Nucleotide sequence of the haptoglobin and haptoglobin-related gene pair. The haptoglobin-related gene contains a retrovirus-like element

Thirty-three kilobase pairs (kb) of human DNA containing the haptoglobin (Hp) and haptoglobin-related (Hpr) gene pair were cloned, and the nucleotide sequence of 21-kb DNA was determined. The two genes are closely linked, with Hpr being 2.2 kb downstream of Hp. Six hundred nucleotides of DNA occur between the two genes that are not found either 5' to the Hp gene or 3' to the Hpr gene. After the duplication event, the first intron of the Hpr gene acquired a 9-kb insert consisting mainly of a retrovirus-like element with a potential primer-binding site homologous to a mouse isoleucine tRNA. The element forms a repeated family in the human genome that I name RTVL-I (retrovirus-like element-isoleucine). In the coding region of the Hpr gene, there are no frameshift or nonsense mutations and its exon-intron splicing sites, 5' flanking and 3' flanking sequences do not show any obvious defects. There are 28 amino acid differences between the decoded amino acid sequences of the Hpr and Hp genes. Sixteen of these differences occur in the hpr beta chain, and all appear to be located on the surface of the molecule in places not thought to be involved in the hemoglobin binding function of haptoglobin. The structure of the Hpr gene suggests that the gene may be expressed and give rise to a functional product.     

Structure and expression of the human haptoglobin locus.

Human genomic clones of the haptoglobin Hp1F and the "haptoglobin related' gene (Hpr) have been isolated. The two genes are adjacent, spanning a region of approximately 21 kb. A comparison of their coding sequences shows that Hpr differs from Hp1F at 28 codons. Northern blot and primer elongation analyses with human liver RNA show that the haptoglobin gene Hp1F appears to be transcribed some 1000-fold less in fetal than in adult liver. In adult liver the amount of Hpr mRNA is at the lower limit of detection, therefore the extent of its expression is at most less than 1000-fold that of the Hp1F gene. No Hpr mRNA can be detected in fetal liver.     

Rh gene evolution in primates: study of intron sequences

By amplification and sequencing of RH gene intron 4 of various primates we demonstrate that an Alu-Sx-like element has been inserted in the RH gene of the common ancestor of humans, apes, Old World monkeys, and New World monkeys. The study of mouse and lemur intron 4 sequences allowed us to precisely define the insertion point of the Alu-Sx element in intron 4 of the RH gene ancestor common to Anthropoidea. Like humans, chimpanzees and gorillas possess two types of RH intron 4, characterized by the presence (human RHCE and ape RHCE-like genes) or absence (human RHD and ape RHD-like genes) of the Alu-Sx element. This led us to conclude that in the RH common ancestor of humans, chimpanzees, and gorillas, a duplication of the common ancestor gene gave rise to two genes, one differing from the other by a 654-bp deletion encompassing an Alu-Sx element. Moreover, most of chimpanzees and some gorillas posses two types of RHD-like intron 4. The introns 4 of type 1 have a length similar to that of human RHD intron 4, whereas introns 4 of type 2 display an insertion of 12 bp. The latest insertion was not found in the human genome (72 individuals tested). The study of RH intron 3 length polymorphism confirmed that, like humans, chimpanzees and gorillas possess two types of intron 3, with the RHD-type intron 3 being 289 bases shorter than the RHCE intron 3. By amplification and sequencing of regions encompassing introns 3 and 4, we demonstrated that chimpanzee and gorilla RH-like genes displayed associations of introns 3 and 4 distinct to those found in man. Altogether, the results demonstrate that, as in humans, chimpanzee and gorilla RH genes experienced intergenic exchanges.     

The haptoglobin-gene deletion responsible for anhaptoglobinemia.

