MBE Advance Access originally published online on July 21, 2007
Molecular Biology and Evolution 2007 24(10):2203-2212; doi:10.1093/molbev/msm146
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Research Articles |
Inactivation of MOXD2 and S100A15A by Exon Deletion during Human Evolution
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* Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD
Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
E-mail: bk{at}nih.gov.
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResults and DiscussionConclusionSupplementary MaterialAcknowledgementsReferences |
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We devised a bioinformatics method for systematic identification of putative human-specific exon-deletion mutations that occurred after the divergence of human and chimpanzee and experimentally verified 2 of the predicted mutations in MOXD2 and S100A15A genes. MOXD2 gene encodes a monooxygenase that is highly conserved in mammals and is mostly expressed in the olfactory epithelium in mouse. The presence of a deletion of the last 2 exons and a polymorphic nonsense mutation in exon 6 suggests that MOXD2 gene is inactive in humans. S100A15A is a member of the S100 family of calcium-binding proteins, the mouse ortholog of which is expressed during epidermal maturation. Human S100A15A gene is likely to be inactive because the start codon–bearing exon is deleted in human. We propose that modification or inactivation of MOXD2 and S100A15A genes have contributed to the loss of certain smell sense in humans and to the development of human skin.
Key Words: human • chimpanzee • exon deletion • gene inactivation
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSupplementary MaterialAcknowledgementsReferences |
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Humans (Homo sapiens) and chimpanzees (Pan troglodytes) diverged from a common ancestor around 6 MYA (Glazko and Nei 2003
; Kumar et al. 2005; Patterson et al. 2006). Since then, humans have acquired various phenotypic traits distinct from other great apes (reviewed in Varki and Altheide 2005). Many of these traits represent reduction or loss of great ape features, such as small jaws and teeth, weakened jaw muscles, decreased smell sensitivity, and reduced body hair. Some of these diminished features have been proposed to be associated with a gain of beneficial traits in humans. For example, smooth hairless skin was suggested to be evolved for enhanced thermoregulation (Folk and Semken 1991) and/or for reduction of parasite loads (Pagel and Bodmer 2003). The human-specific 2-bp deletion in the coding region of MYH16 gene resulting in frameshift and premature termination was suggested to have caused the reduction of jaw muscle and allowed humans to have bigger brains (Stedman et al. 2004). These examples accord with the "less-is-more" model, according to which the gene loss is one of the mechanisms for acquisition of novel phenotypic traits during evolution (Olson 1999; Olson and Varki 2003).
Release of the complete human genome sequence (International Human Genome Sequencing Consortium 2004) and the draft chimpanzee genome sequence (The Chimpanzee Sequencing and Analysis Consortium 2005) enables one to directly compare the 2 genome sequence data for a systematic identification of human-specific genetic changes (Pollard et al. 2006 and reviewed in Li and Saunders 2005; Varki and Altheide 2005; Kehrer-Sawatzki and Cooper 2007). Several strategies were employed to systematically identify genes of which coding region had been disrupted in the human lineage after the divergence of humans and chimpanzees. For examples, human-specific frameshift mutations (Hahn and Lee 2005) and nonsense mutations (Hahn and Lee 2006) were identified, some of which were proposed to lead to a loss or modification of function of the affected genes. Analysis of a nonprocessed human pseudogene data set also yielded a large number of genes that seemed to be specifically pseudogenized in humans (Wang et al. 2006). In this study, we developed a bioinformatics procedure for prediction of exon-deletion mutations in the human genome that might have occurred after the human–chimpanzee split. We then experimentally verified the presence of the deletions in 2 highly plausible candidates, MOXD2 and S100A15A genes. We collected sequence data of orthologs and homologs for each gene for molecular evolutionary analyses. We also discuss possible phenotypic changes that might have been caused by the loss of these genes during human evolution.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSupplementary MaterialAcknowledgementsReferences |
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Data Sets
We downloaded human genome versus chimpanzee genome alignment data, mRNA versus chimpanzee genome alignment data, and other genome annotation data from the University of California Santa Cruz (UCSC) Genome Browser Database (http://genome.ucsc.edu/) (Hinrichs et al. 2006
) as of July 2006. Both of the human and the chimpanzee genomes were March 2006 assemblies. For the selection of nonhuman vertebrate mRNA sequences, we used the National Center for Biotechnology Information (NCBI) Taxonomy Browser database (http://www.ncbi.nlm.nih.gov/Taxonomy/).
