Evolution of the NANOG pseudogene family in the human and chimpanzee genomes

Background 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. Results The NANOG gene and all pseudogenes except NANOGP8 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 NANOGP8 is absent from the chimpanzee genome. Conclusion 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 NANOGP6, NANOGP5, NANOGP3, NANOGP10, NANOGP2, NANOGP9, NANOGP7, NANOGP1, and NANOGP4. All of these pseudogenes were fixed in the genome of the human-chimpanzee common ancestor. NANOGP8 is the most recent pseudogene and it originated exclusively in the human lineage after the human-chimpanzee divergence. NANOGP1 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.


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 [1][2][3]. 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 [4][5][6].
The human chromosomal region containing the NANOG gene has also undergone a tandem duplication resulting in two copies of the NANOG gene on chromosome 12. The two copies are approximately 97% identical and their transcripts are spliced differently [4,5]. Although there is EST-based evidence that both copies are transcribed, Booth and Holland [4] have argued that one of the two copies is an unprocessed pseudogene, which they named NANOGP1. They named the ten processed pseudogenes NANOGP2 through NANOGP11. Two are located on the X chromosome, two on chromosome 6, and one each on chromosomes 2, 7, 9, 10, 14, and 15. NANOGP2 and NANOGP4 through NANOGP10 are full-length or nearly full-length processed pseudogenes lacking introns. NANOGP3 and NANOGP11 are truncated fragments of processed pseudogenes [4,6].
Although several studies are directed at pseudogene families [4,6,10], most focus on the evolution of a single processed pseudogene [11][12][13][14]. The relatively large number of processed NANOG pseudogenes and the recent release of the Build 1.1 assembly of the chimpanzee (Pan troglodytes) genome [15] 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 and discussion
We identified the chimpanzee orthologues of the human NANOG gene and all of its pseudogenes except NANOGP8 using MEGABLAST and BLASTN searches of the Build 1.1 version of the chimpanzee genome assembly. Table 1 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 1.1 assembly. Our data indicate that the NANOG gene and all pseudogenes except NANOGP8 are in their expected orthologous positions in the chimpanzee genome, and that NANOGP8 is not present in the chimpanzee genome.
Chimpanzee orthologues of NANOG and NANOGP1 NANOG [GenBank:NM_024865] is the functional gene in the human genome, whereas NANOGP1 [Gen-Bank:AK097770] is apparently an unprocessed pseudogene derived from tandem duplication of the chromosomal region containing NANOG. However, cDNA and EST data show that NANOGP1 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. [5] designated NANOGP1 as NANOG2 and referred to it as a functional gene, whereas Booth and Holland [4] argued that because of its relatively high degree of divergence from NANOG, and the comparative paucity and ambiguity of transcripts derived from it, NANOGP1 is an unprocessed duplication pseudogene.
MEGABLAST searches of the chimpanzee genome readily identified the orthologues of NANOG and NANOGP1. However, the organization of the chimpanzee orthologue of the human NANOG gene in the chimpanzee Build 1.1 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 ortho- logue are present in the assembly but in two different GenBank accessions. The entire sequences of the 5' UTR, exon 1, and exon 2 are found in the region spanning nucleotides 683046 through 686855 of the chromosome 12 contig [GenBank:NW_114668], in a region on the short arm of chromosome 12 near the telomere at a location orthologous to that of the human NANOG gene at 12p13.31. Introns 1 and 2 of the chimpanzee orthologue are also within this region but large segments of them are unsequenced. The complete sequences of exon 3, intron 3, exon 4, and the 3' UTR of the chimpanzee orthologue are found in nucleotides 3808 though 5350 of another accession [GenBank:NW_115304], which is known to reside on chromosome 12 but has not been placed in the Build 1.1 assembly of this chromosome. Furthermore, exon 4 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 NANOGP1 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 2. Both of these primer combinations amplified PCR fragments of the sizes expected if the gene is intact. We sequenced these fragments (and all other amplified 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 NANOGP1, confirming that our sequences are derived from the intact chimpanzee NANOG gene. Furthermore, our sequences show that the apparent frameshift mutation in exon 4 in the Build  Figure 1, the NANOGP8 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 NANOGP2, NANOGP3, NANOGP4, NANOGP5, NANOGP6, NANOGP7, NANOGP9, NANOGP10, and NANOGP11 in the Build 1.1 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 NANOGP5 and NANOGP9 are present in the chimpanzee genome assembly. A 100-Evidence that the NANOGP8 pseudogene is absent from the chimpanzee genome Figure 1 Evidence that the NANOGP8 pseudogene is absent from the chimpanzee genome. Sequences flanking the human NANOGP8 pseudogene on chromosome 15 are present in chromosome 15 of the chimpanzee genome but the pseudogene is absent. Comparison of the human and chimpanzee sequences shows that the NANOGP8 pseudogene inserted itself into human chromosome 15 without duplication of the surrounding sequences. We attempted to amplify the chimpanzee orthologue of NANOGP9 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 NANOGP9 reading frame (based on the human sequence) and successfully amplified and sequenced a region in NANOGP9 corresponding to positions 43-841 of the 918 nucleotide-pair reading frame in the functional NANOG gene [GenBank:DQ301869]. We verified that the sequence is indeed from NANOGP9 by its high similarity to the human orthologue. This sequence further confirms the presence of NANOGP9 in the chimpanzee genome and it allowed us to compare the sequences of the human and chimpanzee orthologues.