We have found an allelic deletion of the haptoglobin (Hp) gene from an individual with anhaptoglobinemia. The Hp gene cluster consists of coding regions of the alpha chain and beta chain of the haptoglobin gene (Hp) and of the alpha chain and beta chain of the haptoglobin-related gene (Hpr), in tandem from the 5' side. Southern blot and PCR analyses have indicated that the individual with anhaptoglobinemia was homozygous for the gene deletion and that the gene deletion was included at least from the promoter region of Hp to Hpr alpha but not to Hpr beta (Hpdel). In addition, we found seven individuals with hypohaptoglobinemia in three families, and the genotypes of six of the seven individuals were found to be Hp2/Hpdel. The phenotypes and genotypes in one of these three families showed the father to be hypohaptoglobinemic (Hp2) and Hp2/Hpdel, the mother to be Hp2-1 and Hp1/Hp2, one of the two children to be hypohaptoglobinemic (Hp2) and Hp2/Hpdel, and the other child to be Hp1 and Hp1/Hpdel, showing an anomalous inheritance of Hp phenotypes in the child with Hp1. The Hp2/Hpdel individuals had an extremely low level of Hp (mean+/-SD = 0.049+/-0. 043 mg/ml; n=6), compared with the level (1.64+/-1.07 mg/ml) obtained from 52 healthy volunteers having phenotype Hp2, whereas the serum Hp level of an individual with Hp1/Hpdel was 0.50 mg/ml, which was approximately half the level of Hp in control sera from the Hp1 phenotype (1.26+/-0.33 mg/ml; n=9), showing a gene-dosage effect. The other allele (Hp2) of individuals with Hp2/Hpdel was found to have, in all exons, no mutation, by DNA sequencing. On the basis of the present study, the mechanism of anhaptoglobinemia and the mechanism of anomalous inheritance of Hp phenotypes were well explained. However, the mechanism of hypohaptoglobinemia remains unknown.     

Evolution of protamine P1 genes in primates

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine Pls indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10–13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24–29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39–44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest neighbor matrices and trees based on maximum parsimony indicates that PI genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas. are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine Pls are expressed in germ line rather than somatic cells. In contrast to the variability of the exon regions of the protamine P1 genes, the sequence of the single intron is highly conserved.     

Investigation of the RH locus in gorillas and chimpanzees

The human Rh blood-group system is encoded by two homologous genes,RhD andRhCE. TheRH genes in gorillas and chimpanzees were investigated to delineate the phylogeny of the humanRH genes. Southern blot analysis with an exon 7-specific probe suggested that gorillas have more than twoRH genes, as has recently been reported for chimpanzees. Exon 7 was well conserved between humans, gorillas, and chimpanzees, although the exon 7 nucleotide sequences from gorillas were more similar to the humanD gene, whereas the nucleotide sequences of this exon in chimpanzees were more similar to the humanCE gene. The intron between exon 4 and exon 5 is polymorphic and can be used to distinguish the humanD gene from theCE gene. Nucleotide sequencing revealed that the basis for the intron polymorphism is anAlu element inCE which is not present in theD gene. Examination of gorilla and chimpanzee genomic DNA for this intron polymorphism demonstrated that theD intron was present in all the chimpanzees and in all but one gorilla. TheCE intron was found in three of six gorillas, but in none of the seven chimpanzees. Sequence data suggested that theAlu element might have previously been present in the chimpanzeeRH genes but was eliminated by excision or recombination. Conservation of theRhD gene was also apparent from the complete identity between the 3′-noncoding region of the human D cDNA and a gorilla genomic clone, including anAlu element which is present in both species. The data suggest that at least twoRH genes were present in a common ancestor of humans, chimpanzees, and gorillas, and that additionalRH gene duplication has taken place in gorillas and chimpanzees. TheRhCE gene appears to have diverged more thanRhD among primates. In addition, theRhD gene deletion associated with the Rh-negative phenotype in humans seems to have occurred after speciation. Correspondence to: C.M. Westhoff     

Sequence diversification and exon inactivation in the glycophorin A gene family from chimpanzee to human