Bioinformatics Prediction of Human-Specific Exon-Deletion Mutations
First, we collected chimpanzee-only genomic fragments by filtering the database table panTro2.netHg18. The chimpanzee-only regions that fulfilled the following conditions were selected: the region does not align with a syntenic region of the human genome; it is longer than or equal to 100 bp in length; it does not match a nonsyntenic region of the human genome; and the syntenic region of the human genome does not contain a sequencing gap. We obtained 16,524 chimpanzee-only fragments that meet all of these criteria. These fragments may include chimpanzee-specific insertions as well as human-specific deletions.
Next, we collected only those fragments that possibly contain an exon by inspecting whether they overlap with any nonhuman vertebrate mRNA sequence. For the alignment data of the nonhuman and nonchimpanzee vertebrate RefSeqs and mRNAs to the chimpanzee genome, we used the database tables panTro2.xenoRefSeqAli and panTro2.xenoMrna. There were 819 chimpanzee genome fragments that were absent in the human genome and aligned with a nonhuman vertebrate mRNA by at least 10 bp. By using the coding region data of mRNAs derived from the database table hg18.cds, we observed that 33 of these matched a coding region of a nonhuman gene.
As the final step, we manually scrutinized the 33 candidates to collect highly plausible instances. We excluded the cases showing low sequence quality in genome, short or poor alignment between the genome sequences, uncertain orthology between the 2 species, or chimpanzee-specific insertion. In some cases, which were also excluded, the collected chimpanzee-only fragment matched human mRNAs or expressed sequence tags (ESTs), raising the possibility of a miss-assembly of the human genome, false alignment of the 2 genomes, or deletion polymorphism among human population.
Genomic DNA Samples and Polymerase Chain Reaction
Human and nonhuman primate genomic DNA samples were purchased from the Coriell Cell Repositories (http://ccr.coriell.org/). The repository numbers are as follows Mbuti Pygmy, NA10492; Basque, NA15884; Icelandic, NA15756; Northern European, NA17003; Southern Chinese, NA11321; Surui, NA10970; chimpanzee, NG06939; bonobo (Pan paniscus), NG05253; gorilla (Gorilla gorilla), NG05251; Sumatran orangutan (Pongo pygmaeus abelii), NG12256; pigtailed macaque (Macaca nemestrina), NG08452; and common woolly monkey (Lagothrix lagotricha), NG05356. We performed genomic polymerase chain reaction (PCR) by using aforementioned DNA samples in order to detect the predicted deletions of MOXD2 and S100A15A genes. PCR primer sequences are available in supplementary table 1 (Supplementary Material online). The primers used in this study were synthesized by Invitrogen (Carlsbad, CA).
