples include the histone and ribosomal rna genes.
in such cases the expansion of gene families is driven by selection for high expression  <cit> .
concerted evolution is generally maintained by unequal crossover, intergenic gene conversion or other illegitimate recombination mechanisms  <cit> .
conversely, there are multigene families whose members encode diverse functions e.g.
genes encoding immunoglobulin , t cell receptor  and major histocompatibility complex  proteins  <cit> .
such diversity occurs when there is less homogenisation than mutation, due to the evolution of specific programmed mutational mechanisms  <cit> .
in addition, more complex modes exist; for example, the immunoglobulin heavy-chain variable-region  genes encode proteins with identical functions, but exhibit little concerted evolution  <cit> .
instead, their evolution is governed by divergence and a birth-and-death process of gene duplication and dysfunctioning mutations  <cit> .
similar to other families of highly expressed trophoblast-specific genes such as the pregnancy-associated glycoproteins   <cit> , the pregnancy-specific glycoproteins, which are the most abundant foetal proteins in the maternal bloodstream during human late pregnancy, are encoded by multiple tandemly arrayed genes  <cit> .
the psg family of glycoproteins, with the related cea-related cell adhesion molecule  proteins, are part of the immunoglobulin superfamily  <cit> .
the ig domain structure of the human and mouse psgs differs, as follows: human psgs contain one v-like ig domain , c2-like ig domains  and relatively hydrophilic tails , with domain arrangements classified as type i , type iia , type iib , type iii  and type iv   <cit> .
in contrast, mouse psgs typically have three or more n domains followed by a single a domain  <cit> .
the common ancestor of rodent and primate psgs and ceacams was probably similar to ceacam <dig>  which is the only cea family member with an identical gene structure in the human, rat and mouse that encodes all types of extracellular domains present in ceacam and psg proteins.
the time of initial gene duplication is estimated at  <dig> myr  <cit> , approximately the time of rodent-primate divergence.
the independent expansion of human and mouse psg gene families occurred through further gene duplication and exon shuffling events  <cit> .
the independent expansion of psg gene families in rodents and primates indicates convergent evolution, implying that psg function is conserved.
these events can be interpreted in the context of evolutionary theories of parent-offspring and inter-sibling conflicts that promote transcriptional 'arms races' leading to high expression of trophoblast-specific genes that influence maternal investment in offspring  <cit> .
in one scenario, duplicated psg genes are selected because they increase effective psg dosage, thereby enhancing an effect on maternal investment in offspring.
in this context, it is noteworthy that human psg n domains contain putative integrin-binding 'rgd' motifs that are proposed to mediate cell interactions with the extracellular matrix  <cit>  and immune cells  <cit> .
such psg-mediated functions could potentially influence trophoblast invasion or maternal immune cell function.
however, not all human, and none of the mouse, psgs contain an rgd motif  <cit> , suggesting that, if human rgd motifs are functionally significant, there has been diversification of function of some human, and all mouse, psgs, relative to a putative rgd-containing ancestor.
in the context of parent-offspring conflict, such divergence might reflect co-evolution of psgs and their receptors, similar to the co-evolution of ligand / receptor pairs observed in host-pathogen interactions  <cit> .
in this study, we sought to analyse psg evolution to determine the extent and patterns of rodent and primate psg sequence divergence by analysing intraspecific and interspecies dna substitution rates in psg coding regions.
we also sought evidence in support of functionality of rgd and rgd-like tri-peptide motifs in psg amino-terminal effector domains.
RESULTS
pairwise comparisons of all 4-domain mouse psg with all 4-domain human psg full-length amino acid sequences indicates conservation of the amino-terminal n domain
with the exception of mouse psg <dig>  psg <dig> and psg <dig> and human psg <dig> and psg <dig>  all psgs for which full length sequences are available have a structure based on four ig-like domains and a leader sequence that is cleaved during post-translational processing.
the only type of domain found in all rodent and primate psgs is the n domain located at the amino terminus.
indeed, this domain is shared by all members of the extended cea family, suggesting that it may contain important functional motifs.
we sought to test this hypothesis with respect to psg function, by analysing both full-length psg sequences and selected domains of possible functional importance.
alignments of full-length 4-domain human and mouse psg protein sequences were generated with clustalx, followed by pairwise comparisons of all mouse sequences with all human sequences.
mean dayhoff pam <dig> log scores were calculated for each alignment position and grouped by domain.
the scores within each of the four domains were then visualised using box and whisker plots  .
the n domains exhibited significantly higher scores  than the other three domains, with positive scores indicating conservation.
there was no evidence of interspecies conservation of the other domains, which is unsurprising given the known lack of orthology between human a <dig> / mouse n <dig>  human a <dig> / mouse n <dig>  and human b <dig> / mouse a domain pairs.
novel rat psg n <dig> domains identified by database searches
rat n <dig> domain exon sequences were identified in ncbi and ensembl databases.
three novel rat psg genes were identified and named psg <dig>  psg <dig> and psg <dig> in keeping with accepted nomenclature  <cit> .
we also identified a novel psg <dig> splice variant with alternative leader and n <dig> domain exons, situated between the n <dig> and n <dig> domain exons of the published psg <dig> sequence .
both blast and pattern matching methods retrieved the same rat psg genes from different databases; therefore we considered our search to be exhaustive.
all rat psg genes were found to reside on contig nw_ <dig> and this was used for the prediction of remaining exons for each psg gene based on blast generated alignments with mouse psg gene sequences .
the cds sequences of the novel predicted rat psg genes and psg <dig> splice variant are listed in additional file  <dig>  we used our predicted sequences in preference to the publicly available sequences in our analyses.
splitstree analysis reveals relatively high contradiction in rat psg n <dig> domain alignments, compared to mouse
following the preliminary identification of amino-terminal n domain conservation, we planned to use an evolutionary tree building approach to further examine inter-domain relationships in rodent and primate psgs.
however, using split decomposition analysis, mclenachan et al.
