BACKGROUND
mammalian apobec <dig>  proteins have the capacity to potently inhibit the replication of a diverse set of reverse-transcribing mobile genetic elements  <cit> .
susceptible exogenous retroelements include lentiviruses , alpharetroviruses , betaretroviruses , gammaretroviruses , deltaretroviruses , foamy viruses and the hepadnavirus hbv .
susceptible endogenous retroelements include the yeast retrotransposons ty <dig> and ty <dig>  the murine endogenous retroviruses musd and pmv, the murine intracisternal a particle , the porcine endogenous retrovirus perv and, potentially, extinct elements such as chimpanzee pterv <dig> and human herv-k, all of which require long-terminal repeats  for replication  <cit> .
in addition, some a <dig> proteins can also inhibit l <dig> and its obligate parasite alu, retrotransposons that replicate by integration-primed reverse transcription  <cit> .
an overall theme is emerging in which most – if not all – retroelements can be inhibited by at least one a <dig> protein.
however, it is now equally clear that the retroelements of any given species have evolved mechanisms to evade restriction by their host's a <dig> protein.
for instance, hiv and siv use vif to trigger a ubiquitin-dependent degradation mechanism, foamy viruses use a protein called bet for an imprecisely defined inhibitory mechanism and some viruses such as mpmv, htlv and mlv appear to employ a simple avoidance mechanism .
thus, it appears that all 'successful' retroelements have evolved strategies to resist restriction by the a <dig> proteins of their hosts.
the defining feature of the a <dig> family of proteins is a conserved zinc-coordinating dna cytosine deaminase motif, h-x1-e-x25–31-c-x2–4-c .
the a <dig> z domains can be grouped into one of three distinct phylogenetic clusters – z <dig>  z <dig> or z <dig>  .
the z-based classification system, proposed originally by conticello and coworkers  <cit> , was revised recently through a collaborative effort  <cit> .
from hereon, the new a <dig> nomenclature system will be used.
z <dig> and z <dig> proteins have a sw-s/t-c-x2–4-c motif, whereas z <dig> proteins have a tw-s-c-x2-c motif.
z <dig> and z <dig> proteins can be further distinguished by h-x1-e-x5-x-v/i and h-x1-e-x5-w-f motifs, respectively.
z <dig> proteins also have a unique isoleucine within a conserved riy motif located c-terminal to the zinc-coordinating residues.
at least one protein of each of the z classes and nearly all identified a <dig> proteins have exhibited single-strand dna cytosine deaminase activity.
for instance, human a3f, a3g and a3h possess catalytically competent z <dig>  z <dig> and z <dig> domains, respectively .
we previously reported a double-domain a3z2-z <dig> gene  from the artiodactyls, sheep , cattle  and pigs   <cit> .
however, the fact that mammals have varying numbers of a <dig> genes  led us to wonder whether additional a <dig> genes would be present in artiodactyls.
to address this point and to learn more about the evolution and functionality of a <dig> genes in mammals, we sequenced and characterized the full a <dig> repertoire of sheep and pigs.
here, we demonstrated that sheep and cattle actually have three a <dig> genes, a3z <dig>  a3z <dig> and a3z <dig>  with a conserved potential to encode at least four active and distinct proteins .
we further showed that porcine lineage has a deletion of the orthologous a3z <dig> gene and the capacity to encode only three proteins.
these data enabled us to deduce that the common ancestor of artiodactyls and primates possessed an a <dig> repertoire consisting of three z domains .
our data further suggested an evolutionary model in which most of the human a <dig> gene expansion occurred more than  <dig> million years ago, during early primate evolution and possibly even associated with pathogen-induced population bottlenecks.
RESULTS
sheep and cattle have three a <dig> genes with a z1-z2-z <dig> organization
we previously used degenerate pcr, race and database mining to identify a cdna for sheep a3z2-z <dig> .
however, because humans have seven a <dig> genes and mice have only one, we postulated that artiodactyls such as sheep and cattle might have an intermediate number.
to address this possibility unambiguously, we sequenced the entire sheep a <dig> genomic locus.
first, a sheep a3z2-z <dig> cdna was hybridized to a sheep bac library to identify corresponding genomic sequence.
second, hybridization-positive bacs were screened by pcr for those that also contain the conserved flanking genes cbx <dig> and cbx <dig>  one bac was identified that spanned the entire cbx <dig> to cbx <dig> region, and it was sheared, subcloned, shotgun sequenced, assembled and analyzed .
dna sequence analyses revealed that the sheep genomic locus contained another a <dig> gene between cbx <dig> and a3z <dig> .
this gene was called a3z <dig>  because it had sequence characteristics of a z1-type a <dig> protein.
we therefore concluded that sheep have three a <dig> genes and, importantly, that each mammalian a <dig> z-type was present.
this conclusion was supported by the bovine genome assembly, which was released during the course of our studies and showed that cattle also have a sheep-like, three gene a <dig> repertoire .
the predicted a3z <dig> coding sequences of sheep and cattle are 86% identical, consistent with the fact that these two ruminant artiodactyls shared a common ancestor approximately 14– <dig> million years ago   <cit> .
the pig has two a <dig> genes with a z2–z <dig> organization
pcr reactions failed to identify an a3z1-like gene in pigs.
since pigs and cattle/sheep last shared a common ancestor approximately 70– <dig> mya  <cit> , we considered the possibility that the negative pcr result was not a technical failure and that pigs might actually have a different a <dig> repertoire.
again, to unambiguously address this possibility, the pig a <dig> genomic locus was sequenced in entirety.
a porcine bac library was probed with pig a3z2-z <dig> cdna and two hybridization-positive bacs were shotgun sequenced.
the sequence assemblies revealed that pigs have only two a <dig> genes a3z <dig> and a3z <dig> between cbx <dig> and cbx <dig> .
the cattle, sheep and pig a <dig> locus genomic sequences were compared using dotplot analyses .
a  <dig> kb discontinuity was detected between the cow and the pig sequences.
the sheep and pig genomic sequences aligned similarly.
multiple  retroelements were found to flank a3z <dig> in sheep and cattle.
two were particularly close to the ends of the  <dig> kb a3z <dig> region, a line/l <dig> and a sine/trna-glu.
it is possible that one of these elements mediated a simple direct repeat recombination event that deleted the a3z <dig> region in pigs.
however, we were unable to identify such a causative retroelement in the pig genomic sequence.
to begin to address whether the potential a3z <dig> deletion in pigs occurred recently  or whether it was more ancient, we asked whether a non-domesticated, distant relative of the pig, the collared peccary , has an a3z <dig> gene.
lineages leading to present-day domesticated pigs and the peccary diverged approximately 25– <dig> mya  <cit> .
a pan-species, a3z <dig> pcr primer set was developed and used in these experiments.
in contrast to human, african green monkey, horse, cow and sheep genomic dna which yielded a 250– <dig> bp z1-specific pcr products confirmable by dna sequencing, the genomic dna of domesticated pig, the collared peccary, mice and opossum failed to yield a product even after  <dig> cycles .
a highly conserved gene, aldoa, was used as a pcr control to demonstrate the integrity of the genomic dna samples.
interestingly, z <dig> pcr product sequencing and recently released est sequences revealed that the related hoofed mammal, the horse, also has a z1-type a <dig> gene .