Sequence Analysis of MOXD2 and S100A15A Genes
We determined full coding sequences of MOXD2 and S100A15A genes from gorilla and orangutan genomes by direct sequencing of amplified exons. Primer sequences used for amplification of exons are presented in supplementary table 1 (Supplementary Material online). The PCR products were sequenced by Macrogen USA (Rockville, MD). The sequences have been deposited in GenBank (http://www.ncbi.nlm.nih.gov/GenBank/) under accession numbers EF044235 (gorilla MOXD2), EF044236 (Sumatran orangutan MOXD2), EF044237 (gorilla S100A15A), and EF044238 (Sumatran orangutan S100A15A). We also collected mammalian orthologs of MOXD2 and S100A15A by deducing coding sequences from the genome assemblies available in the UCSC Genome Browser Database, mRNAs or genomic sequences in GenBank, and the whole-genome shotgun (WGS) clone sequences from the NCBI Trace Archive (http://www.ncbi.nlm.nih.gov/Traces/). We carried out Blast searches of the nonredundant protein database at NCBI web site (http://www.ncbi.nlm.nih.gov/BLAST/) to gather homologous proteins of MOXD2 and S100A15A, respectively. Presence of a putative signal peptide, a glycosylphosphatidylinositol (GPI) anchor signal, and functional domains of the proteins were inferred by using SignalP (http://www.cbs.dtu.dk/services/SignalP/) (Bendtsen et al. 2004), DGPI (http://129.194.185.165/dgpi/), and Pfam (http://pfam.janelia.org/) (Finn et al. 2006), respectively. A multiple alignment of orthologous and homologous protein sequences was prepared using ClustalW (Thompson et al. 1994) and CHROMA (Goodstadt and Ponting 2001). A phylogenetic analysis was performed by using MEGA3 (Kumar et al. 2004). All the accession numbers analyzed in this study are given in supplementary table 2 (Supplementary Material online).
Molecular Evolutionary Analyses
The estimated nonsynonymous/synonymous mutation ratios (
= dN/dS) were computed using the maximum likelihood method for codons (CODEML program in the PAML package, v3.15 obtained from http://abacus.gene.ucl.ac.uk/software/paml.html) (Yang 1997). We used the 
2 test on the log likelihood difference to decide if the differences in values between different lineages and different models were significant (Yang 1998; Stedman et al. 2004). The probabilities were computed by using the "Chi Square Calculator" (available at http://www.stat.tamu.edu/
west/applets/chisqdemo.html). Coding sequences of MOXD2 and S100A15A genes from human, chimpanzee, gorilla, orangutan, rhesus macaque, and marmoset were aligned by using ClustalW (Thompson et al. 1994). The region that is deleted in human and the stop codon positions have been removed from the alignment prior to analyses.
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Results and Discussion |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSupplementary MaterialAcknowledgementsReferences |
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Identification of Putative Human-Specific Exon-Deletion Mutations
We developed a procedure for identification of putative human-specific exon-deletion mutation candidates as described in the Materials and Methods. We manually inspected 33 candidates to narrow them to 6 highly plausible cases, which we named after the mouse orthologs as ADAM5, CMAH, MOXD2, MSR2, NLRP10, and S100A15A. The exon deletion and subsequent inactivation of the CMAH gene has been previously reported (Chou et al. 1998
). In the cases of ADAM5 and MSR2, the chimpanzee orthologs also seem to be defective due to frameshift and/or nonsense mutations. The human NLRP10 (also known as NALP10) protein which is involved in inflammation (Tschopp et al. 2003) exhibits altered C-terminus when compared with putative chimpanzee ortholog due to a deletion encompassing the 3' part of the last exon and the following flanking region. Comparison of mouse and chimpanzee NLRP10 orthologs showed relatively high divergence at their C-termini. This suggests that a variation of the C-terminus of this protein is common among mammals and not necessarily specific to humans. Although these 3 genes could prove to be interesting, we concentrated on MOXD2 and S100A15A for this study because their respective orthologs from mouse and chimpanzee encode proteins that are highly conserved.
Human MOXD2 and S100A15A Genes Are Predicted to Have a Deletion
MOXD2 is the unannotated human ortholog of mouse Moxd2, which encodes a protein named "monooxygenase, DBH-like 2" (GenBank accession number NM_139296). Analysis of the sequence alignment between human and chimpanzee genome assemblies showed a chimpanzee-only segment of 3658 bp in length (from 142,904,107 to 142,907,764 of the chimpanzee chromosome 7), which matched 2 exons of mouse Moxd2 mRNA (fig. 1A). The corresponding genomic segments of human and chimpanzee were retrieved. The exons of human and chimpanzee MOXD2 genes were identified based on mouse Moxd2 gene. A dot matrix comparison (Sonnhammer and Durbin 1995) of the human and chimpanzee MOXD2 genes revealed a deletion in the human chromosome 7 (fig. 1B). The deletion removed the last 2 of the 13 coding exons including 3' untranslated region (UTR).
