BACKGROUND
processed pseudogenes are derived from reverse transcription of rna molecules followed by insertion of dna copies into the genome.
therefore, for a processed pseudogene to be inherited from one organismal generation to the next, it must be derived from rnas encoded by genes expressed in cells of the germline or the embryonic precursors of these cells.
the homeobox gene nanog is expressed in mammalian embryonic stem cells where its product, a homeobox transcription factor, maintains pluripotency of these cells  <cit> .
therefore, nanog is an excellent candidate as a possible source of inherited processed pseudogenes.
in fact, ten processed pseudogenes derived from nanog are present in the human genome, an unusually large family of inherited processed pseudogenes derived from a single gene  <cit> .
the human chromosomal region containing the nanog gene has also undergone a tandem duplication resulting in two copies of the nanog gene on chromosome  <dig>  the two copies are approximately 97% identical and their transcripts are spliced differently  <cit> .
although there is est-based evidence that both copies are transcribed, booth and holland  <cit>  have argued that one of the two copies is an unprocessed pseudogene, which they named nanogp <dig>  they named the ten processed pseudogenes nanogp <dig> through nanogp <dig>  two are located on the x chromosome, two on chromosome  <dig>  and one each on chromosomes  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  nanogp <dig> and nanogp <dig> through nanogp <dig> are full-length or nearly full-length processed pseudogenes lacking introns.
nanogp <dig> and nanogp <dig> are truncated fragments of processed pseudogenes  <cit> .
studies of unprocessed pseudogene evolution in primates are abundant, dating back to the early 1980s  <cit> .
although several studies are directed at pseudogene families  <cit> , most focus on the evolution of a single processed pseudogene  <cit> .
the relatively large number of processed nanog pseudogenes and the recent release of the build  <dig>  assembly of the chimpanzee  genome  <cit>  provide an excellent opportunity to elucidate the evolutionary history of the nanog gene and its large pseudogene family.
this article compares the human nanog gene and its pseudogenes with their chimpanzee orthologues and from this comparison derives an evolutionary history of this pseudogene family.
RESULTS
we identified the chimpanzee orthologues of the human nanog gene and all of its pseudogenes except nanogp <dig> using megablast and blastn searches of the build  <dig>  version of the chimpanzee genome assembly.
table  <dig> summarizes the chromosomal and genomic locations of the human nanog gene and pseudogenes and their chimpanzee orthologues.
megablast and blastn searches of the chimpanzee genome did not reveal any other nanog sequences, suggesting that no new nanog pseudogenes have arisen in the chimpanzee lineage since it diverged from the human lineage.
however, we cannot rule out the possibility that additional nanog pseudogenes may be present in the chimpanzee genome because unsequenced gaps remain in the build  <dig>  assembly.
our data indicate that the nanog gene and all pseudogenes except nanogp <dig> are in their expected orthologous positions in the chimpanzee genome, and that nanogp <dig> is not present in the chimpanzee genome.
chimpanzee orthologues of nanog and nanogp1
nanog  is the functional gene in the human genome, whereas nanogp <dig>  is apparently an unprocessed pseudogene derived from tandem duplication of the chromosomal region containing nanog.
however, cdna and est data show that nanogp <dig> may be transcriptionally active, albeit at a lower level than nanog, and that its transcripts are spliced differently than those derived from nanog.
hart et al.
<cit>  designated nanogp <dig> as nanog <dig> and referred to it as a functional gene, whereas booth and holland  <cit>  argued that because of its relatively high degree of divergence from nanog, and the comparative paucity and ambiguity of transcripts derived from it, nanogp <dig> is an unprocessed duplication pseudogene.
megablast searches of the chimpanzee genome readily identified the orthologues of nanog and nanogp <dig>  however, the organization of the chimpanzee orthologue of the human nanog gene in the chimpanzee build  <dig>  genome assembly suggests that the gene is either rearranged in the chimpanzee genome, or that the assembly is incorrect within this gene.
all four exons of the orthologue are present in the assembly but in two different genbank accessions.
the entire sequences of the 5' utr, exon  <dig>  and exon  <dig> are found in the region spanning nucleotides  <dig> through  <dig> of the chromosome  <dig> contig , in a region on the short arm of chromosome  <dig> near the telomere at a location orthologous to that of the human nanog gene at 12p <dig> .
introns  <dig> and  <dig> of the chimpanzee orthologue are also within this region but large segments of them are unsequenced.
the complete sequences of exon  <dig>  intron  <dig>  exon  <dig>  and the 3' utr of the chimpanzee orthologue are found in nucleotides  <dig> though  <dig> of another accession , which is known to reside on chromosome  <dig> but has not been placed in the build  <dig>  assembly of this chromosome.
furthermore, exon  <dig> in this accession contains an apparent single nucleotide-pair insertion mutation, resulting in a frameshift and premature termination codon in the reading frame.
to determine if the apparent gene rearrangement and frameshift mutation are present in the chimpanzee nanog gene, or whether these are assembly and sequencing errors, we compared the available sequences of nanog and nanogp <dig> in the chimpanzee assembly and selected pcr primer sequences in regions that differed sufficiently to ensure specific amplification of the nanog gene.
to verify that the amplicons were not derived from processed nanog pseudogenes, all target sequences included at least a portion of a nanog-specific intron.
two primer combinations amplified fragments that include the region of apparent misassembly within intron  <dig>  both of these primer combinations amplified pcr fragments of the sizes expected if the gene is intact.
we sequenced these fragments  of the gene and found that their sequences most closely matched those of the intact human nanog gene and less closely the corresponding sequences in the human pseudogenes, including nanogp <dig>  confirming that our sequences are derived from the intact chimpanzee nanog gene.
furthermore, our sequences show that the apparent frameshift mutation in exon  <dig> in the build  <dig>  assembly is a sequencing error.
our sequencing enabled us to assemble and annotate the genomic sequence of the intact chimpanzee nanog gene .
human nanogp <dig> and its absence in the chimpanzee genome
human nanogp <dig>  is located on human chromosome  <dig> at 15q <dig> .
it is the most recent of the nanog processed pseudogenes and is the only one that carries an alu element found in the 3' utr of the human nanog gene.