two-'toed' hoofed animals such as sheep, cattle and pigs belong to the ungulate order artiodactyla , and one-'toed' hoofed animals such as horses belong to the ungulate order perissodactyla .
since these two ungulate orders diverged approximately 80– <dig> mya  <cit>  and both have species with z1-type a <dig> genes, it is highly likely that the common ancestor also had an a3z <dig> gene .
it is therefore highly unlikely that an a3z <dig> gene independently appeared at the same genomic position in artiodactyls, perissodactyls and primates.
rather, all of the data support a model where a common ancestor of the domesticated pig and the collared peccary experienced a  <dig> kb deletion that resulted in the loss of a3z <dig> .
furthermore, since artiodactyls, perissodactyls and humans shared a common ancestor approximately 80– <dig> mya  <cit> , the presence of z1-type a <dig> genes in both the primate and the artiodactyl limbs of the mammalian tree is also most easily explained by common ancestry.
thus, our combined datasets indicated that this ancestor possessed a full a <dig> z repertoire, with one of each type of z domain , the minimal substrate required to evolve into the present-day eleven z domain human a <dig> locus .
the artiodactyl a3z <dig> and a3z <dig> genes combine to encode  <dig> distinct mrnas and proteins
we previously characterized several activities of the double-domain a3z2-z <dig> protein from cattle, sheep and pigs  <cit> .
while re-confirming the 5' and 3' ends of the a3z2-z <dig> transcripts by race, we discovered two interesting variants that were conserved between these three species.
first, using sheep and cattle pbmc or cell line cdna , 3' race frequently produced a smaller than expected fragment.
the sequence of this fragment indicated the existence of a short  <dig> bp transcript due to premature termination  <dig> or  <dig> nucleotides into intron  <dig> for sheep and cattle, respectively .
this truncated transcript was readily amplified from sheep and cattle pbmcs and represented by existing est sequences .
therefore, this novel transcript was predicted to result in a single-domain z <dig> protein, a3z <dig>  with a length of  <dig> and  <dig> amino acids for sheep and cattle, respectively .
a pig a3z <dig> transcript was also identified by race and est sequences but, in contrast to sheep and cattle, exon  <dig> was spliced to two additional exons before terminating prematurely .
as a consequence, pig a3z <dig> was predicted to be  <dig> amino acids.
these analyses indicated that artiodactyls have the capacity to express a single domain a3z <dig> protein, in contrast to what we had deduced previously  <cit> .
second, 5' race data and cattle and pig est sequences suggested that yet another mechanism served to broaden the coding potential of the artiodactyl a <dig> locus .
several transcripts appeared to originate from the region immediately upstream of a3z <dig>  whereas our prior studies had only detected transcripts originating upsteam of a3z <dig>  <cit> .
a comparison of cdna and genomic sequences revealed the presence of an exon in this location .
transcripts initiating here produced  <dig> ,  <dig>  or  <dig>  nucleotide messages.
the resulting a3z <dig> protein was predicted to be  <dig> residues for sheep and cattle and  <dig> for pigs .
the a3z <dig> mrna data strongly suggested the existence of an internal promoter.
this was supported by cis-regulatory element prediction algorithms, which identified a conserved interferon-stimulated response element  upstream of a3z <dig>  as well as upstream of a3z <dig> .
these isres were strikingly similar to those located in the promoter regions of human a3de, a3f and a3g, supporting the likelihood that interferon-inducibility is a conserved feature of many mammalian a <dig> genes .
these putative isres significant similarity to functional elements in known interferon-inducible genes isg <dig> and isg <dig>  <cit> .
we also predicted binding sites for another well-known transcription factor, sp <dig>  upstream of the a3z <dig> transcription start site.
this activator was also recently reported for human and cat a <dig> genes .
together with our previous data on the double domain a <dig> protein of these artiodactyl species, a3z2-z <dig>  <cit> , these expression and promoter data revealed that two single-domain a <dig> genes can readily encode at least three distinct proteins – a3z <dig>  a3z <dig> and a3z2-z <dig>  a similar strategy may also be used by rodents, which also have an a <dig> gene with z <dig> and z <dig> domains.
a similar modularity was reported recently for the cat a <dig> locus, where two single domain a <dig> genes combined to produce a functional double-domain a <dig> protein  <cit> .
we suggest that combining single-domain a3s to yield functionally unique double-domain proteins may be a general strategy used by many mammals to bolster their a3-dependent innate immune defenses.
all four artiodactyl a <dig> proteins – a3z <dig>  a3z <dig>  a3z <dig> and a3z2-z <dig> – elicit dna cytosine deaminase activity
all currently described a <dig> proteins have elicited single-strand dna cytosine to uracil deaminase activity in one or more assays .
for instance, we showed that the artiodactyl a3z2-z <dig> proteins could catalyze the deamination of e. coli dna and retroviral cdna  <cit> .
however, catalytic mutants indicated that only the n-terminal z <dig> domain of cow, sheep and pig a3z2-z <dig> was active.
this observation contrasted with data for the double-domain human a3b, a3f and a3g proteins, where the c-terminal domain clearly contains the dominant active site .
nevertheless, these datasets suggested that the double-domain a <dig> proteins have separated function, with one domain predominantly serving as a catalytic center and the other as a regulatory center.
however, a recent study with human a3b indicated that both z domains have the potential to be catalytically active  <cit> .
it was therefore reasonable to ask whether the single domain a3z <dig> and a3z <dig> proteins of artiodactyls would be capable of dna cytosine deamination in an e. coli-based activity assay.
elevated frequencies of rifampicin-resistance  mutations in e. coli provide a quantitative measure of the intrinsic a <dig> protein dna cytosine deaminase activity .
in contrast to full-length cow a3z2-z <dig>  which triggered a modest 2-fold increase in the median rifr mutation frequency over the vector control, non-induced levels of cow a3z <dig> caused a large 50-fold increase .
the ptrc99-based vector used in these studies has an iptg-inducible promoter, and induced levels of cow a3z <dig> prevented e. coli growth, presumably through catastrophic levels of dna cytosine deamination.
in contrast, induced levels of sheep or pig a3z <dig> proteins were not lethal, but their expression also caused significant increases in the median rifr mutation frequency .
thus, as anticipated by our prior studies, the a3z <dig> proteins of cattle, sheep and pigs showed intrinsic dna cytosine deaminase activity.
we were therefore surprised that induced levels of the cow single-domain protein a3z <dig> also caused a significant 4-fold increase in the median rifr mutation frequency .
this result contrasted with the related z <dig> protein of humans, a3h, which appeared inactive in this assay .
however, it is worth noting that other z3-type a <dig> proteins, a different human a3h variant, african green monkey a3h, rhesus macaque a3h and cat a3z <dig> , all showed evidence for dna deaminase activity in the e. coli-based mutation assay and/or in retrovirus infectivity assays  <cit> .
thus, our intended human a3h control appears to be the exception rather than the rule and that the single-domain a3z <dig> protein of artiodactyls is capable of dna cytosine deaminase activity.
we also observed that the artiodactyl a3z <dig> protein was capable of robust dna cytosine deaminase activity .
this result was fully anticipated based on the fact that the related z <dig> domain proteins of humans a3a, a3b and a3g are catalytically active  <cit> .