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The S100A15A is an unannotated gene in the human chromosome 1. Initial analysis of the sequence alignment data showed a 1501-bp-long segment in the chimpanzee genome (from 132,521,559 to 132,523,059 of the chimpanzee chromosome 1), which overlaps with an exon of mouse S100a15 mRNA but is missing in the human genome (fig. 1C). The mouse S100a15 gene has 3 exons, the coding region spanning from exon 2 to exon 3. A dot-plot analysis revealed that a deletion in the human S100A15A gene eliminated the start codon-containing exon 2 (fig. 1D). We named this S100A15A in order to distinguish it from the known human gene S100A7a, which is also known as S100A15 because it is commonly thought as orthologous to mouse S100a15 (Wolf et al. 2003
, 2006). However, our sequence comparison shows that true human ortholog of mouse S100a15 is the newly found S100A15A and that mouse has lost the ortholog of S100A7 group of human genes (see below).
Experimental Validation of Exon Deletion in MOXD2 and S100A15A Genes
In order to experimentally verify the predicted deletions in human MOXD2 and S100A15A genes, we performed genomic PCR analysis using human DNA samples and nonhuman primate DNA samples (fig. 2). Six human DNA samples with diverse geographical origin and nonhuman DNA samples isolated from 6 primate species were obtained from the Coriell Cell Repositories. Two pairs of PCR primers were designed to amplify the breakpoint junction and a part of the deleted segment, respectively, for each of MOXD2 and S100A15A genes (see fig. 1B and D for primer locations). PCRs using the primer pair designed to encompass the breakpoint yielded smaller products from genomic DNA of all of the human individuals but larger products from the nonhuman primates for both MOXD2 (fig. 2A) and S100A15A (fig. 2C) genes. The size reduction of the amplified segment clearly demonstrates that the predicted deletion is present in the human genomes. We also tried to amplify the exons that are missing from the human genome. We successfully observed amplified bands from the nonhuman primate species but not from the humans (fig. 2B and D). All amplified products were in the expected size range depending on the presence or absence of the deletion. It is not clear whether the absence of amplified bands in the case of the woolly monkey S100A15A gene is due to an independent deletion or to mismatches in primers which were designed based on intron sequences of the chimpanzee and the rhesus macaque (Macaca mulatta) S100A15A.
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MOXD2 Is a Membrane-Bound Monooxygenase in Olfactory Epithelium
MOXD2 proteins are highly conserved among mammals including opossum (Monodelphis domestica), a marsupial (Supplementary fig. 1, Supplementary Material online). Protein sequence identity between the chimpanzee and the opossum orthologs is 82%. The exceptional level of conservation implies that this protein may play an important role in mammalian biology. MOXD2 contains a signal peptide, a GPI anchor signal, and a catalytic core of the copper monooxygenase, indicating that the protein is a membrane-associated enzyme. MOXD2 shows sequence similarity to dopamine ß-hydroxylase (DBH) and MOXD1 (monooxygenase, DBH-like 1) (see supplementary fig. 1, Supplementary Material online). Sequence comparison, exon organization, and phylogenetic analysis indicate that MOXD2/MOXD1/DBH genes originated from a common ancestor by gene duplication and divergence (fig. 3). The DBH protein, which does not have a GPI anchor signal in its C-terminus, is also attached to the membrane by using the uncleaved signal peptide as the anchor (Feng et al. 1992
; Houhou et al. 1995). Cleavage of the signal peptide also generates soluble form of the enzyme. DBH converts dopamine to norepinephrine in the synaptic vesicles of postganglionic sympathetic neurons, and its mutation causes several psychiatric disorders (Kim et al. 2002; Cubells and Zabetian 2004; Timmers et al. 2004). The MOXD1 protein is composed of the similar domains when compared with MOXD2. The divergence of MOXD1 and MOXD2 seems to have occurred in an ancestral chordate. MOXD1 proteins form tightly membrane-associated oligomeric enzymes that are predicted to hydroxylate a hydrophobic substrate in the endoplasmic reticulum (Xin et al. 2004). The mouse Moxd2 gene is highly expressed in the medial olfactory epithelium (the GNF Expression Atlas 2 [Su et al. 2004] track at the UCSC Genome Browser Database). The specialized expression pattern and the molecular function of homologs suggest that Moxd2 protein may play a role in olfactory sense by converting one neurotransmitter to another or by modifying incoming odorant molecules. Loss of functional MOXD2 might be implicated in the alteration of smell sensitivity of the human.