megablast and blastn searches of the chimpanzee genome failed to reveal the presence of a nanogp <dig> orthologue; all significant hits were to the nanog gene and other nanog pseudogenes.
to determine whether or not nanogp <dig> is indeed absent from the chimpanzee genome, we used  <dig> nucleotides flanking the 5' end and  <dig> nucleotides flanking the 3' end of the human nanogp <dig> pseudogene as queries in a blastn search of the chimpanzee genome.
the search identified highly homologous and contiguous sequences on chimpanzee chromosome  <dig>  spanning nucleotides  <dig> through  <dig> of the chromosome  <dig> contig .
as shown in figure  <dig>  the nanogp <dig> gene is indeed absent from its predicted site in the chimpanzee genome.
other nanog pseudogenes in the chimpanzee genome
we identified the chimpanzee orthologues of the human nanog processed pseudogenes nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  and nanogp <dig> in the build  <dig>  assembly.
all of these pseudogenes are in their predicted chromosomal locations when compared to the human genome.
the complete sequences of all of these pseudogenes except nanogp <dig> and nanogp <dig> are present in the chimpanzee genome assembly.
a 100-nucleotide segment in the 3' utr of nanogp <dig> and a  <dig> nucleotide segment containing the 5' utr and the entire reading frame of nanogp <dig> are unsequenced in the build  <dig>  assembly.
however, the presence of  <dig> nucleotides of the 3' utr, as well as orthologous flanking sequences, confirm the presence of nanogp <dig> at its expected position.
we attempted to amplify the chimpanzee orthologue of nanogp <dig> with primers designed to include small regions of flanking sequence on both ends to fully place it within the genome assembly.
this pseudogene is embedded in repetitive sequences and, although our primers were designed to match what appeared to be small regions of nonrepetitive sequences in the flanking regions, they failed to amplify the target sequence.
we designed primers to match unique sequences near the ends of the nanogp <dig> reading frame  and successfully amplified and sequenced a region in nanogp <dig> corresponding to positions 43– <dig> of the  <dig> nucleotide-pair reading frame in the functional nanog gene .
we verified that the sequence is indeed from nanogp <dig> by its high similarity to the human orthologue.
this sequence further confirms the presence of nanogp <dig> in the chimpanzee genome and it allowed us to compare the sequences of the human and chimpanzee orthologues.
this sequence also resolved a question about the origins of nanogp <dig> and nanogp <dig>  both are located on the x chromosome and both contain a  <dig> nucleotide-pair deletion that does not appear in the alignment when these two pseudogenes are aligned with each other, suggesting that they share this deletion.
these observations imply that nanogp <dig> and nanogp <dig> may be the products of a single insertion event followed by duplication of the chromosomal segment containing the pseudogene.
however, these deletions reside in a region consisting of ten copies of an imperfect  <dig> nucleotide-pair tandem repeat within the reading frame.
the chimpanzee nanogp <dig> orthologue does not contain the deletion present in the human orthologue, whereas the chimpanzee and human orthologues of nanogp <dig> have the same deletion.
this observation indicates that the deletion in human nanogp <dig> occurred after the h/c divergence and its origin is thus independent of the deletion in nanogp <dig>  furthermore, we examined  <dig> nucleotides on both sides of these pseudogenes and found no evidence of a duplication.
we conclude that nanogp <dig> and nanogp <dig> originated independently.
evolution of the nanog gene and pseudogene family
the entire functional nanog gene  and nanogp <dig> are present in both the human and chimpanzee genome assemblies at orthologous chromosomal positions.
in the 3' utr of the nanog gene, there is an alu element, which is missing from nanogp <dig> in both genomes.
therefore, the nanogp <dig> unprocessed pseudogene arose through duplication of the chromosomal region containing nanog before the human-chimpanzee  divergence and before insertion of the alu element into the nanog gene.
because the same alu element is present in both the human and chimpanzee nanog genes, its insertion must also have preceded the h/c divergence.
the processed pseudogenes nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  and nanogp <dig> lack this alu element.
they thus likely arose before its insertion and, therefore, also predate the h/c divergence.
the presence of the nanogp <dig> pseudogene fragment in both the human and chimpanzee genomes likewise shows that its origin preceded h/c divergence.
the human nanogp <dig> pseudogene is highly similar to the nanog gene, is absent from the chimpanzee genome, and contains the same alu element as the nanog gene, indicating that this processed pseudogene is the most recent of the nanog pseudogenes and was inserted into human chromosome  <dig> after the h/c divergence.
based on the assumption of a pseudogene mutation rate of  <dig>  × 10- <dig> mutations per site per year in humans  <cit> , booth and holland  <cit>  estimated the origin of the nanogp <dig> pseudogene as the most recent at  <dig>  million years ago, about the time of the h/c divergence.
our results demonstrate that nanogp <dig> arose after the h/c divergence, and thus are consistent with this date.
booth and holland  <cit>  estimated the origins of the other pseudogenes as ranging from over  <dig> million years ago for nanogp <dig> to  <dig> million years ago for nanogp <dig>  with the caveat that these dates may be inaccurate, and are likely overestimates, because nucleotide substitution rates for pseudogenes are not well calibrated within this range.
booth and holland  <cit>  determined the relative ages of the human nanog pseudogenes by counting the number of mutations in the reading-frame regions of the human nanog pseudogenes when compared to the reading frame of the functional nanog gene, scaling their analysis by counting adjacent deletions as a unit-site size of one to compensate for the reduced opportunity of substitution mutation in deleted regions.
they concluded that nanogp <dig> is the most ancient of the pseudogenes, followed in order of most ancient to most recent by nanogp <dig> or nanogp <dig>  then nanogp <dig>  then nanogp <dig> or nanogp <dig>  then nanogp <dig>  then nanogp <dig>  then nanogp <dig>  and nanogp <dig> as the most recent.
booth and holland's analysis did not distinguish the order of nanogp <dig> and nanogp <dig> relative to each other, nor of nanogp <dig> and nanogp <dig> relative to each other, because of similar degrees of divergence for each of these pairs of pseudogenes from nanog.