however, it is worth noting three observations suggesting that cow a3z <dig> is the most active of all reported a <dig> proteins.
first, we were never able to directionally clone  a3z <dig> of sheep or cattle into ptrc99a, which has a leaky promoter.
second, we were only able to topoisomerase-clone cow a3z <dig> in a direction opposite to the lac promoter .
finally, even with cow a3z <dig> in the promoter-opposing orientation in the topoisomerase cloning plasmid, we observed 100-fold increases in rifr mutation frequency in the e. coli-based mutation assay that were fully dependent on the catalytic glutamate e58a .
to summarize this section, all four of the a <dig> proteins of artiodactyls demonstrated intrinsic dna cytosine deaminase activity.
a3z <dig>  a3z <dig>  a3z <dig> and a3z2-z <dig> differentially localize in cells
fluorescent microscopy was used to examine the subcellular distribution of each of the artiodactyl a <dig> proteins fused to gfp.
like the human a <dig> proteins, which each have unique overall subcellular distributions, we imagined that distinct localization patterns might correlate with differential functions.
for instance, the first column of figure  <dig> shows representative images of live hela cells expressing human a3f-gfp, a3a-gfp, a3c-gfp and a3h-gfp, which predominantly localize to the cytoplasm, cell-wide with a nuclear bias, cell-wide and cell-wide with a clear nucleolar preference, respectively.
cow a3z1-gfp showed an indiscriminate cell-wide distribution similar to that of human a3a-gfp and gfp alone .
as shown previously, cattle and pig a3z2-z3-gfp localize to the cytoplasm, with some cells showing bright aggregates .
cattle and pig a3z <dig> also appeared predominantly cytoplasmic, but a significant fraction clearly penetrated the nuclear compartment .
the subcellular distribution of cattle and pig a3z <dig> differed from the similarly sized z <dig> protein human a3c, which was cell-wide, and it is therefore likely that an active process underlies the cytoplasmic bias of the artiodactyl a3z <dig> proteins.
interestingly, the a3z <dig> proteins of cattle and sheep, like human a3h, localized cell-wide with clear accumulations in the nucleoli .
similar data were obtained using these gfp fusion constructs in live cattle mdbk cells and in live pig pk <dig> cells .
these fluorescent microscopy observations demonstrated that all of the artiodactyl a <dig> proteins can be expressed in mammalian cells and that they have both distinct and overlapping subcellular distributions.
the artiodactyl a <dig> genes show evidence for positive selection
many human, non-human primate and feline a <dig> genes show signs of strong positive selection, which can be interpreted as evidence for a history filled with pathogen conflicts  <cit> .
however, given the relative stability of the artiodactyl a <dig> locus, at least in terms of gene number, we wondered whether the artiodactyl a <dig> genes might be under less intense selective pressure .
this possibility was assessed using two methods to compare the number of mutations that resulted in amino acid replacements to the number that were silent between pairs of artiodactyl species.
this ratio of replacement  to silent  mutations yields an omega  value, which if greater than one is indicative of positive selection, if equal to one of neutral selection and if less than one of negative selection.
we focused these analyses on the single exon that encodes the conserved z domain to minimize potentially confounding effects from recombination.
we first generated a combined phylogeny for each distinct a <dig> z domain and its inferred ancestral sequences .
using the paml free ration model, the artiodactyl a3z <dig> and the a3z <dig> genes appeared to be under a weak negative selection pressure, with ω values uniformly below one .
similarly, since the existence of the last common ancestor of cattle and sheep or of the pig and peccary, the artiodactyl a3z <dig> genes showed evidence for weak negative selection pressure .
however, a comparison of the inferred ancestral ruminant sequence with the inferred porcine sequence yielded a ω value of  <dig> , suggesting that the ancestor of modern day artiodactyls may have experienced intermittent positive selection .
these values were not as high as those for primate a3z <dig> .
moreover, all of these data contrasted sharply with the artiodactyl and primate aid genes, which are under an obvious strong negative selection pressure presumably for essential functions in antibody diversification.
however, because the free ratio model averages all possible sites and has a tendency to underestimate instances of positive selection, we subsequently used paml nssites to do a more focussed examination of artiodactyl a <dig> z domain variation.
several distinct selection models were used , and each yielded significant signs of positive selection .
the z <dig> domain a <dig> genes of sheep, cattle, pig, peccary and horse showed the highest dn/ds ratios, ranging from  <dig>  to  <dig>  and indicating that 22–31% of the residues were subjected to positive selection.
lower but still significant positive dn/ds ratios were obtained for the z <dig> domain a <dig> genes .
moreover, together with available dog and horse z <dig> sequences, the z <dig> a <dig> genes of cattle and sheep showed intermediate degrees of positive selection, with dn/ds ratios of  <dig>  to  <dig>  and  <dig> to 50% of the residues under some degree of positive selection .
thus, similar to most other mammals analyzed to date, the artiodactyl a <dig> genes have been subjected to strong evolutionary pressure .
aonly the sequences of z domain-encoding exons were used in these analyses .
btwo different codon frequency models were used to minimize potentially artificial results.
clikelihood ratio tests were done to compare the null models m <dig> and m <dig> to each positive selection model m <dig> and m <dig>  respectively, using paml nssites.
dtree length provides a measure of nucleotide substitutions per codon along all combined phylogenetic branches.
ethe percentage of all codons influenced by positive selection is indicated in parentheses.
a <dig> z domain distribution in mammals
our studies strongly indicated that the present-day a <dig> locus of sheep and cattle resembles one that existed in the common ancestor of placental mammals, consisting of precisely one of each of the three phylogenetically distinct z domains: z <dig>  z <dig> and z <dig> .
molecular phylogenetic data helped us infer that such a common ancestor existed approximately 100– <dig> mya  <cit> .
however, the bulk of the primate a <dig> gene expansion most likely occurred more recently because the main branches leading to rodents and humans split 90– <dig> mya.
it is therefore likely that rodents lost a z <dig> a <dig> gene after branching off of the main mammalian tree .
moreover, the recently published draft of the rhesus macaque genome helped to further whittle-down when the bulk of the primate-specific expansion occurred, because these animals also possess a human-like a <dig> gene repertoire .
thus, since the human and macaque lineages diverged approximately  <dig> mya  <cit> , the massive expansion from the inferred sheep/cow-like z1-z2-z <dig> a <dig> gene set to a locus resembling the present-day human repertoire must have occurred within a relatively short 65– <dig> million year period .
a minimum of  <dig> recombination events were required to generate the present-day human a <dig> locus from the common ancestor of artiodactyls and primates
the inferred ancestral z1-z2-z <dig> locus was used as a starting point to deduce the most likely evolutionary scenario that transformed it into the much larger eleven z domain human a <dig> repertoire.
two types of recombination events were considered, tandem duplications  and deletions.
self-similarities in the dna sequence of the human a <dig> locus provided strong evidence for prior tandem duplications by unequal crossing-over .
this mode of evolution is also supported by the fact that the human a <dig> locus contains many retroelements that could serve as substrates for homologous recombination  <cit> .
since our present studies showed that the z domains are highly modular and capable of individual function, they were considered as the core units for duplications in our inference procedures .
similarly, deletions could involve one or more z domains and result from unequal crossing-over or intra-chromosomal events.