<cit> , in their study of a subset of human psgs, concluded that it is not possible to accurately determine branch points in an evolutionary tree of human psgs.
split decomposition analysis identifies contradictory relationships within alignment data; for example, there may be a pattern grouping psgx and psgy together, and another pattern grouping psgy and psgz together  <cit> .
this information is normally approximated when drawing evolutionary trees, however split decomposition is a non-approximation method that permits the building of trees with support indicated for relationships based on all patterns in the data.
such analysis can therefore predict to a limited extent the occurrence of sequence homogenisation e.g.
by gene conversion or positive selection.
we performed split decomposition analysis on nucleotide sequences using the splitstree <dig> program  <cit>  on the individual domain exons of mouse psg genes .
for a more complete analysis of n <dig> domains we also performed the analysis using rat n <dig> domain exons, all known human n <dig> domain exons and all known baboon n <dig> domain exons .
we detected no conflicting signals for mouse psg n <dig> domain exons , in contrast to the human n domain exons .
however, our results for human n <dig> domains  differ from those obtained by mclenachan et al.
<cit>  because we observed only two contradictions: i. regarding the relationship of psg <dig> and psg <dig> to each other, and to their nearest neighbours psg <dig> and the common ancestor of psg <dig> and psg <dig> and, ii.
between 'the relationship of psg <dig> to psg <dig> and psg11'.
this discrepancy is probably due to our inclusion of four extra psg n <dig> domain sequences, and the fact that the psg <dig> sequence  used by mclenachan et al.
<cit>  has been updated.
analysis of the mouse n <dig> domains indicates numerous contradictions in the alignments of the psg <dig>  psg <dig>  psg <dig>  psg <dig> and psg <dig> group .
in contrast, the n <dig> domains exhibit no discernable conflicts .
the a domain only showed contradiction within the psg <dig>  psg <dig>  psg <dig>  psg <dig> and psg <dig> group .
examination of the rat psg n <dig> domain exon alignments demonstrated minor contradictions between the common ancestor of psg <dig>  psg <dig> and psg <dig> and that of psg <dig> and psg <dig> .
in contrast to all the other psg n <dig> domains thus compared, the baboon psgs demonstrate considerable conflicting signals as demonstrated by the 'spider's web' appearance of the splitstree graph .
phylogenetic analysis indicates interspecific amino-terminal n domain conservation and identifies potential mouse / rat orthologues
few examples of orthologous relationships between psg sequences have been identified.
in order to compare the relationship between rodent and primate amino-terminal n domain exon coding sequences, an nj tree was produced .
the tree was generated from clustalx alignments of nucleotide sequences, with bootstrapping  <dig> times to test the reliability of branches.
the human and baboon n sequences formed one distinct cluster, the mouse and rat n <dig> sequences formed a second, the mouse n <dig> domains formed a third and the mouse n <dig> domains formed a fourth.
of particular interest was the split between the ancestral n-type domain and the common ancestor of the n <dig> and n <dig> domains.
the confidence of this split was 93% and demonstrates that the mouse n <dig> domains are more closely related to primate n domains than to the mouse n <dig> and n <dig> domains.
a similar comparison of the entire set of mouse and human psg domains confirmed that the interspecific n domain clustering is unique because the human psg a <dig> and a <dig> domains segregated into distinct branches  and the b <dig> domains cluster on a distinct branch .
mouse and rat psg gene coding sequences were analysed using an nj plot which highlighted four putative orthologous relationships, as follows: rat psg <dig> and mouse psg24; rat psg <dig> and mouse psg29; rat psg <dig> and mouse psg32; rat psg <dig> and mouse psg <dig> .
there is also distinct branching of rat psg <dig> with mouse psg <dig> and psg <dig>  the orthologous relationship is also supported for psg <dig> and psg <dig> because both contain five n domains.
psg n domain sequences are generally conserved but alignments reveal specific regions that may be diverging
the crystal structure of mouse ceacam <dig>  has been resolved  <cit> .
comparison of the mouse psg n <dig> domains identifies the predicted β-sheet-forming cfg β-strands as the most variable regions of the n domains .
the cfg face of ceacam n domains has been shown to interact with pathogens and mammalian proteins .
within box  <dig> and box  <dig>  there is considerable variation between mouse n <dig> domains, which is illustrated quantitatively using dayhoff charts .
positive dayhoff scores and generally low standard deviations indicate good conservation of mouse psg n <dig> domains , and even stronger conservation of human psg n domains .
the latter may be explained by homogenisation of human psg gene sequences  <cit> .
dayhoff score analysis using comparisons of all mouse n <dig> domain versus all human n domain clustalx aligned sequences gives an indication, at the amino acid level, of the general pattern of evolution of these domains since the rodent / primate divergence .
again, the majority of residues exhibit good conservation, and relatively little variability is observed between pair-wise comparisons particularly with regard to residues that are involved in protein folding.
the reduction in size of box  <dig> in fig.
<dig> and fig.