we conducted a similar analysis of relative age, with the same scaling for multiple-nucleotide deletions as a single unit site when those deletions were shared by the human and chimpanzee sequences.
we identified mutations that occurred after the h/c divergence as differences between the human and chimpanzee sequences and corrected them to reflect the ancestral sequence at the time of the h/c divergence before completing our analysis.
this correction was especially important for nanogp <dig>  which has accumulated  <dig> mutations since the h/c divergence, compared to 1– <dig> mutations for the other pseudogenes.
we excluded nanogp <dig> from this correction because of its absence in the chimpanzee genome.
also, since nanogp <dig> is a truncated pseudogene with only  <dig> nucleotides within the nanog coding region, we compared only the portions of nanog and the other pseudogenes that aligned with these  <dig> nucleotides when determining the relative age of nanogp <dig>  the pseudogene fragment nanogp <dig> was not included in booth and holland's analysis nor ours because it lacks the entire reading frame and has no significant homology with several of the other processed pseudogenes.
comparison of the sequences after these adjustments results in a relative order that is the same as that determined by booth and holland  <cit> .
also similar to booth and holland's conclusions, our analysis showed that nanogp <dig> and nanogp <dig> were almost identical in the degree of similarity to nanog , and that nanogp <dig> and nanogp <dig> were likewise nearly identical in the degree of divergence from nanog .
thus, like booth and holland  <cit> , we could not conclusively determine the relative orders within each of these two pairs of pseudogenes using this type of analysis.
such an analysis assumes that natural selection has conserved the functional gene's sequence so that the modern sequence of the reading frame represents the source sequence of each of the pseudogenes.
under most circumstances, such an assumption cannot readily be tested.
however, the periodic insertion and fixation of ten nanog pseudogenes with a complete or partial reading frame should have left a record, albeit an imperfect one, of the functional nanog gene-sequence evolution.
if we assume that the reading frame of the functional nanog gene has changed during the time when the pseudogenes were inserted into the genome, the mutational differences in the pseudogenes should consist of three different types: 1) source-gene mutations, defined as those that occurred in the functional nanog gene after the insertion of one pseudogene but before the insertion of another, resulting in a polymorphism between these pseudogenes, 2) post-insertion mutations, defined as those that occurred in a pseudogene after its insertion but before the h/c divergence, and 3) post-h/c divergence mutations, defined as mutations that occurred in the nanog gene and its pseudogenes after the h/c divergence.
we readily identified  <dig> post-h/c divergence mutations in the reading-frame regions of the nanog gene and its pseudogenes, and in all but four cases we were able to determine the mutant and ancestral nucleotides at each site by comparison of the human and chimpanzee orthologues with the nanog gene and the other pseudogenes.
some of the source-gene mutations should be distinguishable from post-insertion pseudogene mutations in our data as a nucleotide that is identical in a set of older pseudogenes, which then changes to a different nucleotide in a set of younger pseudogenes.
moreover, if possible source-gene mutations can be identified, they can be used to reconstruct the evolutionary history of the pseudogene family, and to some extent the evolutionary history of the gene itself.
to reconstruct the evolutionary history of the nanog gene and its pseudogene family with source-gene mutation analysis, we aligned the reading frame of the human and chimpanzee nanog gene with the corresponding sequences in all pseudogenes , and corrected  post-h/c divergence mutations to reflect the ancestral sequence.
we identified sites with possible source-gene mutations as a nucleotide shared by two or more pseudogenes and a different nucleotide shared by two or more additional pseudogenes.
any nucleotide present in a particular position in only one pseudogene was considered as a post-insertion pseudogene mutation.
a total of  <dig> sites  within the reading frame met these criteria for identification of possible source-gene mutations.
we then identified the most parsimonious order of pseudogenes as the one which required the fewest number of source-gene mutations across these  <dig> sites.
the most parsimonious ordering of the nanog pseudogenes  from most ancient to most recent is nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  and nanogp <dig> as the most recent.
the next most parsimonious ordering  is the same as the above order but with the positions of nanogp <dig> and nanogp <dig> reversed.
as a truncated pseudogene, nanogp <dig> contains only  <dig> possible source-gene mutation sites.
of these, only five are informative in distinguishing nanogp <dig> and nanogp <dig>  three supporting nanogp <dig> as the older pseudogene and two supporting nanogp <dig>  sites with only one mutation in a particular order are more likely to represent a true source-gene mutation than sites with multiple mutations, which probably consist of a combination of source-gene and post-insertion mutations.
the three sites,  <dig>   <dig>  and  <dig>  that support nanogp <dig> as the older pseudogene require  <dig>   <dig>  and  <dig> mutations to explain the order, respectively.
the two sites that support nanogp <dig> as the older pseudogene  require  <dig> and  <dig> mutations, respectively, to explain that order, suggesting that the most parsimonious order  is also the most plausible with respect to these two pseudogenes.
additionally, our analysis clarifies the relative order of nanogp <dig> and nanogp <dig> by clearly placing nanogp <dig> as the older of the two .
the only notable discrepancy between the results of source-gene mutation analysis and ordering by overall similarity to the modern nanog gene is the relative placement of nanogp <dig> and nanogp <dig>  in the latter analysis, the functional nanog gene is more similar to nanogp <dig>  than it is to nanogp <dig> , implying that nanogp <dig> is the older pseudogene.
however, source-gene mutation analysis places nanogp <dig> as the more recent of the two.
examination of the mutations that distinguish nanogp <dig> from nanog provides compelling evidence that nanogp <dig> is indeed the older pseudogene.
nanogp <dig> is an unprocessed pseudogene that arose from duplication of a segment of chromosome  <dig>  and thus may have remained functional for an undetermined period of time after its formation.
as booth and holland  <cit>  pointed out, nanogp <dig> cannot use the same initiation codon as nanog because a mutation at position  <dig> in the reading frame produced a premature termination codon after only eight amino acids.
this mutation is present in both the human and chimpanzee orthologues indicating that it preceded the h/c divergence.
booth and holland noted, however, that of the three characterized human transcripts from nanogp <dig>  two are alternatively spliced to remove all of exon  <dig>  so that the nanogp <dig> reading frame begins at a position corresponding to the 58th amino acid in the protein encoded by nanog, which is an internal methionine in the nanog protein.