an 8-event model for human a <dig> z domain history is shown in figure  <dig> .
this model can be appreciated by considering the present-day human locus and then working backward in time using highly similar local sequences within the a <dig> locus, which provide 'footprints' for recent recombination events.
first, full-length a3a and the z <dig> domain of a3b are 97% identical, and they are flanked by nearly homologous ~ <dig>  kb regions .
these footprints strongly suggested that a recent duplication of two consecutive ancestral domains  gave rise to present-day a3b .
second, we inferred that this recent duplication resulted in a vestigial z <dig> domain upstream of a3c, which was subsequently deleted prior to the divergence of human and chimpanzee lineages .
such a deletion event was supported by the fact that ~ <dig> kb regions of 92% identical dna reside upstream of the present-day a3b and a3c z <dig> domains .
third, a 92% similarity between two regions  encompassing the a3de and a3f genes suggested they originated from a recent duplication.
moreover, a similar level of identity was found between two other regions  encompassing the z <dig> domains of a3f and a3g.
this strongly supported a common ancestral origin for the n-terminal domains of the a3de, a3f and a3g genes .
the likelihood of these four relatively recent events suggested that the ancestral locus configuration prior to event  <dig>  was a key intermediate in the evolution of the primate a <dig> locus .
unequal crossing-over events prior to the ancestral intermediate were harder to infer because the footprints have been erased by sequence divergence.
we therefore developed an algorithm to compute the minimal series of duplication and deletion events that could have generated this intermediate locus from the z1-z2-z <dig> ancestor.
three minimal scenarios were found and each involved  <dig> events.
however, when phylogenetic data were considered, only one scenario was plausible and it involved a 2-domain duplication, a 3-domain duplication and two single domain deletions .
thus, together with the events detailed above, we inferred that the current human a <dig> repertoire is the product of  <dig> recombination events –  <dig> duplications and  <dig> deletions.
theoretically, models with as few as  <dig> events are possible if the likely intermediate locus configuration is ignored.
however, these models are also untenable as they clash with phylogenetic and local sequence alignment data.
it should be noted that  <dig> events represent only a lower bound to explain the evolution of the a <dig> human locus.
scenarios involving more than  <dig> events could also lead to the same domain organization, and some events may have left no observable trace in the human lineage.
thus, this lower bound could increase when the complete a <dig> locus sequence of more mammals, and especially more primates, comes available.
finally, it is worth emphasizing that most  of the  <dig> recombination events modeled here happened in the  <dig> to  <dig> million year period between the points when the rodent and old world monkey  lineages split from the phylogenetic branch that led to humans .
discussion
the present studies were initiated to gain a better understanding of the full a <dig> repertoire of three artiodactyl lineages – cattle, pigs and sheep – and to achieve insights into the mechanism and timing of the a <dig> gene expansion in mammals.
we demonstrated that sheep and cattle have three a <dig> genes, a3z <dig> a3z <dig> and a3z <dig>  however, the latter two genes and their counterpart in pigs have the unique ability to produce a double-domain protein a3z2-z <dig>  in addition to single-domain polypeptides.
thus, the a <dig> proteome of these species is more formidable than gene number alone would indicate.
our studies also help highlight the important point that, although a <dig> proteins consist of either one or two conserved z domains, each of these domains can function and evolve independently.
prior to the present studies, it was clear that most  placental mammals had z2- and z3-type a <dig> domains .
it was far less clear how broadly the z <dig> domain distributed.
here, we presented two critical lines of evidence strongly indicating that the z <dig> distribution is equally broad and, importantly, that the common ancestor of placental mammals had a z1-z2-z <dig> a <dig> gene repertoire, similar to that of present-day sheep and cattle.
first, the sheep and cattle a <dig> genomic sequences demonstrated the presence of a z1-type a <dig> gene outside of the primate phylogenetic branches .
second, our pan-species z <dig> pcr data, public est data and draft genomic sequences from horses and dogs combined to show that a a3z <dig> gene exists in other parts of the artiodactyl-containing phylogentic branch set.
these data supported a model in which the common ancestor of the primate- and the artiodactyl-containing mammalian super-orders, euarchontoglires and laurasiatheria, respectively, had a a3z <dig> gene and precisely one of each of the three conserved z domain types .
we have therefore established a critical foundation for understanding the function and evolutionary history of the a <dig> repertoire of any other placental mammal.
it is noteworthy that our pan-species z <dig> pcr analyses failed to generate product from opossum genomic dna and that the recently released opossum and platypus genomic sequences lack a <dig> genes .
this is unlikely to be a gap in the dna sequence assemblies because, like non-mammalian vertebrates, dna and protein searches clearly revealed the a3-flanking genes cbx <dig> and cbx <dig> in both animals .
thus, unfortunately, these two interesting non-placental mammals are unlikely to provide significant insights into the earliest stages of a <dig> gene evolution .
perhaps data from the other two placental mammal super-orders, afrotheria and xenarthra , will help shed light on earlier stages of a <dig> gene evolution, when presumably an aid-like gene transposed between cbx <dig> and cbx <dig> and duplicated to give rise to the ancestral z1-z2-z <dig> locus.
nevertheless, because all current data indicate that the a <dig> genes are specific to placental mammals, we hypothesize that a unique role of these genes may relate to the placenta itself, where the a <dig> proteins may function to help protect the developing fetus from potentially harmful retrotransposition events and/or retroviral infections.
a growing body of evidence indicates that the sole function of the a <dig> genes of mammals is to provide an innate immune defense to retrovirus and retrotransposon mobilization.
this is supported by the fact that the single a <dig> gene of mice is dispensable and that many of the mammalian a <dig> genes show evidence for a strong diversifying selection .
although the reason are presently unknown, a large a <dig> repertoire is clearly more important for some mammals than it is for others.
humans, chimpanzees and rhesus macaques have  <dig> z domains, approximately 3- to 4-fold more than any other known non-primate mammal .
indeed, our studies indicated that the ancestors of humans and chimpanzees experienced at least eight z domain recombination events, which is more than the total combined number of events for other known mammals.
therefore, despite the fact that the artiodactyl a <dig> genes show evidence for positive selection, their relative stability in copy number suggests that a considerable disadvantage – such as the potential to mutate genomic dna – may outweigh the innate benefit of having numerous a3s to combat potentially invasive retroelements.
this possibility may very well relate directly to an emerging trend in mammals, which is the frequent loss of a a3z <dig> gene which encodes a protein that can penetrate the nuclear compartment .
an a3z <dig> deletion was shown here for pigs, inferred here for cats and mice/rats, and demonstrated recently for some human populations .
finally, a major question is what selective pressure drove the a <dig> expansion from an ancestral z1-z2-z <dig> repertoire to the present day human z1-z2-z1-6-z1-z <dig> repertoire?
we propose that large-scale events such as gene expansions were selected by extremely pathogenic or lethal retroviral epidemics, because rare expansions would have been easily lost amongst a population of non-expanded alleles.
a powerful selective pressure such as a lethal epidemic has the potential to produce a population bottleneck such that mostly  pathogen-resistant individuals would survive .
such powerful selective pressures would have the potential to promote and perhaps even cause speciation events.
we further predict that such events may be marked by changes in a <dig> z domain copy number.
it is therefore quite plausible that at least some of the eight recombination events required to transform the ancestral z1-z2-z <dig> repertoire into the present day human z1-z2-z1-6-z1-z <dig> repertoire may have protected our human ancestors from ancient retroviral infections and thereby facilitated the evolution of primates .