<dig> is explained by deletions of mouse dna sequences, requiring exclusion of the corresponding amino acids from the analysis.
to gain further insight into mouse psg n domain exon evolution, the n <dig>  n <dig> and n <dig> domain exons of mouse psg genes , the n <dig> domain exons of rat psg genes  and the n domain exons of human psg genes  were analysed in the following comparisons: mn <dig> vs mn2; mn <dig> vs mn3; mn <dig> vs mn3; mn <dig> vs rn1; mn <dig> vs hn.
synonymous  and non-synonymous  substitutions per synonymous and non-synonymous site, respectively, were determined in each case for all combinations of psg gene pairwise comparisons, and box and whisker plots were generated from the data .
the majority of data points derived from individual comparisons lie under the 45° line of equivalence where dn = ds, and most variation in the comparisons lies within the values of ds .
when the data are presented as box and whisker plots, the values are indicative of conservation, with median values ranging from  <dig>  –  <dig>  .
the higher values for median dn/ds in the mn <dig> vs rn <dig> comparison appear to be the result of a tighter ds distribution as observed in fig.
11a, with values not exceeding one substitution per synonymous site in any pairwise comparison.
in view of the sequence variations in the cfg face, which are visible in alignments , against a background of overall conservation, as estimated from dn/ds analysis, we sought to determine whether the dn/ds values were higher in the cfg face than the abed face of the n <dig> domain.
nucleotide sequence alignments were generated using all mouse psg n <dig> domain exons , and the nucleotides present in the three sections comprising the cfg face  were separated from those comprising the abed face.
the two new sets of data were analysed individually to determine mean dn and ds values from pairwise comparisons of all sequences within each dataset .
a plot of dn vs ds for the abed face of the mouse n <dig>  n <dig> and n <dig> domains  demonstrates a distribution of pairwise-alignment data points which overwhelmingly lie below the line of equivalence.
however, a similar plot generated from analysis of the cfg face has data points distributed approximately equally on both sides of the line of equivalence .
this is due predominately to a higher number of non-synonymous substitutions.
the values of dn/ds obtained for the cfg face in the n <dig>  n <dig> and n <dig> domains of the mouse and the n <dig> domain of the rat are all significantly greater than the values obtained for the abed face .
the dn/ds values obtained for the mouse n <dig>  n <dig> and n <dig> domain cfg faces equal or exceed  <dig> , with the highest median value of  <dig>  observed in the n <dig> domain.
the rat n <dig> domains are more conserved, with dn/ds values derived from both the cfg and abed faces under  <dig>  on average.
evidence of conservation of rgd-like motifs in mouse n <dig> domains
within box  <dig> of the cfg face  there is evidence of conservation of putative integrin-interacting rgd-like motifs in the mouse n <dig> domain, which may have functional significance.
to investigate this possibility further, a survey of all mouse, rat, baboon and human psg rgd, and related, motifs was compiled .
extant primate and rodent psg rgd-like motifs are linked in sequence space by an rgd motif encoded by the sequence cga gga gat which, incidentally, is not observed in any of the extant psg coding sequences.
the most commonly observed motif, rgd, is encoded by cga ggt gat, and the majority of variants are closely related to this sequence.
in rodents, rge and hge are the most commonly observed motifs.
however, the ngk motif, which is not an rgd-like motif as we have defined it, is well represented, and is separated in sequence space from hge by a transition and a transversion.
of the seventeen aligned mouse psg n <dig> domain exon sequences, 53% possess a tri-peptide at the site of the rgd-like motif belonging to the rgd-like 5-1- <dig> tri-group .
for comparative purposes, tri-groups were determined for tri-peptide motifs at fifty random positions within the alignment.
the number of most commonly represented tri-groups at each position was expressed as a percentage of the number of aligned sequences, and the mean and standard deviation was determined to indicate the mean maximal tri-group representation for the  <dig> random alignment positions.
the control value obtained was  <dig>  ±  <dig> %; the value of 53% of 5-1- <dig> tri-groups at the rgd site therefore lies within the control range, albeit  <dig> % below the mean value.
however, a more revealing statistic is derived from aligning the mouse n <dig> domains with the mouse n <dig> and n <dig> domains , compared to aligning the mouse n <dig> domains with the human n domains.
in the former comparison  the most commonly represented tri-group is 4-2- <dig>  with 27% representation.
this tri-group is not rgd-like and its representation is lower than the mean maximal tri-group representation of  <dig>  ±  <dig> % determined for fifty random alignment positions.
however, when the mouse n <dig> domain is aligned with the human n domain, the most commonly represented tri-group is the rgd-like 5-1- <dig> group which has 59% representation, comparable to the mean maximal tri-group representation of  <dig>  ±  <dig> %.
discussion
we recently collated the full-length coding sequences of the entire mouse psg gene family  <cit> .
in the present study we aimed to identify evolutionary signals embedded in psg gene and psg protein sequences to determine whether psg protein function has diverged between the rodent and primate lineages, and to attempt to understand the reasons for the independent expansions of rodent and primate psg gene families.
mouse and human psg protein amino-terminal n domains exhibit different patterns of evolution.
mclenachan et al.