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The deletion in the human MOXD2 gene removes 3' UTR and the poly(A) signal as well as coding region in the last 2 terminal exons. Loss of the 3' UTR regulatory elements may interfere with proper mRNA processing and/or translation. In addition to the deletion, the gene suffers a nonsense mutation (codon 305, CGA/TGA) within exon 6, which is polymorphic in human population (http://www.ncbi.nlm.nih.gov/projects/SNP/; the Single Nucleotide Polymorphism database [dbSNP] accession number rs4376428). The dbSNP record indicates that the nonsense allele is rather frequent in Asians but scarce in Africans. The presence of the secondary mutation strengthens the notion that the gene is inactive in humans. However, because the promoter and the preceding exons may be intact in alleles without a nonsense mutation in exon 6, we cannot rule out the possibility that the gene is still transcribed and even produces the truncated proteins. The expression of the gene in the pooled human tissue has been observed as ESTs, for examples, DY654834 and DY655575.
MOXD2 Genes Are Mutated in Higher Primates
When we analyzed MOXD2/Moxd2 orthologs of mammalian species derived from various sources including sequences of amplified genomic fragments and the WGS clones at the NCBI Trace Archive, we found more cases of disruptive mutations in higher primates. Besides the deletion and the nonsense mutations in the human, we observed 2 nonsense mutations and a splice site mutation in the orangutan MOXD2 and a frameshift mutation in the rhesus macaque ortholog (supplementary fig. 1, Supplementary Material online). The 2 nonsense mutations, one in exon 8 (codon 402, TAA) and the other in exon 13 (codon 570, TAG), were detected in both Sumatran orangutans and Bornean orangutans (Pongo pygmaeus pygmaeus). The second nonsense mutation exhibits polymorphism in Sumatran orangutans: CAG/TAG. The splice donor consensus sequence GT of intron 3 was absent in Sumatran orangutans due to a 2-bp deletion but present in their Bornean cousins. The frequent incidence of such deleterious mutations in the orangutan MOXD2 gene indicates that the gene also became not in use in the early stage of the orangutan evolution and is now decaying. It is uncertain whether the independent inactivation of the gene in orangutan features the same phenotypic change as in human.
The rhesus macaque genome assembly (January 2006 freeze) and the WGS clone sequences examined reveal a 13-nt deletion in the last exon of the MOXD2 gene, resulting in frameshift and premature termination. The altered open reading frame would encode a protein with 586 residues. Other primate genes without a mutation would produce proteins with 618 amino acids. The mutation should not modify the enzymatic core of the protein but abolishes the GPI anchor sequence at its C-terminus. Therefore, the enzyme would be soluble, if not attached to the membrane by using a signal anchor as in the case of DBH. It is not clear whether the altered form of MOXD2 protein in the rhesus macaque confers similar enzymatic activity and biological role as in other species.