CONCLUSIONS
the a <dig> locus of sheep and cattle consists of three genes, a3z <dig>  a3z <dig> and a3z <dig>  and the potential to encode four functional proteins, three directly and one  by read-through transcription and alternative splicing.
the a <dig> locus of pigs experienced a deletion and therefore lacks a3z <dig>  the artiodactyl a <dig> repertoire demonstrates a unique modularity centered upon the conserved zinc-coordinating motifs.
dna deaminase activity data and subcellular localization studies suggest that this modularity may also correspond to a broader functionality.
all of the data combined to indicate that the common ancestor of artiodactyls and humans possessed a sheep/cattle-like a <dig> gene set, with the organization and capacity to evolve into the present day repertoires.
the remarkable a <dig> gene expansion in the primate lineage – from the three ancestral genes  to the present-day eleven z-domain human repertoire – was predicted to require a minimum of eight recombination events, most of which may have been required to thwart an ancient retroviral infections.

 mammalian apobec proteins have the capacity to potently inhibit the replication of a diverse set of reverse-transcribing mobile genetic elements. susceptible exogenous retroelements include lentiviruses, alpharetroviruses, betaretroviruses, gammaretroviruses, deltaretroviruses, foamy viruses and the hepadnavirus hbv. susceptible endogenous retroelements include the yeast retrotransposons ty and ty the murine endogenous retroviruses musd and pmv, the murine intracisternal a particle, the porcine endogenous retrovirus perv and, potentially, extinct elements such as chimpanzee pterv and human herv-k, all of which require long-terminal repeats for replication. in addition, some a proteins can also inhibit l and its obligate parasite alu, retrotransposons that replicate by integration-primed reverse transcription. an overall theme is emerging in which most – if not all – retroelements can be inhibited by at least one a protein. however, it is now equally clear that the retroelements of any given species have evolved mechanisms to evade restriction by their host's a protein. for instance, hiv and siv use vif to trigger a ubiquitin-dependent degradation mechanism, foamy viruses use a protein called bet for an imprecisely defined inhibitory mechanism and some viruses such as mpmv, htlv and mlv appear to employ a simple avoidance mechanism. thus, it appears that all'successful' retroelements have evolved strategies to resist restriction by the a proteins of their hosts. the defining feature of the a family of proteins is a conserved zinc-coordinating dna cytosine deaminase motif, h-x1-e-x25–31-c-x2–4-c. the a z domains can be grouped into one of three distinct phylogenetic clusters – z z or z. the z-based classification system, proposed originally by conticello and coworkers, was revised recently through a collaborative effort. from hereon, the new a nomenclature system will be used. z and z proteins have a sw-s/t-c-x2–4-c motif, whereas z proteins have a tw-s-c-x2-c motif. z and z proteins can be further distinguished by h-x1-e-x5-x-v/i and h-x1-e-x5-w-f motifs, respectively. z proteins also have a unique isoleucine within a conserved riy motif located c-terminal to the zinc-coordinating residues. at least one protein of each of the z classes and nearly all identified a proteins have exhibited single-strand dna cytosine deaminase activity. for instance, human a3f, a3g and a3h possess catalytically competent z z and z domains, respectively. we previously reported a double-domain a3z2-z gene from the artiodactyls, sheep, cattle and pigs. however, the fact that mammals have varying numbers of a genes led us to wonder whether additional a genes would be present in artiodactyls. to address this point and to learn more about the evolution and functionality of a genes in mammals, we sequenced and characterized the full a repertoire of sheep and pigs. here, we demonstrated that sheep and cattle actually have three a genes, a3z a3z and a3z with a conserved potential to encode at least four active and distinct proteins. we further showed that porcine lineage has a deletion of the orthologous a3z gene and the capacity to encode only three proteins. these data enabled us to deduce that the common ancestor of artiodactyls and primates possessed an a repertoire consisting of three z domains. our data further suggested an evolutionary model in which most of the human a gene expansion occurred more than million years ago, during early primate evolution and possibly even associated with pathogen-induced population bottlenecks.  sheep and cattle have three a genes with a z1-z2-z organization we previously used degenerate pcr, race and database mining to identify a cdna for sheep a3z2-z. however, because humans have seven a genes and mice have only one, we postulated that artiodactyls such as sheep and cattle might have an intermediate number. to address this possibility unambiguously, we sequenced the entire sheep a genomic locus. first, a sheep a3z2-z cdna was hybridized to a sheep bac library to identify corresponding genomic sequence. second, hybridization-positive bacs were screened by pcr for those that also contain the conserved flanking genes cbx and cbx one bac was identified that spanned the entire cbx to cbx region, and it was sheared, subcloned, shotgun sequenced, assembled and analyzed. dna sequence analyses revealed that the sheep genomic locus contained another a gene between cbx and a3z. this gene was called a3z because it had sequence characteristics of a z1-type a protein. we therefore concluded that sheep have three a genes and, importantly, that each mammalian a z-type was present. this conclusion was supported by the bovine genome assembly, which was released during the course of our studies and showed that cattle also have a sheep-like, three gene a repertoire. the predicted a3z coding sequences of sheep and cattle are 86% identical, consistent with the fact that these two ruminant artiodactyls shared a common ancestor approximately 14– million years ago. the pig has two a genes with a z2–z organization pcr reactions failed to identify an a3z1-like gene in pigs. since pigs and cattle/sheep last shared a common ancestor approximately 70– mya, we considered the possibility that the negative pcr result was not a technical failure and that pigs might actually have a different a repertoire. again, to unambiguously address this possibility, the pig a genomic locus was sequenced in entirety. a porcine bac library was probed with pig a3z2-z cdna and two hybridization-positive bacs were shotgun sequenced. the sequence assemblies revealed that pigs have only two a genes a3z and a3z between cbx and cbx. the cattle, sheep and pig a locus genomic sequences were compared using dotplot analyses. a kb discontinuity was detected between the cow and the pig sequences. the sheep and pig genomic sequences aligned similarly. multiple retroelements were found to flank a3z in sheep and cattle. two were particularly close to the ends of the kb a3z region, a line/l and a sine/trna-glu. it is possible that one of these elements mediated a simple direct repeat recombination event that deleted the a3z region in pigs. however, we were unable to identify such a causative retroelement in the pig genomic sequence. to begin to address whether the potential a3z deletion in pigs occurred recently or whether it was more ancient, we asked whether a non-domesticated, distant relative of the pig, the collared peccary, has an a3z gene. lineages leading to present-day domesticated pigs and the peccary diverged approximately 25– mya. a pan-species, a3z pcr primer set was developed and used in these experiments. in contrast to human, african green monkey, horse, cow and sheep genomic dna which yielded a 250– bp z1-specific pcr products confirmable by dna sequencing, the genomic dna of domesticated pig, the collared peccary, mice and opossum failed to yield a product even after cycles. a highly conserved gene, aldoa, was used as a pcr control to demonstrate the integrity of the genomic dna samples. interestingly, z pcr product sequencing and recently released est sequences revealed that the related hoofed mammal, the horse, also has a z1-type a gene. two-'toed' hoofed animals such as sheep, cattle and pigs belong to the ungulate order artiodactyla, and one-'toed' hoofed animals such as horses belong to the ungulate order perissodactyla. since these two ungulate orders diverged approximately 80– mya and both have species with z1-type a genes, it is highly likely that the common ancestor also had an a3z gene. it is therefore highly unlikely that an a3z gene independently appeared at the same genomic position in artiodactyls, perissodactyls and primates. rather, all of the data support a model where a common ancestor of the domesticated pig and the collared peccary experienced a kb deletion that resulted