GCCTTCAAGCATCTGTTTAA
This sequence also resolved a question about the origins of NANOGP9 and NANOGP10. Both are located on the X chromosome and both contain a 15 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 NANOGP9 and NANOGP10 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 15 nucleotide-pair tandem repeat within the reading frame. The chimpanzee NANOGP9 orthologue does not contain the deletion present in the human orthologue, whereas the chimpanzee and human orthologues of NANOGP10 have the same deletion. This observation indicates that the deletion in human NANOGP9 occurred after the H/C divergence and its origin is thus independent of the deletion in NANOGP10. Furthermore, we examined 5000 nucleotides on both sides of these pseudogenes and found no evidence of a duplication. We conclude that NANOGP9 and NANOGP10 originated independently.

Evolution of the NANOG gene and pseudogene family
The entire functional NANOG gene (according to our sequencing data) and NANOGP1 are present in both the human and chimpanzee genome assemblies at ortholo-gous chromosomal positions. In the 3' UTR of the NANOG gene, there is an Alu element, which is missing from NANOGP1 in both genomes. Therefore, the NANOGP1 unprocessed pseudogene arose through duplication of the chromosomal region containing NANOG before the human-chimpanzee (H/C) 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 NANOGP2, NANOGP3, NANOGP4, NANOGP5, NANOGP6, NANOGP7, NANOGP9, and NANOGP10 lack this Alu element. They thus likely arose before its insertion and, therefore, also predate the H/C divergence. The presence of the NANOGP11 pseudogene fragment in both the human and chimpanzee genomes likewise shows that its origin preceded H/C divergence.
The human NANOGP8 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 15 after the H/C divergence.
Based on the assumption of a pseudogene mutation rate of 1.25 × 10 -9 mutations per site per year in humans [16,17] 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 NANOGP10, which has accumulated 20 mutations since the H/C divergence, compared to 1-10 mutations for the other pseudogenes. We excluded NANOGP8 from this correction because of its absence in the chimpanzee genome. Also, since NANOGP3 is a truncated pseudogene with only 254 nucleotides within the NANOG coding region, we compared only the portions of NANOG and the other pseudogenes that aligned with these 254 nucleotides when determining the relative age of NANOGP3. The pseudogene fragment NANOGP11 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 [4]. Also similar to Booth and Holland's conclusions, our analysis showed that NANOGP3 and NANOGP5 were almost identical in the degree of similarity to NANOG (88.6% and 88.2%, respectively), and that NANOGP2 and NANOGP9 were likewise nearly identical in the degree of divergence from NANOG (94.6% and 94.4%, respectively). Thus, like Booth and Holland [4], 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) postinsertion 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 88 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 (except NANOGP11, which lacks the reading frame), and corrected (in all but four cases) post-H/C divergence mutations to reflect the ancestral sequence. We identified sites with possible sourcegene 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 68 sites (out of 918) 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 68 sites.
The most parsimonious ordering of the NANOG pseudogenes (154 possible source-gene mutations across 68 sites) from most ancient to most recent is NANOGP6, NANOGP5, NANOGP3, NANOGP10, NANOGP2, NANOGP9, NANOGP7, NANOGP1, NANOGP4, and NANOGP8 as the most recent. The next most parsimonious ordering (156 mutations) is the same as the above order but with the positions of NANOGP5 and NANOGP3 reversed. As a truncated pseudogene, NANOGP3 contains only 19 possible source-gene mutation sites. Of these, only five are informative in distinguishing NANOGP3 and NANOGP5, three supporting NANOGP5 as the older pseudogene and two supporting NANOGP3. 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, 399, 531, and 568, that support NANOGP5 as the older pseudogene require 1, 2, and 1 mutations to explain the order, respectively. The two sites that support NANOGP3 as the older pseudogene (sites 390 and 566) require 5 and 4 mutations, respectively, to explain that order, suggesting that the most parsimonious order (NANOGP5 older than NANOGP3) is also the most plausible with respect to these two pseudogenes. Additionally, our analysis clarifies the relative order of NANOGP2 and NANOGP9 by clearly placing NANOGP2 as the older of the two (reversing their positions in the order requires 168 mutations).