S100A15A Is Involved in Skin Differentiation
We compared orthologous S100A15A proteins deduced from various sequence data of mammalian species (supplementary fig. 2, Supplementary Material online). The orthologous protein sequences were well conserved even in a marsupial species. The chimpanzee and the opossum orthologs share 68% sequence identity. Unlike the case of MOXD2 genes, we do not find any mutation causing coding disruption in nonhuman S100A15A genes examined. The S100A15A protein contains 2 calcium-binding domains and belongs to the subfamily A of the family of S100 proteins (supplementary fig. 2, Supplementary Material online). S100 proteins are involved in cell growth and differentiation, cell cycle regulation, and metabolic control (Donato 2003). Many members are implicated in human diseases, including skin disease, as well as cancer pathogenesis (Heizmann et al. 2002; Eckert et al. 2004; Emberley et al. 2004).
More than 20 genes encoding S100 proteins exist as a cluster in a genomic locus of each mammalian genome (e.g., on human chromosome 1q21.3 and on mouse chromosome 3qF1, according to the UCSC Genome Browser Database) (Marenholz et al. 2004; Ravasi et al. 2004). The multigenic nature and lineage-dependent expansion or pseudogenization of some members make nomenclature of the S100 protein genes and correct assignment of orthologs complicated (Marenholz et al. 2004, 2006). For example, the mouse S100a15 protein was considered as the ortholog of human S100A7a and S100A7c (also known as Psoriasin) in recent studies (Webb et al. 2005; Eckert and Lee 2006; Wolf et al. 2006) and was renamed as S100a7a (Marenholz et al. 2006). However, a phylogenetic analysis of mammalian S100A proteins including newly identified S100A15A orthologs indicates that S100A7 and S100A15A are distinct from each other even though they are the closest members among S100A proteins (fig. 4). Human and chimpanzee S100A7a and S100A7c show 32–33% amino acid sequence identity, respectively, with mouse S100a15, whereas the full-length chimpanzee S100A15A and mouse S100a15 share 84% amino acid identity. The S100A15A and S100A7 genes, which are tandemly located in the genome, seem to have been formed by a segmental duplication before the mammal emerged because the opossum, a marsupial species, also possesses S100A15A. The S100A7 proteins are found in horse and cow but not in mouse, suggesting that it was lost during rodent evolution. As a result, the mouse S100a15 and the human S100A7 are the most similar members between the 2 species, despite their low level of sequence identity, that are still functioning in the cells of each species. However, they must be considered evolutionarily paralogous on which disparate selection pressure might have been exerted. In order to build a more accurate evolutionary history and a better orthology relationship of S100A proteins, more number of genomes need to be inspected, including rhesus macaque, mouse, rat, cow, and dog.
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Mouse S100a15 mRNAs and/or proteins were detected during skin maturation, especially in differentiating cells of the hair follicles and the cornified layer (Webb et al. 2005
; Wolf et al. 2006). It is also expressed in mouse mammary gland and is upregulated during mammary tumorigenesis (Webb et al. 2005) Mouse S100a15 may have similar function to human S100A7 because they are the closest members in the S100A protein family. However, the 2 proteins have low sequence similarity even in the same species, for example, the sequence identity between S100A7a and S100A15A in chimpanzee is 33%. Chimpanzee S100A7a and S100A7c share 94% sequence identity. The conservation levels of the S100A7 orthologs and S100A15A orthologs among mammals are quite different: the sequence identity between the S100A15A orthologs of chimpanzee and cow is 87% but that between chimpanzee S100A7a and cow S100A7 is 62%. Accelerated divergence among S100A7 proteins and duplicative expansion of S100A7 genes in primates suggests that S100A7 may confer specialized function in some mammalian lineages such as primates, whereas S100A15A retains more basal function for all mammalian species. Thus, although S100A7 may have substituted some of the functions of S100A15A, the substitution may not have been complete, in which case the loss of S100A15A gene function might effect human-specific features in the structure or physiology of human skin.