if nanogp <dig> did indeed remain functional after its formation, we would expect natural selection to conserve the sequence within its reading frame when compared to nanog.
after correction to the ancestral sequence for post-h/c divergence mutations,  <dig> mutations distinguish nanogp <dig> from the nanog reading frame, and they are nonrandomly distributed.
twelve are clustered in a 121-nucleotide region entirely within exon  <dig> of the nanog gene, a region removed during splicing in two characterized nanogp <dig> transcripts.
of the three mutations in nanogp1's apparent reading frame, two are nonsynonymous and one is synonymous.
a nonsynonymous mutation at position  <dig> is a guanine-to-thymine substitution that results in a lysine-to-asparagine substitution in the protein.
comparison with the human and chimpanzee sequences of the other pseudogenes reveals that this is a source-gene mutation that supports nanogp <dig> as being older than nanogp <dig>  comparison of this polymorphism to the sequences of the other pseudogenes reveals that the guanine in nanogp <dig>  and therefore the lysine in the protein, are ancestral, and that the source-gene mutation occurred after duplication of nanogp <dig> but before insertion of nanogp <dig>  interestingly, booth and holland  <cit>  found through experimental sequencing that this particular mutation  is polymorphic in modern humans, suggesting that neither lysine nor asparagine is detrimental to protein function at this position.
the other nonsynonymous mutation is a cytosine-to-thymine substitution at position  <dig>  resulting in a proline-to-leucine substitution in the protein.
because proline and leucine have similar biochemical properties, this mutation is also not likely to adversely affect protein function.
the nanog gene and all other pseudogenes in both the human and chimpanzee genomes have a cytosine residue at this position, indicating that this is a post-duplication mutation in nanogp <dig>
the single synonymous mutation in the apparent reading frame is at position  <dig>  which lies within the homeobox region.
this is clearly a source-gene mutation that also supports the ordering of nanogp <dig> as being older than nanogp <dig>  only nanog, nanogp <dig>  and nanogp <dig> have a cytosine at this position; all other pseudogenes, including nanogp <dig>  have a thymine at this position.
taken in the aggregate, these observations strongly support the hypothesis that nanogp <dig> remained functional after duplication and, therefore, was subject to selection-driven conservation of its reading frame.
they also raise the possibility that nanogp <dig> may retain some functionality or that its loss of function may be evolutionarily recent.
nucleotide polymorphisms at possible source-gene mutation sites may represent true source-gene mutations or post-insertion pseudogene mutations.
sites in which a single mutation separates a set of older pseudogenes from a set of younger pseudogenes are the most plausible sites for identification of true source-gene mutations.
in the most parsimonious ordering,  <dig> of the  <dig> sites contained a single possible source-gene mutation .
twenty of these mutations are nonsynonymous and nine are synonymous.
if a mutation is indeed a true source-gene mutation, the amino acid it encodes may be reflected in the nanog proteins of other vertebrates.
to determine if this is the case, we used the amino acid sequence of the polypeptide encoded by the human nanog gene  as a query for a blastp search of the protein database of all organisms.
proteins from six species displayed full-length or nearly full length homology to the nanog protein: crab-eating macaque , house mouse , norway rat , domestic cattle , domestic goat , and domestic dog .
we excluded a match to a computationally generated hypothetical protein in chimpanzee  because it is derived from the dna sequence of chimpanzee nanogp <dig>
as shown in figure  <dig>  several of the putative source-gene mutations and their inferred effect on amino acid sequence in the human/chimpanzee nanog pseudogene family are consistent with the corresponding amino acids in the nanog proteins of other eutherian mammals.
for example, at site  <dig> in the reading frame, an adenine-to-guanine substitution in the nanog gene apparently occurred after the insertion of nanogp <dig> but before the insertion of nanogp <dig>  resulting in an asparagine-to-aspartic acid substitution in amino-acid residue  <dig> of the polypeptide.
the dog, cattle, and rat proteins have asparagine at this position, whereas the macaque, chimpanzee, and human have aspartic acid at this position.
similar patterns of congruence between amino acid substitution and amino acid sequences in other mammals is evident at positions 250– <dig>   <dig>   <dig>   <dig>   <dig>  and 820– <dig> of the reading frame .
another feature of the putative source-gene mutations is the paucity of amino acid substitutions at source-gene mutation sites within the homeobox region  indicative of high source-gene sequence conservation in this region.
six possible source-gene mutation sites are present within the homeobox region .
five of these six sites have only synonymous mutations.
the single nonsynonymous mutation is at position  <dig>  with thymine present in nanogp <dig> and nanogp <dig> and cytosine present in all other pseudogenes and the nanog gene, resulting in a leucine-to-phenylalanine substitution in the nanogp <dig> and nanogp <dig> sequences.
these thymines may be independent post-insertion mutations or they could be a source-gene mutation that reverted to its original sequence after the insertion of nanogp <dig>
pseudogene mutations can be used to estimate the dates of origin for individual pseudogenes.
however, only post-insertion mutations not subject to purifying selection are reliable indicators of the age of a pseudogene.
our analysis shows that, in the case of the nanog pseudogene family, source-gene mutations are present and may contribute to a significant number of polymorphisms in the pseudogenes.
although some source-gene and post-insertion mutations may be readily distinguished based on their patterns when the pseudogenes are ordered, others may not be so easily discerned.
even when post-insertion mutations can be reliably identified, pseudogene evolution rates have not been well calibrated prior to the h/c divergence, as pointed out by booth and holland  <cit> .
for these reasons, we have avoided age estimations in this study, focusing instead on the relative order of nanog pseudogene origins.
CONCLUSIONS
a synthesis of the results from this article with those of booth and holland  <cit>  produces a straightforward evolutionary history of the nanog pseudogene family in the human and chimpanzee genomes.
nanogp <dig> is the most ancient of the pseudogenes followed in order of most ancient to most recent by the processed pseudogenes nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  nanogp <dig>  and nanogp <dig>  before insertion of nanogp <dig>  the region on chromosome  <dig> containing nanog underwent a duplication producing nanogp <dig>  which remained functional and subject to selection-driven conservation of its reading frame.
all of these events, and the resulting fixation of their products in the genome, preceded the h/c divergence.
following the h/c divergence, nanogp <dig> inserted itself into chromosome  <dig> in the human lineage.