In humans, the allelic diversity of MNSs glycophorins (GP) occurs mainly through the recombinational modulation of silent exons (pseudoexons) in duplicated genes. To address the origin of such a mechanism, structures of GPA, GPB, and GPE were determined in chimpanzee, the only higher primate known to have achieved a three-gene framework as in humans. Pairwise comparison of the chimpanzee and human genes revealed a high degree of sequence identity and similar exon-intron organization. However, the chimpanzee GPA gene lacks a completely formed M- or N-defining sequence as well as a consensus sequence for the Asn-linked glycosylation. In the case of the GPB gene, exon III is expressed in the chimpanzee but silenced, as a pseudoexon, in the human. Therefore, the protein product in the chimpanzee bears a larger extracellular domain than in the human. For the GPE genes, exon III and exon IV have been inactivated by identical donor splice-site mutations in the two species. Nevertheless, the chimpanzee GPE-like mRNA appeared to be transcribed from a GPB/E composite gene containing no 24-bp insertion sequence in exon V for the transmembrane domain. These results suggest a divergent processing of exonic units from chimpanzee to human in which the inactivation of GPB exon III preserved a limited sequence repertoire for diversification of human glycophorins.Correspondence to: O.O. Blumenfeld     

Junctions between genes in the haptoglobin gene cluster of primates.

To investigate the nature of the recombination that generated the haptoglobin three-gene cluster in Old World primates, we sequenced the region between the second gene (HPR) and the third gene (HPP) in chimpanzees (15 kb), as well as the region 3' to the cluster in humans (14 kb). Comparison to the previously sequenced human haptoglobin (HP) and HPR genes showed that the junction point between HP and HPR in humans (junction 1) was not identical to the junction point between the HPR and HPP genes of the chimpanzee (junction 2). An Alu sequence was found at each junction, but both Alu sequences lacked short direct repeats of the flanking genomic DNA. The lack of direct repeats implies that both junction Alu sequences are the products of recombination between different Alu elements. In addition, other insertion and deletion events are clustered in the regions near the junction Alu sequences. The observation that Alu sequences define the junctions between genes in the haptoglobin gene cluster emphasizes the importance of Alu sequences in the evolution of multigene families.     

The evolutionary history of human and chimpanzee Y-chromosome gene loss

Recent studies have suggested that gene gain and loss may contribute significantly to the divergence between humans and chimpanzees. Initial comparisons of the human and chimpanzee Y-chromosomes indicate that chimpanzees have a disproportionate loss of Y-chromosome genes, which may have implications for the adaptive evolution of sex-specific as well as reproductive traits, especially because one of the genes lost in chimpanzees is critically involved in spermatogenesis in humans. Here we have characterized Y-chromosome sequences in gorilla, bonobo, and several chimpanzee subspecies for 7 chimpanzee gene-disruptive mutations. Our analyses show that 6 of these gene-disruptive mutations predate chimpanzee-bonobo divergence at approximately 1.8 MYA, which indicates significant Y-chromosome change in the chimpanzee lineage relatively early in the evolutionary divergence of humans and chimpanzees.     

Chimpanzee apolipoprotein H (beta2-glycoprotein I): report on the gene structure, a common polymorphism, and a high prevalence of antiphospholipid antibodies

Apolipoprotein H (apoH, protein; APOH, gene) is a 50-kDa glycoprotein that binds to negatively charged substrates, including phospholipids. ApoH is a main target antigen for the binding of antiphospholipid antibodies that are associated with thrombotic events. We have previously characterized the structural organization of the human APOH gene. Because of the significant structural homology between the human and chimpanzee genomes, we have employed oligonucleotides from the human APOH gene sequence to amplify chimpanzee DNA covering the entire transcribed region together with flanking sequence in the 5' region. As in humans, the chimpanzee APOH gene consists of eight exons and seven introns and encodes for a 326-amino-acid protein. The deduced amino acid and nucleotide sequence show 99.4% and 99.6% similarity between human and chimpanzee APOH, respectively. Using isoelectric focusing (IEF) and immunoblotting, we screened 155 chimpanzees (128 unrelated captured parents and 27 captive-born offspring) for the apoH protein polymorphism. The most common IEF pattern in chimpanzees was identical to a previously described APOH*3 allele in humans. In addition, an anodally shifted pattern was observed in chimpanzees with an allele frequency of 0.168, and the corresponding allele was designated as APOH*4. DNA sequencing of APOH*4 carriers revealed a missense mutation in exon 6 (A-->G) at codon 210, which replaces the amino acid lysine by glutamic acid. This mutation does not affect the binding of apoH to cardiolipin as revealed by cardiolipin/enzyme-linked immunosorbent assay (ELISA). We also evaluated the prevalence of anti-apoH antibodies in chimpanzee plasma by using human-apoH-based ELISA and the association of the Lys210Glu mutation with the occurrence of anti-apoH antibodies. The prevalence of anti-apoH antibodies in chimpanzees (64%) was found to be unusually high compared with that found in humans. However, the Lys210Glu mutation showed no association with the occurrence of anti-apoH antibodies. The prevalence of anti-apoH antibodies in chimpanzees may serve as a useful animal model for the human antiphospholipid syndrome, where these antibodies are associated with clinical manifestations.     