<cit>  analysed the evolution of a subset of human psgs using split decomposition analysis and found, in individual comparisons of n, a <dig>  b <dig> and c domain exons, strong contradictions in alignments, which they suggested was due to gene conversion and/or positive selection.
our similar analysis of an expanded set of human psg sequences revealed a detectable, but less marked, degree of homogenisation.
analysis of mouse n and a domain exons showed that, in general, there is less evidence of purifying selection compared to the human, although there are examples of gene conversions as described previously for the closely related psg <dig> and psg <dig> genes  <cit> .
detailed analysis of alignments using plots of dayhoff scores confirmed the difference between mouse and human n domain evolution.
using dn/ds analysis for interspecies comparisons, we found that the psg protein amino-terminal n and n <dig> domains are relatively conserved, consistent with conservation of function in rodents and primates.
however, inspection of mouse psg n <dig> domain alignments, and scrutiny of corresponding dayhoff scores, revealed regions of apparently poor conservation.
these regions correspond to the cfg face within the n <dig> domain of ceacam <dig>  in the ceacam family, the cfg face interacts with pathogens and mammalian proteins.
comparisons of dn/ds values obtained from the cfg and abed faces of mouse n <dig>  n <dig> and n <dig> domains confirmed that the cfg face has evolved more rapidly than the abed face in all three domains.
the greatest effect was observed in the n <dig> domain exon with a doubling of the dn/ds ratio in the cfg face compared with the abed face.
the dn/ds ratio of  <dig>  suggests weak positive selection on the cfg face of the n <dig> domain.
the increase in the dn/ds ratio appears to be mainly due to an increase in the dn value, indicative of diversification.
the high dn/ds values for the cfg face in the n <dig> and n <dig> domains, which are not known to interact with ligands, could be due to a low contribution of these sequences to the structural integrity of the igv-like domain.
interestingly, the rat n <dig> domain cfg face does not appear to have evolved as rapidly as the mouse n <dig> domain, with a dn/ds ratio of  <dig> .
this observation, combined with the relatively smaller number of psg genes identified in the rat  and the higher level of gene homogenisation implied by split decomposition analysis suggests that the rat psg gene family has not expanded or diversified as extensively as the mouse.
however, we cannot exclude the possibility that further rat psg genes may yet be identified because there may be under-representation in the wgs database  <cit> .
notwithstanding this possibility, there has clearly been ongoing turnover of the psg gene family in all of the lineages analysed, as there are no known human orthologues of rat and mouse psgs, and only four potential orthologous relationships between known rat and mouse psgs.
these findings suggest partial conservation of psg n domain function across rodent and primate lineages.
however, the relaxed constraint on the cfg face of mouse psgs suggests diversification of binding partners or modification of existing ligand-binding kinetics, analogous to the ceacams.
this observation receives experimental support from the recent observation that treatment of mouse macrophages in vitro with recombinant mouse psg17n, or human psg <dig> or psg <dig>  induces cytokine expression; however, only in the case of mouse psg17n does this depend on cd <dig> receptor expression  <cit> .
divergence of psg function is also suggested by differences in the level and developmental timing of expression of different mouse psgs  <cit> , expansion of n domain number in psg <dig>  psg <dig> and psg <dig>  <cit> , and loss of secretory signals in psg <dig> and in the brain-specific splice variant of psg <dig>
as noted above, the only psg receptor identified to date is the integrin-associated tetraspanin, cd <dig>  which binds the n <dig> domain of mouse psg <dig> but not, apparently, to human psgs  <cit> .
however, a peptide containing the rgd motif from the human psg <dig> n domain binds to a receptor on a promonocytic cell line suggesting that some human psgs may effect their functions through an integrin-type receptor  <cit> .
in this context, the high frequency of the rgd motif on an exposed loop in primate psg n domains  may be significant.
rodent psg n <dig> domains do not have an rgd motif, but have a high frequency of the rgd-like motifs rge, hge and hae on the cfg face.
under the null hypothesis that these motifs are unlikely to underpin structural integrity of the n <dig> domain and are therefore free of constraint, our analysis reveals evidence of unexpected conservation of rgd-like motifs in the n <dig> domain, which have been lost in the n <dig> and n <dig> domains.
given the high transition and transversion rates in the n <dig> domain and the fact that the mouse n <dig>  n <dig> and n <dig> domains share a common ancestor after the divergence of the rodent / primate lineages, the conservation of rgd-like motifs exclusively in the n <dig> domain may have functional significance.
we note that the rge motif in the context of the poem protein induced apoptosis of mc3t3-e <dig> cells in vitro  <cit> .
we speculate that certain rge or rge-like motifs may elicit weak cell attachment, followed by apoptosis – a combination of properties, reminiscent of snake venom disintegrins  <cit> , that could have important functional implications in the context of the extensive tissue remodelling that occurs during placentation  <cit> .
in summary, our data are consistent with experimental evidence indicating functional convergence of rodent and primate psgs, in spite of the independent expansions of the gene families in the two lineages.
in the context of parent-offspring conflict, the homogenisation of human psg sequences is consistent with the theory that placental hormones encoded by multigene families are monofunctional and selected for high expression, possibly due to coevolution with physiologically conflicting maternal mechanisms  <cit> .
however, the evidence for positive selection on the cfg face of the n <dig> domain implies divergent evolution of rodent psgs.
allied to the evidence for functionality of putative integrin-interacting rgd-like motifs in rodents, a scenario can be envisaged whereby the different rgd-like motifs observed in human and baboon psgs also suggest some degree of functional divergence in these species.
CONCLUSIONS
our analysis provides evidence for conservation of rodent and primate psg amino-terminal n domains, with ongoing independent expansion of the gene families in the two lineages.
there has been some diversification of the cfg face of mouse n <dig> domains, a region that includes putative integrin-interacting rgd-like motifs.
our analysis provides reassurance that the mouse psg gene family is a suitable model system for the analysis of human psg gene function.