 processed pseudogenes are derived from reverse transcription of rna molecules followed by insertion of dna copies into the genome. therefore, for a processed pseudogene to be inherited from one organismal generation to the next, it must be derived from rnas encoded by genes expressed in cells of the germline or the embryonic precursors of these cells. the homeobox gene nanog is expressed in mammalian embryonic stem cells where its product, a homeobox transcription factor, maintains pluripotency of these cells. therefore, nanog is an excellent candidate as a possible source of inherited processed pseudogenes. in fact, ten processed pseudogenes derived from nanog are present in the human genome, an unusually large family of inherited processed pseudogenes derived from a single gene. the human chromosomal region containing the nanog gene has also undergone a tandem duplication resulting in two copies of the nanog gene on chromosome the two copies are approximately 97% identical and their transcripts are spliced differently. although there is est-based evidence that both copies are transcribed, booth and holland have argued that one of the two copies is an unprocessed pseudogene, which they named nanogp they named the ten processed pseudogenes nanogp through nanogp two are located on the x chromosome, two on chromosome and one each on chromosomes and nanogp and nanogp through nanogp are full-length or nearly full-length processed pseudogenes lacking introns. nanogp and nanogp are truncated fragments of processed pseudogenes. studies of unprocessed pseudogene evolution in primates are abundant, dating back to the early 1980s. although several studies are directed at pseudogene families, most focus on the evolution of a single processed pseudogene. the relatively large number of processed nanog pseudogenes and the recent release of the build assembly of the chimpanzee genome provide an excellent opportunity to elucidate the evolutionary history of the nanog gene and its large pseudogene family. this article compares the human nanog gene and its pseudogenes with their chimpanzee orthologues and from this comparison derives an evolutionary history of this pseudogene family.  we identified the chimpanzee orthologues of the human nanog gene and all of its pseudogenes except nanogp using megablast and blastn searches of the build version of the chimpanzee genome assembly. table summarizes the chromosomal and genomic locations of the human nanog gene and pseudogenes and their chimpanzee orthologues. megablast and blastn searches of the chimpanzee genome did not reveal any other nanog sequences, suggesting that no new nanog pseudogenes have arisen in the chimpanzee lineage since it diverged from the human lineage. however, we cannot rule out the possibility that additional nanog pseudogenes may be present in the chimpanzee genome because unsequenced gaps remain in the build assembly. our data indicate that the nanog gene and all pseudogenes except nanogp are in their expected orthologous positions in the chimpanzee genome, and that nanogp is not present in the chimpanzee genome. chimpanzee orthologues of nanog and nanogp1 nanog is the functional gene in the human genome, whereas nanogp is apparently an unprocessed pseudogene derived from tandem duplication of the chromosomal region containing nanog. however, cdna and est data show that nanogp may be transcriptionally active, albeit at a lower level than nanog, and that its transcripts are spliced differently than those derived from nanog. hart et al. designated nanogp as nanog and referred to it as a functional gene, whereas booth and holland argued that because of its relatively high degree of divergence from nanog, and the comparative paucity and ambiguity of transcripts derived from it, nanogp is an unprocessed duplication pseudogene. megablast searches of the chimpanzee genome readily identified the orthologues of nanog and nanogp however, the organization of the chimpanzee orthologue of the human nanog gene in the chimpanzee build genome assembly suggests that the gene is either rearranged in the chimpanzee genome, or that the assembly is incorrect within this gene. all four exons of the orthologue are present in the assembly but in two different genbank accessions. the entire sequences of the 5' utr, exon and exon are found in the region spanning nucleotides through of the chromosome contig, in a region on the short arm of chromosome near the telomere at a location orthologous to that of the human nanog gene at 12p. introns and of the chimpanzee orthologue are also within this region but large segments of them are unsequenced. the complete sequences of exon intron exon and the 3' utr of the chimpanzee orthologue are found in nucleotides though of another accession, which is known to reside on chromosome but has not been placed in the build assembly of this chromosome. furthermore, exon in this accession contains an apparent single nucleotide-pair insertion mutation, resulting in a frameshift and premature termination codon in the reading frame. to determine if the apparent gene rearrangement and frameshift mutation are present in the chimpanzee nanog gene, or whether these are assembly and sequencing errors, we compared the available sequences of nanog and nanogp in the chimpanzee assembly and selected pcr primer sequences in regions that differed sufficiently to ensure specific amplification of the nanog gene. to verify that the amplicons were not derived from processed nanog pseudogenes, all target sequences included at least a portion of a nanog-specific intron. two primer combinations amplified fragments that include the region of apparent misassembly within intron both of these primer combinations amplified pcr fragments of the sizes expected if the gene is intact. we sequenced these fragments of the gene and found that their sequences most closely matched those of the intact human nanog gene and less closely the corresponding sequences in the human pseudogenes, including nanogp confirming that our sequences are derived from the intact chimpanzee nanog gene. furthermore, our sequences show that the apparent frameshift mutation in exon in the build assembly is a sequencing error. our sequencing enabled us to assemble and annotate the genomic sequence of the intact chimpanzee nanog gene. human nanogp and its absence in the chimpanzee genome human nanogp is located on human chromosome at 15q. it is the most recent of the nanog processed pseudogenes and is the only one that carries an alu element found in the 3' utr of the human nanog gene. megablast and blastn searches of the chimpanzee genome failed to reveal the presence of a nanogp orthologue; all significant hits were to the nanog gene and other nanog pseudogenes. to determine whether or not nanogp is indeed absent from the chimpanzee genome, we used nucleotides flanking the 5' end and nucleotides flanking the 3' end of the human nanogp pseudogene as queries in a blastn search of the chimpanzee genome. the search identified highly homologous and contiguous sequences on chimpanzee chromosome spanning nucleotides through of the chromosome contig. as shown in figure the nanogp gene is indeed absent from its predicted site in the chimpanzee genome. other nanog pseudogenes in the chimpanzee genome we identified the chimpanzee orthologues of the human nanog processed pseudogenes nanogp nanogp nanogp nanogp nanogp nanogp nanogp nanogp and nanogp in the build assembly. all of these pseudogenes are in their predicted chromosomal locations when compared to the human genome. the