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 NANOGP1 and NANOGP4. In the latter analysis, the functional NANOG gene is more similar to NANOGP1 (98.6%) than it is to NANOGP4 (96.4%), implying that NANOGP4 is the older pseudogene. However, source-gene mutation analysis places NANOGP4 as the more recent of the two. Examination of the mutations that distinguish NANOGP1 from NANOG provides compelling evidence that NANOGP1 is indeed the older pseudogene. NANOGP1 is an unprocessed pseudogene that arose from duplication of a segment of chromosome 12, and thus may have remained functional for an undetermined period of time after its formation. As Booth and Holland [4] pointed out, NANOGP1 cannot use the same initiation codon as NANOG because a mutation at position 25 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 NANOGP1, two are alternatively spliced to remove all of exon 1, so that the NANOGP1 reading frame begins at a position corresponding to the 58 th amino acid in the protein encoded by NANOG, which is an internal methionine in the NANOG protein. If NANOGP1 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, 15 mutations distinguish NANOGP1 from the NANOG reading frame, and they are nonrandomly distributed. Twelve are clustered in a 121nucleotide region entirely within exon 1 of the NANOG gene, a region removed during splicing in two characterized NANOGP1 transcripts. Of the three mutations in NANOGP1's apparent reading frame, two are nonsynonymous and one is synonymous. A nonsynonymous mutation at position 246 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 NANOGP1 as being older than NANOGP4. Comparison of this polymorphism to the sequences of the other pseudogenes reveals that the guanine in NANOGP1, and therefore the lysine in the protein, are ancestral, and that the source-gene mutation occurred after duplication of NANOGP1 but before insertion of NANOGP4. Interestingly, Booth and Holland [4] found through experimental sequencing that this particular mutation (and amino acid substitution) 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-tothymine substitution at position 477, 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 NANOGP1.
The single synonymous mutation in the apparent reading frame is at position 384, which lies within the homeobox region. This is clearly a source-gene mutation that also supports the ordering of NANOGP1 as being older than NANOGP4. Only NANOG, NANOGP4, and NANOGP8 have a cytosine at this position; all other pseudogenes, including NANOGP1, have a thymine at this position.
Taken in the aggregate, these observations strongly support the hypothesis that NANOGP1 remained functional after duplication and, therefore, was subject to selectiondriven conservation of its reading frame. They also raise the possibility that NANOGP1 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, 29 of the 68 sites contained a single possible source-gene mutation (Figure 2). 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. As shown in Figure 2, several of the putative source-gene mutations and their inferred effect on amino acid Potential single source-gene mutations in the most parsimonious ordering of the NANOG pseudogenes by source-gene muta-tion analysis  Eight Species 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 52 in the reading frame, an adenine-toguanine substitution in the NANOG gene apparently occurred after the insertion of NANOGP10 but before the insertion of NANOGP2, resulting in an asparagine-toaspartic acid substitution in amino-acid residue 18 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 250-251, 275, 568, 713, 817, and 820-821 of the reading frame ( Figure 2).

T T -C C C C C C C C 821 A A -A A A A T T T T 903 G G -G/A A A A A A A
Another feature of the putative source-gene mutations is the paucity of amino acid substitutions at source-gene mutation sites within the homeobox region (positions 283-462 in the reading frame) indicative of high sourcegene sequence conservation in this region. Six possible source-gene mutation sites are present within the homeobox region (three of which are single-mutation sites depicted in Figure 2). Five of these six sites have only synonymous mutations. The single nonsynonymous mutation is at position 358, with thymine present in NANOGP7 and NANOGP9 and cytosine present in all other pseudogenes and the NANOG gene, resulting in a leucine-to-phenylalanine substitution in the NANOGP7 and NANOGP9 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 NANOGP7.
Pseudogene mutations can be used to estimate the dates of origin for individual pseudogenes. However, only postinsertion 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 [4]. For these reasons, we have avoided age estimations in this study, focusing instead on the relative order of NANOG pseudogene origins.