 in tandemly repeated gene families, in which all members share a common function, there is a tendency for concerted evolution that is characterised by homogenisation of gene sequences. classical examples include the histone and ribosomal rna genes. in such cases the expansion of gene families is driven by selection for high expression. concerted evolution is generally maintained by unequal crossover, intergenic gene conversion or other illegitimate recombination mechanisms. conversely, there are multigene families whose members encode diverse functions e.g. genes encoding immunoglobulin, t cell receptor and major histocompatibility complex proteins. such diversity occurs when there is less homogenisation than mutation, due to the evolution of specific programmed mutational mechanisms. in addition, more complex modes exist; for example, the immunoglobulin heavy-chain variable-region genes encode proteins with identical functions, but exhibit little concerted evolution. instead, their evolution is governed by divergence and a birth-and-death process of gene duplication and dysfunctioning mutations. similar to other families of highly expressed trophoblast-specific genes such as the pregnancy-associated glycoproteins, the pregnancy-specific glycoproteins, which are the most abundant foetal proteins in the maternal bloodstream during human late pregnancy, are encoded by multiple tandemly arrayed genes. the psg family of glycoproteins, with the related cea-related cell adhesion molecule proteins, are part of the immunoglobulin superfamily. the ig domain structure of the human and mouse psgs differs, as follows: human psgs contain one v-like ig domain, c2-like ig domains and relatively hydrophilic tails, with domain arrangements classified as type i, type iia, type iib, type iii and type iv. in contrast, mouse psgs typically have three or more n domains followed by a single a domain. the common ancestor of rodent and primate psgs and ceacams was probably similar to ceacam which is the only cea family member with an identical gene structure in the human, rat and mouse that encodes all types of extracellular domains present in ceacam and psg proteins. the time of initial gene duplication is estimated at myr, approximately the time of rodent-primate divergence. the independent expansion of human and mouse psg gene families occurred through further gene duplication and exon shuffling events. the independent expansion of psg gene families in rodents and primates indicates convergent evolution, implying that psg function is conserved. these events can be interpreted in the context of evolutionary theories of parent-offspring and inter-sibling conflicts that promote transcriptional'arms races' leading to high expression of trophoblast-specific genes that influence maternal investment in offspring. in one scenario, duplicated psg genes are selected because they increase effective psg dosage, thereby enhancing an effect on maternal investment in offspring. in this context, it is noteworthy that human psg n domains contain putative integrin-binding'rgd' motifs that are proposed to mediate cell interactions with the extracellular matrix and immune cells. such psg-mediated functions could potentially influence trophoblast invasion or maternal immune cell function. however, not all human, and none of the mouse, psgs contain an rgd motif, suggesting that, if human rgd motifs are functionally significant, there has been diversification of function of some human, and all mouse, psgs, relative to a putative rgd-containing ancestor. in the context of parent-offspring conflict, such divergence might reflect co-evolution of psgs and their receptors, similar to the co-evolution of ligand / receptor pairs observed in host-pathogen interactions. in this study, we sought to analyse psg evolution to determine the extent and patterns of rodent and primate psg sequence divergence by analysing intraspecific and interspecies dna substitution rates in psg coding regions. we also sought evidence in support of functionality of rgd and rgd-like tri-peptide motifs in psg amino-terminal effector domains.  pairwise comparisons of all 4-domain mouse psg with all 4-domain human psg full-length amino acid sequences indicates conservation of the amino-terminal n domain with the exception of mouse psg psg and psg and human psg and psg all psgs for which full length sequences are available have a structure based on four ig-like domains and a leader sequence that is cleaved during post-translational processing. the only type of domain found in all rodent and primate psgs is the n domain located at the amino terminus. indeed, this domain is shared by all members of the extended cea family, suggesting that it may contain important functional motifs. we sought to test this hypothesis with respect to psg function, by analysing both full-length psg sequences and selected domains of possible functional importance. alignments of full-length 4-domain human and mouse psg protein sequences were generated with clustalx, followed by pairwise comparisons of all mouse sequences with all human sequences. mean dayhoff pam log scores were calculated for each alignment position and grouped by domain. the scores within each of the four domains were then visualised using box and whisker plots. the n domains exhibited significantly higher scores than the other three domains, with positive scores indicating conservation. there was no evidence of interspecies conservation of the other domains, which is unsurprising given the known lack of orthology between human a / mouse n human a / mouse n and human b / mouse a domain pairs. novel rat psg n domains identified by database searches rat n domain exon sequences were identified in ncbi and ensembl databases. three novel rat psg genes were identified and named psg psg and psg in keeping with accepted nomenclature. we also identified a novel psg splice variant with alternative leader and n domain exons, situated between the n and n domain exons of the published psg sequence. both blast and pattern matching methods retrieved the same rat psg genes from different databases; therefore we considered our search to be exhaustive. all rat psg genes were found to reside on contig nw_ and this was used for the prediction of remaining exons for each psg gene based on blast generated alignments with mouse psg gene sequences. the cds sequences of the novel predicted rat psg genes and psg splice variant are listed in additional file we used our predicted sequences in preference to the publicly available sequences in our analyses. splitstree analysis reveals relatively high contradiction in rat psg n domain alignments, compared to mouse following the preliminary identification of amino-terminal n domain conservation, we planned to use an evolutionary tree building approach to further examine inter-domain relationships in rodent and primate psgs. however, using split decomposition