complete sequences of all of these pseudogenes except nanogp and nanogp are present in the chimpanzee genome assembly. a 100-nucleotide segment in the 3' utr of nanogp and a nucleotide segment containing the 5' utr and the entire reading frame of nanogp are unsequenced in the build assembly. however, the presence of nucleotides of the 3' utr, as well as orthologous flanking sequences, confirm the presence of nanogp at its expected position. we attempted to amplify the chimpanzee orthologue of nanogp with primers designed to include small regions of flanking sequence on both ends to fully place it within the genome assembly. this pseudogene is embedded in repetitive sequences and, although our primers were designed to match what appeared to be small regions of nonrepetitive sequences in the flanking regions, they failed to amplify the target sequence. we designed primers to match unique sequences near the ends of the nanogp reading frame and successfully amplified and sequenced a region in nanogp corresponding to positions 43– of the nucleotide-pair reading frame in the functional nanog gene. we verified that the sequence is indeed from nanogp by its high similarity to the human orthologue. this sequence further confirms the presence of nanogp in the chimpanzee genome and it allowed us to compare the sequences of the human and chimpanzee orthologues. this sequence also resolved a question about the origins of nanogp and nanogp both are located on the x chromosome and both contain a nucleotide-pair deletion that does not appear in the alignment when these two pseudogenes are aligned with each other, suggesting that they share this deletion. these observations imply that nanogp and nanogp may be the products of a single insertion event followed by duplication of the chromosomal segment containing the pseudogene. however, these deletions reside in a region consisting of ten copies of an imperfect nucleotide-pair tandem repeat within the reading frame. the chimpanzee nanogp orthologue does not contain the deletion present in the human orthologue, whereas the chimpanzee and human orthologues of nanogp have the same deletion. this observation indicates that the deletion in human nanogp occurred after the h/c divergence and its origin is thus independent of the deletion in nanogp furthermore, we examined nucleotides on both sides of these pseudogenes and found no evidence of a duplication. we conclude that nanogp and nanogp originated independently. evolution of the nanog gene and pseudogene family the entire functional nanog gene and nanogp are present in both the human and chimpanzee genome assemblies at orthologous chromosomal positions. in the 3' utr of the nanog gene, there is an alu element, which is missing from nanogp in both genomes. therefore, the nanogp unprocessed pseudogene arose through duplication of the chromosomal region containing nanog before the human-chimpanzee divergence and before insertion of the alu element into the nanog gene. because the same alu element is present in both the human and chimpanzee nanog genes, its insertion must also have preceded the h/c divergence. the processed pseudogenes nanogp nanogp nanogp nanogp nanogp nanogp nanogp and nanogp lack this alu element. they thus likely arose before its insertion and, therefore, also predate the h/c divergence. the presence of the nanogp pseudogene fragment in both the human and chimpanzee genomes likewise shows that its origin preceded h/c divergence. the human nanogp pseudogene is highly similar to the nanog gene, is absent from the chimpanzee genome, and contains the same alu element as the nanog gene, indicating that this processed pseudogene is the most recent of the nanog pseudogenes and was inserted into human chromosome after the h/c divergence. based on the assumption of a pseudogene mutation rate of × 10- mutations per site per year in humans, booth and holland estimated the origin of the nanogp pseudogene as the most recent at million years ago, about the time of the h/c divergence. our results demonstrate that nanogp arose after the h/c divergence, and thus are consistent with this date. booth and holland estimated the origins of the other pseudogenes as ranging from over million years ago for nanogp to million years ago for nanogp with the caveat that these dates may be inaccurate, and are likely overestimates, because nucleotide substitution rates for pseudogenes are not well calibrated within this range. booth and holland determined the relative ages of the human nanog pseudogenes by counting the number of mutations in the reading-frame regions of the human nanog pseudogenes when compared to the reading frame of the functional nanog gene, scaling their analysis by counting adjacent deletions as a unit-site size of one to compensate for the reduced opportunity of substitution mutation in deleted regions. they concluded that nanogp is the most ancient of the pseudogenes, followed in order of most ancient to most recent by nanogp or nanogp then nanogp then nanogp or nanogp then nanogp then nanogp then nanogp and nanogp as the most recent. booth and holland's analysis did not distinguish the order of nanogp and nanogp relative to each other, nor of nanogp and nanogp relative to each other, because of similar degrees of divergence for each of these pairs of pseudogenes from nanog. we conducted a similar analysis of relative age, with the same scaling for multiple-nucleotide deletions as a single unit site when those deletions were shared by the human and chimpanzee sequences. we identified mutations that occurred after the h/c divergence as differences between the human and chimpanzee sequences and corrected them to reflect the ancestral sequence at the time of the h/c divergence before completing our analysis. this correction was especially important for nanogp which has accumulated mutations since the h/c divergence, compared to 1– mutations for the other pseudogenes. we excluded nanogp from this correction because of its absence in the chimpanzee genome. also, since nanogp is a truncated pseudogene with only nucleotides within the nanog coding region, we compared only the portions of nanog and the other pseudogenes that aligned with these nucleotides when determining the relative age of nanogp the pseudogene fragment nanogp was not included in booth and holland's analysis nor ours because it lacks the entire reading frame and has no significant homology with several of the other processed pseudogenes. comparison of the sequences after these adjustments results in a relative order that is the same as that determined by booth and holland. also similar to booth and holland's conclusions, our analysis showed that nanogp and nanogp were almost identical in the degree of similarity to nanog, and that nanogp and nanogp were likewise nearly identical in the degree of divergence from nanog. thus, like booth and holland, we could not conclusively determine the relative orders within each of these two pairs of pseudogenes using this type of analysis. such an analysis assumes that natural selection has conserved the functional gene's sequence so that the modern sequence of the reading frame represents the source sequence of each of the pseudogenes. under most circumstances, such an assumption cannot readily be tested. however, the periodic insertion and fixation of ten nanog pseudogenes with a complete or partial reading frame should have left a record, albeit an imperfect one, of the functional nanog gene-sequence evolution. if we assume that the reading frame of the functional nanog gene