Comparison of chimpanzee and human leukocyte Ig-like receptor genes reveals framework and rapidly evolving genes.

The leukocyte receptor complex (LRC) on human chromosome 19 contains related Ig superfamily killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LIR) genes. Previously, we discovered much difference in the KIR genes between humans and chimpanzees, primate species estimated to have approximately 98.8% genomic sequence similarity. Here, the common chimpanzee LIR genes are identified, characterized, and compared with their human counterparts. From screening a chimpanzee splenocyte cDNA library, clones corresponding to nine different chimpanzee LIRs were isolated and sequenced. Analysis of genomic DNA from 48 unrelated chimpanzees showed 42 to have all nine LIR genes, and six animals to lack just one of the genes. In structural diversity and functional type, the chimpanzee LIRs cover the range of human LIRs. Although both species have the same number of inhibitory LIRs, humans have more activating receptors, a trend also seen for KIRs. Four chimpanzee LIRs are clearly orthologs of human LIRs. Five other chimpanzee LIRs have paralogous relationships with clusters of human LIRs and have undergone much recombination. Like the human genes, chimpanzee LIR genes appear to be organized into two duplicated blocks, each block containing two orthologous genes. This organization provides a conserved framework within which there are clusters of faster evolving genes. Human and chimpanzee KIR genes have an analogous arrangement. Whereas both KIR and LIR genes can exhibit greater interspecies differences than the genome average, within each species the LIR gene family is more conserved than the KIR gene family.     

Reexamination of the African hominoid trichotomy with additional sequences from the primate beta-globin gene cluster.

Additional DNA sequence information from a range of primates, including 13.7 kb from pygmy chimpanzee (Pan paniscus), was added to data sets of beta-globin gene cluster sequence alignments that span the gamma 1, gamma 2, and psi eta loci and their flanking and intergenic regions. This enlarged body of data was used to address the issue of whether the ancestral separations of gorilla, chimpanzee, and human lineages resulted from only one trichotomous branching or from two dichotomous branching events. The degree of divergence, corrected for superimposed substitutions, seen in the beta-globin gene cluster between human alleles is about a third to a half that observed between two species of chimpanzee and about a fourth that between human and chimpanzee. The divergence either between chimpanzee and gorilla or between human and gorilla is slightly greater than that between human and chimpanzee, suggesting that the ancestral separations resulted from two closely spaced dichotomous branchings. Maximum parsimony analysis further strengthened the evidence that humans and chimpanzees share the longest common ancestry. Support for this human-chimpanzee clade is statistically significant at P = 0.002 over a human-gorilla clade or a chimpanzee-gorilla clade. An analysis of expected and observed homoplasy revealed that the number of sequence changes uniquely shared by human and chimpanzee lineages is too large to be attributed to homoplasy. Molecular clock calculations that accommodated lineage variations in rates of molecular evolution yielded hominoid branching times that ranged from 17-19 million years ago (MYA) for the separation of gibbon from the other hominoids to 5-7 MYA for the separation of chimpanzees from humans. Based on the relatively late dates and mounting corroborative evidence from unlinked nuclear genes and mitochondrial DNA for the close sister grouping of humans and chimpanzees, a cladistic classification would place all apes and humans in the same family. Within this family, gibbons would be placed in one subfamily and all other extant hominoids in another subfamily. The later subfamily would be divided into a tribe for orangutans and another tribe for gorillas, chimpanzees, and humans. Finally, gorillas would be placed in one subtribe with chimpanzees and humans in another, although this last division is not as strongly supported as the other divisions.     