analysis, mclenachan et al., in their study of a subset of human psgs, concluded that it is not possible to accurately determine branch points in an evolutionary tree of human psgs. split decomposition analysis identifies contradictory relationships within alignment data; for example, there may be a pattern grouping psgx and psgy together, and another pattern grouping psgy and psgz together. this information is normally approximated when drawing evolutionary trees, however split decomposition is a non-approximation method that permits the building of trees with support indicated for relationships based on all patterns in the data. such analysis can therefore predict to a limited extent the occurrence of sequence homogenisation e.g. by gene conversion or positive selection. we performed split decomposition analysis on nucleotide sequences using the splitstree program on the individual domain exons of mouse psg genes. for a more complete analysis of n domains we also performed the analysis using rat n domain exons, all known human n domain exons and all known baboon n domain exons. we detected no conflicting signals for mouse psg n domain exons, in contrast to the human n domain exons. however, our results for human n domains differ from those obtained by mclenachan et al. because we observed only two contradictions: i. regarding the relationship of psg and psg to each other, and to their nearest neighbours psg and the common ancestor of psg and psg and, ii. between'the relationship of psg to psg and psg11'. this discrepancy is probably due to our inclusion of four extra psg n domain sequences, and the fact that the psg sequence used by mclenachan et al. has been updated. analysis of the mouse n domains indicates numerous contradictions in the alignments of the psg psg psg psg and psg group. in contrast, the n domains exhibit no discernable conflicts. the a domain only showed contradiction within the psg psg psg psg and psg group. examination of the rat psg n domain exon alignments demonstrated minor contradictions between the common ancestor of psg psg and psg and that of psg and psg. in contrast to all the other psg n domains thus compared, the baboon psgs demonstrate considerable conflicting signals as demonstrated by the'spider's web' appearance of the splitstree graph. phylogenetic analysis indicates interspecific amino-terminal n domain conservation and identifies potential mouse / rat orthologues few examples of orthologous relationships between psg sequences have been identified. in order to compare the relationship between rodent and primate amino-terminal n domain exon coding sequences, an nj tree was produced. the tree was generated from clustalx alignments of nucleotide sequences, with bootstrapping times to test the reliability of branches. the human and baboon n sequences formed one distinct cluster, the mouse and rat n sequences formed a second, the mouse n domains formed a third and the mouse n domains formed a fourth. of particular interest was the split between the ancestral n-type domain and the common ancestor of the n and n domains. the confidence of this split was 93% and demonstrates that the mouse n domains are more closely related to primate n domains than to the mouse n and n domains. a similar comparison of the entire set of mouse and human psg domains confirmed that the interspecific n domain clustering is unique because the human psg a and a domains segregated into distinct branches and the b domains cluster on a distinct branch. mouse and rat psg gene coding sequences were analysed using an nj plot which highlighted four putative orthologous relationships, as follows: rat psg and mouse psg24; rat psg and mouse psg29; rat psg and mouse psg32; rat psg and mouse psg. there is also distinct branching of rat psg with mouse psg and psg the orthologous relationship is also supported for psg and psg because both contain five n domains. psg n domain sequences are generally conserved but alignments reveal specific regions that may be diverging the crystal structure of mouse ceacam has been resolved. comparison of the mouse psg n domains identifies the predicted β-sheet-forming cfg β-strands as the most variable regions of the n domains. the cfg face of ceacam n domains has been shown to interact with pathogens and mammalian proteins. within box and box there is considerable variation between mouse n domains, which is illustrated quantitatively using dayhoff charts. positive dayhoff scores and generally low standard deviations indicate good conservation of mouse psg n domains, and even stronger conservation of human psg n domains. the latter may be explained by homogenisation of human psg gene sequences. dayhoff score analysis using comparisons of all mouse n domain versus all human n domain clustalx aligned sequences gives an indication, at the amino acid level, of the general pattern of evolution of these domains since the rodent / primate divergence. again, the majority of residues exhibit good conservation, and relatively little variability is observed between pair-wise comparisons particularly with regard to residues that are involved in protein folding. the reduction in size of box in fig. and fig. is explained by deletions of mouse dna sequences, requiring exclusion of the corresponding amino acids from the analysis. to gain further insight into mouse psg n domain exon evolution, the n n and n domain exons of mouse psg genes, the n domain exons of rat psg genes and the n domain exons of human psg genes were analysed in the following comparisons: mn vs mn2; mn vs mn3; mn vs mn3; mn vs rn1; mn vs hn. synonymous and non-synonymous substitutions per synonymous and non-synonymous site, respectively, were determined in each case for all combinations of psg gene pairwise comparisons, and box and whisker plots were generated from the data. the majority of data points derived from individual comparisons lie under the 45° line of equivalence where dn = ds, and most variation in the comparisons lies within the values of ds. when the data are presented as box and whisker plots, the values are indicative of conservation, with median values ranging from –. the higher values for median dn/ds in the mn vs rn comparison appear to be the result of a tighter ds distribution as observed in fig. 11a, with values not exceeding one substitution per synonymous site in any pairwise comparison. in view of the sequence variations in the cfg face, which are visible in alignments, against a background of overall conservation, as estimated from dn/ds analysis, we sought to determine whether the dn/ds values were higher in the cfg face than the abed face of the n domain. nucleotide sequence alignments were generated using all mouse psg n domain exons, and the nucleotides present in the three sections comprising the cfg face were separated from those comprising the abed face. the two new sets of data were analysed individually to determine mean dn and ds values