in the loss of a3z. furthermore, since artiodactyls, perissodactyls and humans shared a common ancestor approximately 80– mya, the presence of z1-type a genes in both the primate and the artiodactyl limbs of the mammalian tree is also most easily explained by common ancestry. thus, our combined datasets indicated that this ancestor possessed a full a z repertoire, with one of each type of z domain, the minimal substrate required to evolve into the present-day eleven z domain human a locus. the artiodactyl a3z and a3z genes combine to encode distinct mrnas and proteins we previously characterized several activities of the double-domain a3z2-z protein from cattle, sheep and pigs. while re-confirming the 5' and 3' ends of the a3z2-z transcripts by race, we discovered two interesting variants that were conserved between these three species. first, using sheep and cattle pbmc or cell line cdna, 3' race frequently produced a smaller than expected fragment. the sequence of this fragment indicated the existence of a short bp transcript due to premature termination or nucleotides into intron for sheep and cattle, respectively. this truncated transcript was readily amplified from sheep and cattle pbmcs and represented by existing est sequences. therefore, this novel transcript was predicted to result in a single-domain z protein, a3z with a length of and amino acids for sheep and cattle, respectively. a pig a3z transcript was also identified by race and est sequences but, in contrast to sheep and cattle, exon was spliced to two additional exons before terminating prematurely. as a consequence, pig a3z was predicted to be amino acids. these analyses indicated that artiodactyls have the capacity to express a single domain a3z protein, in contrast to what we had deduced previously. second, 5' race data and cattle and pig est sequences suggested that yet another mechanism served to broaden the coding potential of the artiodactyl a locus. several transcripts appeared to originate from the region immediately upstream of a3z whereas our prior studies had only detected transcripts originating upsteam of a3z. a comparison of cdna and genomic sequences revealed the presence of an exon in this location. transcripts initiating here produced, or nucleotide messages. the resulting a3z protein was predicted to be residues for sheep and cattle and for pigs. the a3z mrna data strongly suggested the existence of an internal promoter. this was supported by cis-regulatory element prediction algorithms, which identified a conserved interferon-stimulated response element upstream of a3z as well as upstream of a3z. these isres were strikingly similar to those located in the promoter regions of human a3de, a3f and a3g, supporting the likelihood that interferon-inducibility is a conserved feature of many mammalian a genes. these putative isres significant similarity to functional elements in known interferon-inducible genes isg and isg. we also predicted binding sites for another well-known transcription factor, sp upstream of the a3z transcription start site. this activator was also recently reported for human and cat a genes. together with our previous data on the double domain a protein of these artiodactyl species, a3z2-z, these expression and promoter data revealed that two single-domain a genes can readily encode at least three distinct proteins – a3z a3z and a3z2-z a similar strategy may also be used by rodents, which also have an a gene with z and z domains. a similar modularity was reported recently for the cat a locus, where two single domain a genes combined to produce a functional double-domain a protein. we suggest that combining single-domain a3s to yield functionally unique double-domain proteins may be a general strategy used by many mammals to bolster their a3-dependent innate immune defenses. all four artiodactyl a proteins – a3z a3z a3z and a3z2-z – elicit dna cytosine deaminase activity all currently described a proteins have elicited single-strand dna cytosine to uracil deaminase activity in one or more assays. for instance, we showed that the artiodactyl a3z2-z proteins could catalyze the deamination of e. coli dna and retroviral cdna. however, catalytic mutants indicated that only the n-terminal z domain of cow, sheep and pig a3z2-z was active. this observation contrasted with data for the double-domain human a3b, a3f and a3g proteins, where the c-terminal domain clearly contains the dominant active site. nevertheless, these datasets suggested that the double-domain a proteins have separated function, with one domain predominantly serving as a catalytic center and the other as a regulatory center. however, a recent study with human a3b indicated that both z domains have the potential to be catalytically active. it was therefore reasonable to ask whether the single domain a3z and a3z proteins of artiodactyls would be capable of dna cytosine deamination in an e. coli-based activity assay. elevated frequencies of rifampicin-resistance mutations in e. coli provide a quantitative measure of the intrinsic a protein dna cytosine deaminase activity. in contrast to full-length cow a3z2-z which triggered a modest 2-fold increase in the median rifr mutation frequency over the vector control, non-induced levels of cow a3z caused a large 50-fold increase. the ptrc99-based vector used in these studies has an iptg-inducible promoter, and induced levels of cow a3z prevented e. coli growth, presumably through catastrophic levels of dna cytosine deamination. in contrast, induced levels of sheep or pig a3z proteins were not lethal, but their expression also caused significant increases in the median rifr mutation frequency. thus, as anticipated by our prior studies, the a3z proteins of cattle, sheep and pigs showed intrinsic dna cytosine deaminase activity. we were therefore surprised that induced levels of the cow single-domain protein a3z also caused a significant 4-fold increase in the median rifr mutation frequency. this result contrasted with the related z protein of humans, a3h, which appeared inactive in this assay. however, it is worth noting that other z3-type a proteins, a different human a3h variant, african green monkey a3h, rhesus macaque a3h and cat a3z, all showed evidence for dna deaminase activity in the e. coli-based mutation assay and/or in retrovirus infectivity assays. thus, our intended human a3h control appears to be the exception rather than the rule and that the single-domain a3z protein of artiodactyls is capable of dna cytosine deaminase activity. we also observed that the artiodactyl a3z protein was capable of robust dna cytosine deaminase activity. this result was fully anticipated based on the fact that the related z domain proteins of humans a3a, a3b and a3g are catalytically active. however, it is worth noting three observations suggesting that cow a3z is the most active of all reported a proteins. first, we were never able to directionally clone a3z of sheep or cattle into ptrc99a, which has a leaky promoter. second, we were only able to topoisomerase-clone cow a3z in a direction opposite to the lac promoter. finally, even with cow a3z in the promoter-opposing orientation in the topoisomerase cloning plasmid, we observed 100-fold increases in rifr mutation frequency in the e. coli-based mutation assay that were fully dependent on the catalytic glutamate e58a. to summarize this section, all four of the a proteins of artiodactyls demonstrated intrinsic dna cytosine deaminase activity. a3z a3z a3z and a3z2-z differentially localize in cells fluorescent microscopy was used to examine the subcellular distribution of each of the artiodactyl a proteins fused to gfp. like the human a proteins, which each have unique overall subcellular distributions, we imagined that distinct localization patterns might correlate with differential functions. for instance, the first column of figure shows representative images of live hela cells expressing human a3f-gfp, a3a-gfp, a3c-gfp and a3h-gfp, which predominantly localize to the cytoplasm, cell-wide with a nuclear bias, cell-wide and cell-wide with a clear nucleolar preference, respectively. cow a3z1-gfp showed an indiscriminate cell-wide distribution similar to that of human a3a-gfp and gfp alone. as shown previously, cattle and pig a3z2-z3-gfp localize to the cytoplasm, with some cells showing bright aggregates. cattle and pig a3z also appeared predominantly cytoplasmic, but a significant fraction clearly penetrated the nuclear compartment. the subcellular distribution of cattle and pig a3z differed from the similarly sized z protein human a3c, which was cell-wide, and it is therefore likely that an active process underlies the cytoplasmic bias of the artiodactyl a3z proteins. interestingly, the a3z proteins of cattle and sheep, like human a3h, localized cell-wide with clear accumulations in the nucleoli. similar data were obtained using these gfp fusion constructs in live cattle mdbk cells and