has changed during the time when the pseudogenes were inserted into the genome, the mutational differences in the pseudogenes should consist of three different types: 1) source-gene mutations, defined as those that occurred in the functional nanog gene after the insertion of one pseudogene but before the insertion of another, resulting in a polymorphism between these pseudogenes, 2) post-insertion mutations, defined as those that occurred in a pseudogene after its insertion but before the h/c divergence, and 3) post-h/c divergence mutations, defined as mutations that occurred in the nanog gene and its pseudogenes after the h/c divergence. we readily identified post-h/c divergence mutations in the reading-frame regions of the nanog gene and its pseudogenes, and in all but four cases we were able to determine the mutant and ancestral nucleotides at each site by comparison of the human and chimpanzee orthologues with the nanog gene and the other pseudogenes. some of the source-gene mutations should be distinguishable from post-insertion pseudogene mutations in our data as a nucleotide that is identical in a set of older pseudogenes, which then changes to a different nucleotide in a set of younger pseudogenes. moreover, if possible source-gene mutations can be identified, they can be used to reconstruct the evolutionary history of the pseudogene family, and to some extent the evolutionary history of the gene itself. to reconstruct the evolutionary history of the nanog gene and its pseudogene family with source-gene mutation analysis, we aligned the reading frame of the human and chimpanzee nanog gene with the corresponding sequences in all pseudogenes, and corrected post-h/c divergence mutations to reflect the ancestral sequence. we identified sites with possible source-gene mutations as a nucleotide shared by two or more pseudogenes and a different nucleotide shared by two or more additional pseudogenes. any nucleotide present in a particular position in only one pseudogene was considered as a post-insertion pseudogene mutation. a total of sites within the reading frame met these criteria for identification of possible source-gene mutations. we then identified the most parsimonious order of pseudogenes as the one which required the fewest number of source-gene mutations across these sites. the most parsimonious ordering of the nanog pseudogenes from most ancient to most recent is nanogp nanogp nanogp nanogp nanogp nanogp nanogp nanogp nanogp and nanogp as the most recent. the next most parsimonious ordering is the same as the above order but with the positions of nanogp and nanogp reversed. as a truncated pseudogene, nanogp contains only possible source-gene mutation sites. of these, only five are informative in distinguishing nanogp and nanogp three supporting nanogp as the older pseudogene and two supporting nanogp sites with only one mutation in a particular order are more likely to represent a true source-gene mutation than sites with multiple mutations, which probably consist of a combination of source-gene and post-insertion mutations. the three sites, and that support nanogp as the older pseudogene require and mutations to explain the order, respectively. the two sites that support nanogp as the older pseudogene require and mutations, respectively, to explain that order, suggesting that the most parsimonious order is also the most plausible with respect to these two pseudogenes. additionally, our analysis clarifies the relative order of nanogp and nanogp by clearly placing nanogp as the older of the two. the only notable discrepancy between the results of source-gene mutation analysis and ordering by overall similarity to the modern nanog gene is the relative placement of nanogp and nanogp in the latter analysis, the functional nanog gene is more similar to nanogp than it is to nanogp, implying that nanogp is the older pseudogene. however, source-gene mutation analysis places nanogp as the more recent of the two. examination of the mutations that distinguish nanogp from nanog provides compelling evidence that nanogp is indeed the older pseudogene. nanogp is an unprocessed pseudogene that arose from duplication of a segment of chromosome and thus may have remained functional for an undetermined period of time after its formation. as booth and holland pointed out, nanogp cannot use the same initiation codon as nanog because a mutation at position in the reading frame produced a premature termination codon after only eight amino acids. this mutation is present in both the human and chimpanzee orthologues indicating that it preceded the h/c divergence. booth and holland noted, however, that of the three characterized human transcripts from nanogp two are alternatively spliced to remove all of exon so that the nanogp reading frame begins at a position corresponding to the 58th amino acid in the protein encoded by nanog, which is an internal methionine in the nanog protein. if nanogp did indeed remain functional after its formation, we would expect natural selection to conserve the sequence within its reading frame when compared to nanog. after correction to the ancestral sequence for post-h/c divergence mutations, mutations distinguish nanogp from the nanog reading frame, and they are nonrandomly distributed. twelve are clustered in a 121-nucleotide region entirely within exon of the nanog gene, a region removed during splicing in two characterized nanogp transcripts. of the three mutations in nanogp1's apparent reading frame, two are nonsynonymous and one is synonymous. a nonsynonymous mutation at position is a guanine-to-thymine substitution that results in a lysine-to-asparagine substitution in the protein. comparison with the human and chimpanzee sequences of the other pseudogenes reveals that this is a source-gene mutation that supports nanogp as being older than nanogp comparison of this polymorphism to the sequences of the other pseudogenes reveals that the guanine in nanogp and therefore the lysine in the protein, are ancestral, and that the source-gene mutation occurred after duplication of nanogp but before insertion of nanogp interestingly, booth and holland found through experimental sequencing that this particular mutation is polymorphic in modern humans, suggesting that neither lysine nor asparagine is detrimental to protein function at this position. the other nonsynonymous mutation is a cytosine-to-thymine substitution at position resulting in a proline-to-leucine substitution in the protein. because proline and leucine have similar biochemical properties, this mutation is also not likely to adversely affect protein function. the nanog gene and all other pseudogenes in both the human and chimpanzee genomes have a cytosine residue at this position, indicating that this is a post-duplication mutation in nanogp the single synonymous mutation in the apparent reading frame is at position which lies within the homeobox region. this is clearly a source-gene mutation that also supports the ordering of nanogp as being older than nanogp only nanog, nanogp and nanogp have a cytosine at this position; all other pseudogenes, including nanogp have a thymine at this position. taken in the aggregate, these observations strongly support the hypothesis that nanogp remained functional after duplication and, therefore, was subject to selection-driven conservation of its reading frame. they also raise the possibility that nanogp may retain some functionality or that its loss of function may be evolutionarily recent. nucleotide polymorphisms at possible source-gene mutation