from pairwise comparisons of all sequences within each dataset. a plot of dn vs ds for the abed face of the mouse n n and n domains demonstrates a distribution of pairwise-alignment data points which overwhelmingly lie below the line of equivalence. however, a similar plot generated from analysis of the cfg face has data points distributed approximately equally on both sides of the line of equivalence. this is due predominately to a higher number of non-synonymous substitutions. the values of dn/ds obtained for the cfg face in the n n and n domains of the mouse and the n domain of the rat are all significantly greater than the values obtained for the abed face. the dn/ds values obtained for the mouse n n and n domain cfg faces equal or exceed, with the highest median value of observed in the n domain. the rat n domains are more conserved, with dn/ds values derived from both the cfg and abed faces under on average. evidence of conservation of rgd-like motifs in mouse n domains within box of the cfg face there is evidence of conservation of putative integrin-interacting rgd-like motifs in the mouse n domain, which may have functional significance. to investigate this possibility further, a survey of all mouse, rat, baboon and human psg rgd, and related, motifs was compiled. extant primate and rodent psg rgd-like motifs are linked in sequence space by an rgd motif encoded by the sequence cga gga gat which, incidentally, is not observed in any of the extant psg coding sequences. the most commonly observed motif, rgd, is encoded by cga ggt gat, and the majority of variants are closely related to this sequence. in rodents, rge and hge are the most commonly observed motifs. however, the ngk motif, which is not an rgd-like motif as we have defined it, is well represented, and is separated in sequence space from hge by a transition and a transversion. of the seventeen aligned mouse psg n domain exon sequences, 53% possess a tri-peptide at the site of the rgd-like motif belonging to the rgd-like 5-1- tri-group. for comparative purposes, tri-groups were determined for tri-peptide motifs at fifty random positions within the alignment. the number of most commonly represented tri-groups at each position was expressed as a percentage of the number of aligned sequences, and the mean and standard deviation was determined to indicate the mean maximal tri-group representation for the random alignment positions. the control value obtained was ± %; the value of 53% of 5-1- tri-groups at the rgd site therefore lies within the control range, albeit % below the mean value. however, a more revealing statistic is derived from aligning the mouse n domains with the mouse n and n domains, compared to aligning the mouse n domains with the human n domains. in the former comparison the most commonly represented tri-group is 4-2- with 27% representation. this tri-group is not rgd-like and its representation is lower than the mean maximal tri-group representation of ± % determined for fifty random alignment positions. however, when the mouse n domain is aligned with the human n domain, the most commonly represented tri-group is the rgd-like 5-1- group which has 59% representation, comparable to the mean maximal tri-group representation of ± %. discussion we recently collated the full-length coding sequences of the entire mouse psg gene family. in the present study we aimed to identify evolutionary signals embedded in psg gene and psg protein sequences to determine whether psg protein function has diverged between the rodent and primate lineages, and to attempt to understand the reasons for the independent expansions of rodent and primate psg gene families. mouse and human psg protein amino-terminal n domains exhibit different patterns of evolution. mclenachan et al. analysed the evolution of a subset of human psgs using split decomposition analysis and found, in individual comparisons of n, a b and c domain exons, strong contradictions in alignments, which they suggested was due to gene conversion and/or positive selection. our similar analysis of an expanded set of human psg sequences revealed a detectable, but less marked, degree of homogenisation. analysis of mouse n and a domain exons showed that, in general, there is less evidence of purifying selection compared to the human, although there are examples of gene conversions as described previously for the closely related psg and psg genes. detailed analysis of alignments using plots of dayhoff scores confirmed the difference between mouse and human n domain evolution. using dn/ds analysis for interspecies comparisons, we found that the psg protein amino-terminal n and n domains are relatively conserved, consistent with conservation of function in rodents and primates. however, inspection of mouse psg n domain alignments, and scrutiny of corresponding dayhoff scores, revealed regions of apparently poor conservation. these regions correspond to the cfg face within the n domain of ceacam in the ceacam family, the cfg face interacts with pathogens and mammalian proteins. comparisons of dn/ds values obtained from the cfg and abed faces of mouse n n and n domains confirmed that the cfg face has evolved more rapidly than the abed face in all three domains. the greatest effect was observed in the n domain exon with a doubling of the dn/ds ratio in the cfg face compared with the abed face. the dn/ds ratio of suggests weak positive selection on the cfg face of the n domain. the increase in the dn/ds ratio appears to be mainly due to an increase in the dn value, indicative of diversification. the high dn/ds values for the cfg face in the n and n domains, which are not known to interact with ligands, could be due to a low contribution of these sequences to the structural integrity of the igv-like domain. interestingly, the rat n domain cfg face does not appear to have evolved as rapidly as the mouse n domain, with a dn/ds ratio of. this observation, combined with the relatively smaller number of psg genes identified in the rat and the higher level of gene homogenisation implied by split decomposition analysis suggests that the rat psg gene family has not expanded or diversified as extensively as the mouse. however, we cannot exclude the possibility that further rat psg genes may yet be identified because there may be under-representation in the wgs database. notwithstanding this possibility, there has clearly been ongoing turnover of the psg gene family in all of the lineages analysed, as there are no known human orthologues of rat and mouse psgs, and only four potential orthologous relationships between known rat and mouse psgs. these findings suggest partial conservation of psg n domain function across rodent and primate lineages. however, the relaxed constraint on the cfg face of mouse psgs suggests diversification of binding partners or modification of existing ligand-binding kinetics, analogous to the ceacams. this observation