Conclusion
A synthesis of the results from this article with those of Booth and Holland [4] produces a straightforward evolu-tionary history of the NANOG pseudogene family in the human and chimpanzee genomes. NANOGP6 is the most ancient of the pseudogenes followed in order of most ancient to most recent by the processed pseudogenes NANOGP5, NANOGP3, NANOGP10, NANOGP2, NANOGP9, NANOGP7, and NANOGP4. Before insertion of NANOGP4, the region on chromosome 12 containing NANOG underwent a duplication producing NANOGP1, which remained functional and subject to selectiondriven 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, NANOGP8 inserted itself into chromosome 15 in the human lineage.

DNA amplification, cloning, and sequencing
We obtained chimpanzee DNA (individual PR00226) from the Integrated Primate Biomaterials and Information Resource (IPBIR) of the Coriell Institute for Medical Research (Camden, NJ, USA). We selected sequences for PCR primers specific to the chimpanzee NANOG gene by comparing the NANOG and NANOGP1 sequences from the Build 1.1 assembly and selecting sites with at least two variant nucleotides, with a variant nucleotide on the 3' end of each primer. We selected primer sequences for the NANOGP9 reading frame by identifying sites that contained two variants unique to human NANOGP9 with a variant nucleotide on the 3' end of each primer. All oligonucleotide primers were manufactured by Integrated DNA Technologies, (Coralville, IA, USA). We amplified DNA using AccuprimeT Hi-Fidelity Taq polymerase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendation at 2.5 mM MgCl 2 . The PCR amplification protocol consisted of an initial denaturation step of 1.5 min at 94°C, followed by 35 cycles of amplification consisting of 30 s denaturation at 94°C, 30 s for primer annealing at 57°C and between 1 and 5 min of extension at 68°C, depending on the anticipated product size (1 min/1 kb). We cloned the resulting amplicon using the pGEM-T Easy Vector System II (Promega, Madison, WI, USA), and identified recombinant clones by standard blue/white screening methods with IPTG and X-Gal. We purified plasmid DNA from each selected recombinant clone using a GenEluteTM plasmid miniprep Kit (Sigma, St. Louis, MO, USA) and quantified the DNA using a spectrophotometer. Isolated plasmid DNA was sequenced bidirectionally from M13 (F/R) primers. A 3,889 nucleotide-pair clone containing exon 1, intron 1 and part of exon 2 of the NANOG gene was sequenced by primer walking. DNA sequencing was performed at the Brigham Young University DNA Sequencing Center (Provo, UT, USA) using standard ABI Prism Taq dye-terminator cyclesequencing methodology. DNA sequence chromatograms were analyzed with the Contig Express program in the Vector NTI software suite (InforMax, Frederick, MD, USA).

DNA sequence analysis
To initially identify the locations and DNA sequences of the NANOG gene and its pseudogenes in the chimpanzee genome, we used the GenBank entries for the human NANOG gene and its 11 pseudogenes as queries for MEGABLAST searches of the chimpanzee genome Build 1.1 assembly with default settings including filtering for repetitive sequences. After identifying the genes and pseudogenes in the chimpanzee genome, we copied the sequences and alignments then confirmed and refined them with the "align two sequences" (bl2seq) BLAST tool with a word size of seven and filtering disabled. We further refined the alignments manually, especially on the ends of the sequences where word-size limitations failed at times to identify true alignments.
We copied flanking DNA sequences on both sides of the human NANOGP8 and NANOGP9 pseudogenes and used them as MEGABLAST queries with default settings and filtering to search the chimpanzee genome to confirm whether or not these pseudogenes were present. After MEGABLAST identified the sequences, we refined alignments with the bl2seq tool with a word size of seven and filtering disabled and with manual refinements.
To facilitate determination of the evolutionary order of pseudogene origin, we copied the reading frame of the functional human NANOG gene and used it as a query in the bl2seq tool with a word size of seven and filtering disabled to determine the best alignment with the corresponding regions of each of the pseudogenes except NANOGP11, which does not include the reading-frame region. We used these alignments to generate a multiple alignment of the reading-frame region of human and chimpanzee orthologues of the NANOG gene and all pseudogenes except NANOGP11. This multiple alignment allowed us to identify post-H/C divergence mutations and correct them to reflect the ancestral sequences, and to identify and distinguish between potential source-gene mutations and post-insertion mutations in the pseudogenes, as described in the results and discussion section.