sites may represent true source-gene mutations or post-insertion pseudogene mutations. sites in which a single mutation separates a set of older pseudogenes from a set of younger pseudogenes are the most plausible sites for identification of true source-gene mutations. in the most parsimonious ordering, of the sites contained a single possible source-gene mutation. twenty of these mutations are nonsynonymous and nine are synonymous. if a mutation is indeed a true source-gene mutation, the amino acid it encodes may be reflected in the nanog proteins of other vertebrates. to determine if this is the case, we used the amino acid sequence of the polypeptide encoded by the human nanog gene as a query for a blastp search of the protein database of all organisms. proteins from six species displayed full-length or nearly full length homology to the nanog protein: crab-eating macaque, house mouse, norway rat, domestic cattle, domestic goat, and domestic dog. we excluded a match to a computationally generated hypothetical protein in chimpanzee because it is derived from the dna sequence of chimpanzee nanogp as shown in figure several of the putative source-gene mutations and their inferred effect on amino acid sequence in the human/chimpanzee nanog pseudogene family are consistent with the corresponding amino acids in the nanog proteins of other eutherian mammals. for example, at site in the reading frame, an adenine-to-guanine substitution in the nanog gene apparently occurred after the insertion of nanogp but before the insertion of nanogp resulting in an asparagine-to-aspartic acid substitution in amino-acid residue of the polypeptide. the dog, cattle, and rat proteins have asparagine at this position, whereas the macaque, chimpanzee, and human have aspartic acid at this position. similar patterns of congruence between amino acid substitution and amino acid sequences in other mammals is evident at positions 250– and 820– of the reading frame. another feature of the putative source-gene mutations is the paucity of amino acid substitutions at source-gene mutation sites within the homeobox region indicative of high source-gene sequence conservation in this region. six possible source-gene mutation sites are present within the homeobox region. five of these six sites have only synonymous mutations. the single nonsynonymous mutation is at position with thymine present in nanogp and nanogp and cytosine present in all other pseudogenes and the nanog gene, resulting in a leucine-to-phenylalanine substitution in the nanogp and nanogp sequences. these thymines may be independent post-insertion mutations or they could be a source-gene mutation that reverted to its original sequence after the insertion of nanogp pseudogene mutations can be used to estimate the dates of origin for individual pseudogenes. however, only post-insertion mutations not subject to purifying selection are reliable indicators of the age of a pseudogene. our analysis shows that, in the case of the nanog pseudogene family, source-gene mutations are present and may contribute to a significant number of polymorphisms in the pseudogenes. although some source-gene and post-insertion mutations may be readily distinguished based on their patterns when the pseudogenes are ordered, others may not be so easily discerned. even when post-insertion mutations can be reliably identified, pseudogene evolution rates have not been well calibrated prior to the h/c divergence, as pointed out by booth and holland. for these reasons, we have avoided age estimations in this study, focusing instead on the relative order of nanog pseudogene origins.  a synthesis of the results from this article with those of booth and holland produces a straightforward evolutionary history of the nanog pseudogene family in the human and chimpanzee genomes. nanogp is the most ancient of the pseudogenes followed in order of most ancient to most recent by the processed pseudogenes nanogp nanogp nanogp nanogp nanogp nanogp and nanogp before insertion of nanogp the region on chromosome containing nanog underwent a duplication producing nanogp which remained functional and subject to selection-driven conservation of its reading frame. all of these events, and the resulting fixation of their products in the genome, preceded the h/c divergence. following the h/c divergence, nanogp inserted itself into chromosome in the human lineage. 
comparison of its sequence to the functional nanog gene's reading frame suggests that this apparent pseudogene remained functional after duplication and, therefore, was subject to selection-driven conservation of its reading frame, and that it may retain some functionality or that its loss of function may be evolutionarily recent. the nanog gene is expressed in mammalian embryonic stem cells where it maintains cellular pluripotency. this article compares the nanog gene and its pseudogenes in the human and chimpanzee genomes and derives an evolutionary history of this pseudogene family. the nanog gene and all pseudogenes except nanogp are present at their expected orthologous chromosomal positions in the chimpanzee genome when compared to the human genome, indicating that their origins predate the human-chimpanzee divergence. analysis of flanking dna sequences demonstrates that nanogp is absent from the chimpanzee genome. based on the most parsimonious ordering of inferred source-gene mutations, the deduced evolutionary origins for the nanog pseudogene family in the human and chimpanzee genomes, in order of most ancient to most recent, are nanogp nanogp nanogp nanogp nanogp nanogp nanogp nanogp and nanogp all of these pseudogenes were fixed in the genome of the human-chimpanzee common ancestor. nanogp is the most recent pseudogene and it originated exclusively in the human lineage after the human-chimpanzee divergence. an unusually large family of pseudogenes arose from it with one unprocessed and ten processed pseudogenes in the human genome.
 the nanog gene is expressed in mammalian embryonic stem cells where it maintains cellular pluripotency. an unusually large family of pseudogenes arose from it with one unprocessed and ten processed pseudogenes in the human genome. this article compares the nanog gene and its pseudogenes in the human and chimpanzee genomes and derives an evolutionary history of this pseudogene family.  the nanog gene and all pseudogenes except nanogp are present at their expected orthologous chromosomal positions in the chimpanzee genome when compared to the human genome, indicating that their origins predate the human-chimpanzee divergence. analysis of flanking dna sequences demonstrates that nanogp is absent from the chimpanzee genome.  based on the most parsimonious ordering of inferred source-gene mutations, the deduced evolutionary origins for the nanog pseudogene family in the human and chimpanzee genomes, in order of most ancient to most recent, are nanogp nanogp nanogp nanogp nanogp nanogp nanogp nanogp and nanogp all of these pseudogenes were fixed in the genome of the human-chimpanzee common ancestor. nanogp is the most recent pseudogene and it originated exclusively in the human lineage after the human-chimpanzee divergence. nanogp is apparently an unprocessed pseudogene. comparison of its sequence to the functional nanog gene's reading frame suggests that this apparent pseudogene remained functional after duplication and, therefore, was subject to selection-driven conservation of its reading frame, and that it may retain some functionality or that its loss of function may be evolutionarily recent. 