receives experimental support from the recent observation that treatment of mouse macrophages in vitro with recombinant mouse psg17n, or human psg or psg induces cytokine expression; however, only in the case of mouse psg17n does this depend on cd receptor expression. divergence of psg function is also suggested by differences in the level and developmental timing of expression of different mouse psgs, expansion of n domain number in psg psg and psg, and loss of secretory signals in psg and in the brain-specific splice variant of psg as noted above, the only psg receptor identified to date is the integrin-associated tetraspanin, cd which binds the n domain of mouse psg but not, apparently, to human psgs. however, a peptide containing the rgd motif from the human psg n domain binds to a receptor on a promonocytic cell line suggesting that some human psgs may effect their functions through an integrin-type receptor. in this context, the high frequency of the rgd motif on an exposed loop in primate psg n domains may be significant. rodent psg n domains do not have an rgd motif, but have a high frequency of the rgd-like motifs rge, hge and hae on the cfg face. under the null hypothesis that these motifs are unlikely to underpin structural integrity of the n domain and are therefore free of constraint, our analysis reveals evidence of unexpected conservation of rgd-like motifs in the n domain, which have been lost in the n and n domains. given the high transition and transversion rates in the n domain and the fact that the mouse n n and n domains share a common ancestor after the divergence of the rodent / primate lineages, the conservation of rgd-like motifs exclusively in the n domain may have functional significance. we note that the rge motif in the context of the poem protein induced apoptosis of mc3t3-e cells in vitro. we speculate that certain rge or rge-like motifs may elicit weak cell attachment, followed by apoptosis – a combination of properties, reminiscent of snake venom disintegrins, that could have important functional implications in the context of the extensive tissue remodelling that occurs during placentation. in summary, our data are consistent with experimental evidence indicating functional convergence of rodent and primate psgs, in spite of the independent expansions of the gene families in the two lineages. in the context of parent-offspring conflict, the homogenisation of human psg sequences is consistent with the theory that placental hormones encoded by multigene families are monofunctional and selected for high expression, possibly due to coevolution with physiologically conflicting maternal mechanisms. however, the evidence for positive selection on the cfg face of the n domain implies divergent evolution of rodent psgs. allied to the evidence for functionality of putative integrin-interacting rgd-like motifs in rodents, a scenario can be envisaged whereby the different rgd-like motifs observed in human and baboon psgs also suggest some degree of functional divergence in these species.  our analysis provides evidence for conservation of rodent and primate psg amino-terminal n domains, with ongoing independent expansion of the gene families in the two lineages. there has been some diversification of the cfg face of mouse n domains, a region that includes putative integrin-interacting rgd-like motifs. our analysis provides reassurance that the mouse psg gene family is a suitable model system for the analysis of human psg gene function. 
phylogenetic tree analyses indicate that the primate n domains and the rodent n domains exhibit a higher degree of conservation than that observed in a comparison of the mouse n and n domains, or mouse n and n domains. relative conservation of primate n domains and rodent n domains suggests that, despite independent gene family expansions and structural diversification, mouse and human psgs retain conserved functions. the high non-synonymous substitution rates observed in the cfg face of the mouse n domain, within a context of overall conservation, suggests divergence of function of mouse psgs. we analysed the evolution of the mouse psg sequences, and compared them to rat, human and baboon psgs to attempt to understand the evolution of this complex gene family. in contrast to most primate psg n domains, rodent psg n domains do not contain an rgd tri-peptide motif, but do contain rgd-like sequences, which are not conserved in rodent n and n domains. partial conservation of rgd and rgd-like tri-peptides in primate and rodent n and n domains, respectively, supports a role for these motifs in psg function. rodent and primate pregnancy-specific glycoprotein gene families have expanded independently from a common ancestor and are expressed virtually exclusively in placental trophoblasts.
 rodent and primate pregnancy-specific glycoprotein gene families have expanded independently from a common ancestor and are expressed virtually exclusively in placental trophoblasts. however, within each species, it is unknown whether multiple paralogs have been selected for diversification of function, or for increased dosage of monofunctional psg. we analysed the evolution of the mouse psg sequences, and compared them to rat, human and baboon psgs to attempt to understand the evolution of this complex gene family.  phylogenetic tree analyses indicate that the primate n domains and the rodent n domains exhibit a higher degree of conservation than that observed in a comparison of the mouse n and n domains, or mouse n and n domains. compared to human and baboon psg n domain exons, mouse and rat psg n domain exons have undergone less sequence homogenisation. the high non-synonymous substitution rates observed in the cfg face of the mouse n domain, within a context of overall conservation, suggests divergence of function of mouse psgs. the rat psg family appears to have undergone less expansion than the mouse, exhibits lower divergence rates and increased sequence homogenisation in the cfg face of the n domain. in contrast to most primate psg n domains, rodent psg n domains do not contain an rgd tri-peptide motif, but do contain rgd-like sequences, which are not conserved in rodent n and n domains.  relative conservation of primate n domains and rodent n domains suggests that, despite independent gene family expansions and structural diversification, mouse and human psgs retain conserved functions. human psg gene family expansion and homogenisation suggests that evolution occurred in a concerted manner that maintains similar functions of psgs, whilst increasing gene dosage of the family as a whole. in the mouse, gene family expansion, coupled with local diversification of the cfg face, suggests selection both for increased gene dosage and diversification of function. partial conservation of rgd and rgd-like tri-peptides in primate and rodent n and n domains, respectively, supports a role for these motifs in psg function. 