in live pig pk cells. these fluorescent microscopy observations demonstrated that all of the artiodactyl a proteins can be expressed in mammalian cells and that they have both distinct and overlapping subcellular distributions. the artiodactyl a genes show evidence for positive selection many human, non-human primate and feline a genes show signs of strong positive selection, which can be interpreted as evidence for a history filled with pathogen conflicts. however, given the relative stability of the artiodactyl a locus, at least in terms of gene number, we wondered whether the artiodactyl a genes might be under less intense selective pressure. this possibility was assessed using two methods to compare the number of mutations that resulted in amino acid replacements to the number that were silent between pairs of artiodactyl species. this ratio of replacement to silent mutations yields an omega value, which if greater than one is indicative of positive selection, if equal to one of neutral selection and if less than one of negative selection. we focused these analyses on the single exon that encodes the conserved z domain to minimize potentially confounding effects from recombination. we first generated a combined phylogeny for each distinct a z domain and its inferred ancestral sequences. using the paml free ration model, the artiodactyl a3z and the a3z genes appeared to be under a weak negative selection pressure, with ω values uniformly below one. similarly, since the existence of the last common ancestor of cattle and sheep or of the pig and peccary, the artiodactyl a3z genes showed evidence for weak negative selection pressure. however, a comparison of the inferred ancestral ruminant sequence with the inferred porcine sequence yielded a ω value of, suggesting that the ancestor of modern day artiodactyls may have experienced intermittent positive selection. these values were not as high as those for primate a3z. moreover, all of these data contrasted sharply with the artiodactyl and primate aid genes, which are under an obvious strong negative selection pressure presumably for essential functions in antibody diversification. however, because the free ratio model averages all possible sites and has a tendency to underestimate instances of positive selection, we subsequently used paml nssites to do a more focussed examination of artiodactyl a z domain variation. several distinct selection models were used, and each yielded significant signs of positive selection. the z domain a genes of sheep, cattle, pig, peccary and horse showed the highest dn/ds ratios, ranging from to and indicating that 22–31% of the residues were subjected to positive selection. lower but still significant positive dn/ds ratios were obtained for the z domain a genes. moreover, together with available dog and horse z sequences, the z a genes of cattle and sheep showed intermediate degrees of positive selection, with dn/ds ratios of to and to 50% of the residues under some degree of positive selection. thus, similar to most other mammals analyzed to date, the artiodactyl a genes have been subjected to strong evolutionary pressure. aonly the sequences of z domain-encoding exons were used in these analyses. btwo different codon frequency models were used to minimize potentially artificial results. clikelihood ratio tests were done to compare the null models m and m to each positive selection model m and m respectively, using paml nssites. dtree length provides a measure of nucleotide substitutions per codon along all combined phylogenetic branches. ethe percentage of all codons influenced by positive selection is indicated in parentheses. a z domain distribution in mammals our studies strongly indicated that the present-day a locus of sheep and cattle resembles one that existed in the common ancestor of placental mammals, consisting of precisely one of each of the three phylogenetically distinct z domains: z z and z. molecular phylogenetic data helped us infer that such a common ancestor existed approximately 100– mya. however, the bulk of the primate a gene expansion most likely occurred more recently because the main branches leading to rodents and humans split 90– mya. it is therefore likely that rodents lost a z a gene after branching off of the main mammalian tree. moreover, the recently published draft of the rhesus macaque genome helped to further whittle-down when the bulk of the primate-specific expansion occurred, because these animals also possess a human-like a gene repertoire. thus, since the human and macaque lineages diverged approximately mya, the massive expansion from the inferred sheep/cow-like z1-z2-z a gene set to a locus resembling the present-day human repertoire must have occurred within a relatively short 65– million year period. a minimum of recombination events were required to generate the present-day human a locus from the common ancestor of artiodactyls and primates the inferred ancestral z1-z2-z locus was used as a starting point to deduce the most likely evolutionary scenario that transformed it into the much larger eleven z domain human a repertoire. two types of recombination events were considered, tandem duplications and deletions. self-similarities in the dna sequence of the human a locus provided strong evidence for prior tandem duplications by unequal crossing-over. this mode of evolution is also supported by the fact that the human a locus contains many retroelements that could serve as substrates for homologous recombination. since our present studies showed that the z domains are highly modular and capable of individual function, they were considered as the core units for duplications in our inference procedures. similarly, deletions could involve one or more z domains and result from unequal crossing-over or intra-chromosomal events. an 8-event model for human a z domain history is shown in figure. this model can be appreciated by considering the present-day human locus and then working backward in time using highly similar local sequences within the a locus, which provide'footprints' for recent recombination events. first, full-length a3a and the z domain of a3b are 97% identical, and they are flanked by nearly homologous ~ kb regions. these footprints strongly suggested that a recent duplication of two consecutive ancestral domains gave rise to present-day a3b. second, we inferred that this recent duplication resulted in a vestigial z domain upstream of a3c, which was subsequently deleted prior to the divergence of human and chimpanzee lineages. such a deletion event was supported by the fact that ~ kb regions of 92% identical dna reside upstream of the present-day a3b and a3c z domains. third, a 92% similarity between two regions encompassing the a3de and a3f genes suggested they originated from a recent duplication. moreover, a similar level of identity was found between two other regions encompassing the z domains of a3f and a3g. this strongly supported a common ancestral origin for the n-terminal domains of the a3de, a3f and a3g genes. the likelihood of these four relatively recent events suggested that the ancestral locus configuration prior to event was a key intermediate in the evolution of the primate a locus. unequal crossing-over events prior to the ancestral intermediate were harder to infer because the footprints have been erased by sequence divergence. we therefore developed an algorithm to compute the minimal series of duplication and deletion events that could have generated this intermediate locus from the z1-z2-z ancestor. three minimal scenarios were found and each involved events. however, when phylogenetic data were considered, only one scenario was plausible and it involved a 2-domain duplication, a 3-domain duplication and two single domain deletions. thus, together with the events detailed above, we inferred that the current human a repertoire is the product of recombination events – duplications and deletions. theoretically, models with as few as events are possible if the likely intermediate locus configuration is ignored. however, these models are also untenable as they clash with phylogenetic and local sequence alignment data. it should be noted that events represent only a lower bound to explain the evolution of the a human locus. scenarios involving more than events could also lead to the same domain organization, and some events may have left no observable trace in the human lineage. thus, this lower bound could increase when the complete a locus sequence of more mammals, and especially more primates, comes available. finally, it is worth emphasizing that most of the recombination events modeled here happened in the to million year period between the points when the rodent and old world monkey lineages split from the phylogenetic branch that led to humans. discussion the present studies were initiated to gain a better understanding of the full a repertoire of three artiodactyl lineages – cattle, pigs and sheep – and to achieve insights into the mechanism and timing of the a gene expansion in mammals. we demonstrated that sheep and cattle have three a genes, a3z a3z and a3z however, the latter two genes and their counterpart in pigs have the unique ability to produce a double-domain protein a3z2-z in addition to single-domain polypeptides. thus, the a proteome of these species is more formidable than gene number alone would indicate. our studies also help highlight the important point that, although a proteins consist of either one or two conserved z domains, each of these domains can function and evolve independently. prior to the present studies, it was clear that most placental mammals had z2- and z3-type a domains. it was far less clear how broadly the z domain distributed. here, we presented two critical lines of evidence strongly indicating that the z distribution is equally broad and, importantly, that the common ancestor of placental mammals had a z1-z2-z a gene repertoire, similar to that of present-day sheep and cattle. first, the sheep and cattle a genomic sequences demonstrated the presence of a z1-type a gene outside of the primate phylogenetic branches. second, our pan-species z pcr data, public est data and draft genomic sequences from horses and dogs combined to show that a a3z gene exists in other parts of the artiodactyl-containing phylogentic branch set. these data supported a model in which the common ancestor of the primate- and the artiodactyl-containing mammalian super-orders, euarchontoglires and laurasiatheria, respectively, had a a3z gene and precisely one of each of the three conserved z domain types. we have therefore established a critical foundation for understanding the function and evolutionary history of the a repertoire of any other placental mammal. it is noteworthy that our pan-species z pcr analyses failed to generate product from opossum genomic dna and that the recently released opossum and platypus genomic sequences lack a genes. this is unlikely to be a gap in the dna sequence assemblies because, like non-mammalian vertebrates, dna and protein searches clearly revealed the a3-flanking genes cbx and cbx in both animals. thus, unfortunately, these two interesting non-placental mammals are unlikely to provide significant insights into the earliest stages of a gene evolution. perhaps data from the other two placental mammal super-orders, afrotheria and xenarthra, will help shed light on earlier stages of a gene evolution, when presumably an aid-like gene transposed between cbx and cbx and duplicated to give rise to the ancestral z1-z2-z locus. nevertheless, because all current data indicate that the a genes are specific to placental mammals, we hypothesize that a unique role of these genes may relate to the placenta itself, where the a proteins may function to help protect the developing fetus from potentially harmful retrotransposition events and/or retroviral infections. a growing body of evidence indicates that the sole function of the a genes of mammals is to provide an innate immune defense to retrovirus and retrotransposon mobilization. this is supported by the fact that the single a gene of mice is dispensable and that many of the mammalian a genes show evidence for a strong diversifying selection. although the reason are presently unknown, a large a repertoire is clearly more important for some mammals than it is for others. humans, chimpanzees and rhesus macaques have z domains, approximately 3- to 4-fold more than any other known non-primate mammal. indeed, our studies indicated that the ancestors of humans and chimpanzees experienced at least eight z domain recombination events, which is more than the total combined number of events for other known mammals. therefore, despite the fact that the artiodactyl a genes show evidence for positive selection, their relative stability in copy number suggests that a considerable disadvantage – such as the potential to mutate genomic dna – may outweigh the innate benefit of having numerous a3s to combat potentially invasive retroelements. this possibility may very well relate directly to an emerging trend in mammals, which is the frequent loss of a a3z gene which encodes a protein that can penetrate the nuclear compartment. an a3z deletion was shown here for pigs, inferred here for cats and mice/rats, and demonstrated recently for some human populations. finally, a major question is what selective pressure drove the a expansion from an ancestral z1-z2-z repertoire to the present day human z1-z2-z1-6-z1-z repertoire? we propose that large-scale events such as gene expansions were selected by extremely pathogenic or lethal retroviral epidemics, because rare expansions would have been easily lost amongst a population of non-expanded alleles. a powerful selective pressure such as a lethal epidemic has the potential to produce a population bottleneck such that mostly pathogen-resistant individuals would survive. such powerful selective pressures would have the potential to promote and perhaps even cause speciation events. we further predict that such events may be marked by changes in a z domain copy number. it is therefore quite plausible that at least some of the eight recombination events required to transform the ancestral z1-z2-z repertoire into the present day human z1-z2-z1-6-z1-z repertoire may have protected our human ancestors from ancient retroviral infections and thereby facilitated the evolution of primates.  the a locus of sheep and cattle consists of three genes, a3z a3z and a3z and the potential to encode four functional proteins, three directly and one by read-through transcription and alternative splicing. the a locus of pigs experienced a deletion and therefore lacks a3z the artiodactyl a repertoire demonstrates a unique modularity centered upon the conserved zinc-coordinating motifs. dna deaminase activity data and subcellular localization studies suggest that this modularity may also correspond to a broader functionality. all of the data combined to indicate that the common ancestor of artiodactyls and humans possessed a sheep/cattle-like a gene set, with the organization and capacity to evolve into the present day repertoires. the remarkable a gene expansion in the primate lineage – from the three ancestral genes to the present-day eleven z-domain human repertoire – was predicted to require a minimum of eight recombination events, most of which may have been required to thwart an ancient retroviral infections. 
this mammalian ancestor therefore possessed the minimal a gene set, z1-z2-z required to evolve through a remarkable series of eight recombination events into the present day eleven z domain human repertoire. to gain insights into the mechanism and timing of a gene expansion and into the functional modularity of these genes, we analyzed the genomic sequences, expressed cdnas and activities of the full a repertoire of three artiodactyl lineages: sheep, cattle and pigs. these data, together with phylogenetic analyses, indicated that a similar, functionally modular a repertoire existed in the common ancestor of artiodactyls and primates. in all three species, read-through transcription and alternative splicing also produced a catalytically active double domain a3z2-z protein that had a distinct cytoplasmic localization.
 apobec proteins deaminate dna cytosines and block the replication of retroviruses and retrotransposons. each a gene encodes a protein with one or two conserved zinc-coordinating motifs. the presence of one a gene in mice and seven in humans, a3a-h, suggests extraordinary evolutionary flexibility. to gain insights into the mechanism and timing of a gene expansion and into the functional modularity of these genes, we analyzed the genomic sequences, expressed cdnas and activities of the full a repertoire of three artiodactyl lineages: sheep, cattle and pigs.  sheep and cattle have three a genes, a3z a3z and a3z whereas pigs only have two, a3z and a3z a comparison between domestic and wild pigs indicated that a3z was deleted in the pig lineage. in all three species, read-through transcription and alternative splicing also produced a catalytically active double domain a3z2-z protein that had a distinct cytoplasmic localization. thus, the three a genes of sheep and cattle encode four conserved and active proteins. these data, together with phylogenetic analyses, indicated that a similar, functionally modular a repertoire existed in the common ancestor of artiodactyls and primates. this mammalian ancestor therefore possessed the minimal a gene set, z1-z2-z required to evolve through a remarkable series of eight recombination events into the present day eleven z domain human repertoire.  the dynamic recombination-filled history of the mammalian a genes is consistent with the modular nature of the locus and a model in which most of these events were selected by ancient pathogenic retrovirus infections. 