Complex Evolutionary History of MOXD2 and S100A15A in Great Apes
To perform molecular evolutionary analyses, we prepared multiple sequence alignment of human, chimpanzee, orangutan, gorilla, rhesus macaque, and marmoset coding sequences of MOXD2 and S100A15A genes. The alignments include 1492 bp for MOXD2 and 174 bp for S100A15A. The nonsynonymous/synonymous substitution ratios ( = dN/dS) were computed based on the codon maximum likelihood method (Yang 1998; Stedman et al. 2004) by using CODEML program in the PAML package (Yang 1997). The estimated numbers of nonsynonymous (N) and synonymous (S) substitutions were calculated along each branch.
Analysis of MOXD2 orthologs revealed an elevated nonsynonymous change (N > S) in each of the branches leading to human, gorilla, and orangutan genes (fig. 5A). The estimated values for these branches are 2.4709, 1.4315, and 0.5157, respectively (see supplementary table 3A [Supplementary Material online] for all values). According to the log likelihood ratio test (Yang 1998), values for the human (H) and the gorilla (G) are significantly different from that of background value 0 (fig. 5A and test numbers 1 and 3 in supplementary table 3B; Supplementary Material online). They are larger than 1, but we cannot reject the null hypothesis that they are equal to 1 (test numbers 7 and 8 in supplementary table 3B, Supplementary Material online), indicating that these genes are not under strongly positive or negative (purifying) selection. It is also notable that there is no nonsynonymous substitution along the branch either for the human–chimpanzee ancestor or for the chimpanzee, indicating a strong purifying selection in these branches.
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= 3.84). Double asterisk indicates extremely significant (P < 0.01; Comparative analysis of S100A15A orthologs also reveals accelerated nonsynonymous substitutions in the human and the gorilla lineages that are statistically significant compared with the background level (fig. 5B and supplementary tables 4A and 4B; Supplementary Material online). The
value of human S100A15A (H) is infinite because the number of synonymous substitution is 0. However, again we cannot reject the null hypothesis that it is equal to 1 (test number 7 in supplementary table 4B, Supplementary Material online). Strong purifying selection is hinted at in the orangutan, the human–chimpanzee ancestor, and the human–chimpanzee–gorilla ancestor branches. It is important to note that the small size of S100A15A sequence analyzed (174 bp) may restrict computation of reliable values.
Possible Mechanism for Exon-Deletion Mutation
A mechanism known as Alu recombination–mediated deletion has been reported to play a role in the human-specific inactivation of the CMAH (Hayakawa et al. 2001) and other genes (Sen et al. 2006). However, no Alu repeats or other repetitive elements are present near the deletion boundaries of the MOXD2 and the S100A15A genes in the human and the chimpanzee genome. The cleaved and rejoined regions do not show any other special sequence features such as inverted repeats in either of the cases. Sequence alignment between the human and the chimpanzee genomic sequences surrounding the breakpoint junction shows no mismatched or additional sequences in each of the cases, indicating that the genomic fragment was cleanly removed. We assume that the deletion in the MOXD2 and the S100A15A genes were mediated by DNA double-strand break and nonhomologous end joining (Cahill et al. 2006).
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Conclusion |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSupplementary MaterialAcknowledgementsReferences |
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In summary, we developed a semiautomatic procedure for systematic identification of human-specific exon-deletion mutations and experimentally verified those in the MOXD2 and the S100A15A genes. The MOXD2 gene encodes a monooxygenase of which mouse ortholog is highly expressed in the olfactory sensory organ. Inactivation of this gene in humans may have resulted in an alteration of the olfactory sense. A preferential expression of mouse S100a15 gene in the skin suggests that the loss of this protein in humans may contribute to the structural or physiological difference of the human skin from other mammals.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSupplementary MaterialAcknowledgementsReferences |
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Supplementary tables 1–4 and figures 1 and 2 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionSupplementary MaterialAcknowledgementsReferences |
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This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. Funding to pay the Open Access publication charges for this article was provided by the Intramural Research Program of the National Institutes of Health.
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Footnotes |
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1 Present address: Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul, South Korea
2 Present address: Center for Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea
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