Random nucleotide substitutions in primate nonfunctional gene for L-gulono-gamma-lactone oxidase, the missing enzyme in L-ascorbic acid biosynthesis

Humans and other primates have no functional gene for L-gulono-gamma-lactone oxidase that catalyzes the last step of L-ascorbic acid biosynthesis. The 164-nucleotide sequence of exon X of the gene was compared among human, chimpanzee, orangutan, and macaque, and it was found that nucleotide substitutions had occurred at random throughout the sequence with a single nucleotide deletion, indicating that the primate L-gulono-gamma-lactone oxidase genes are a typical example of pseudogene.     

Individual Variation in Levels of Haptoglobin-Related Protein in Children from Gabon

Background

Haptoglobin related protein (Hpr) is a key component of trypanosome lytic factors (TLF), a subset of high-density lipoproteins (HDL) that form the first line of human defence against African trypanosomes. Hpr, like haptoglobin (Hp) can bind to hemoglobin (Hb) and it is the Hpr-Hb complexes which bind to these parasites allowing uptake of TLF. This unique form of innate immunity is primate-specific. To date, there have been no population studies of plasma levels of Hpr, particularly in relation to hemolysis and a high prevalence of ahaptoglobinemia as found in malaria endemic areas.

Methods and Principal Findings

We developed a specific enzyme-linked immunosorbent assay to measure levels of plasma Hpr in Gabonese children sampled during a period of seasonal malaria transmission when acute phase responses (APR), malaria infection and associated hemolysis were prevalent. Median Hpr concentration was 0.28 mg/ml (range 0.03–1.1). This was 5-fold higher than that found in Caucasian children (0.049 mg/ml, range 0.002–0.26) with no evidence of an APR. A general linear model was used to investigate associations between Hpr levels, host polymorphisms, parasitological factors and the acute phase proteins, Hp, C-reactive protein (CRP) and albumin. Levels of Hpr were associated with Hp genotype, decreased with age and were higher in females. Hpr concentration was strongly correlated with that of Hp, but not CRP.

Conclusions/Significance

Individual variation in Hpr levels was related to Hp level, Hp genotype, demographics, malaria status and the APR. The strong correlations between plasma levels of Hp and Hpr suggest that they are regulated by similar mechanisms. These population-based observations indicate that a more dynamic view of the relative roles of Hpr and Hpr-Hb complexes needs to be considered in understanding innate immunity to African trypanosomes and possibly other pathogens including the newly discovered Plasmodium spp of humans and primates.     

Similar numbers but different repertoires of olfactory receptor genes in humans and chimpanzees

Animals recognize their external world through the detection of tens of thousands of chemical odorants. Olfactory receptor (OR) genes encode proteins for detecting odorant molecules and form the largest multigene family in mammals. It is known that humans have fewer OR genes and a higher fraction of OR pseudogenes than mice or dogs. To investigate whether these features are human specific or common to all higher primates, we identified nearly complete sets of OR genes from the chimpanzee and macaque genomes and compared them with the human OR genes. In contrast to previous studies, here we show that the number of OR genes ( approximately 810) and the fraction of pseudogenes (51%) in chimpanzees are very similar to those in humans, though macaques have considerably fewer OR genes. The pseudogenization rates and the numbers of genes affected by positive selection are also similar between humans and chimpanzees. Moreover, the most recent common ancestor between humans and chimpanzees had a larger number of functional OR genes (>500) and a lower fraction of pseudogenes (41%) than its descendents, suggesting that the OR gene repertoires are in a phase of deterioration in both lineages. Interestingly, despite the close evolutionary relationship between the 2 species, approximately 25% of their functional gene repertoires are species specific due to massive gene losses. These findings suggest that the tempo of evolution of OR genes is similar between humans and chimpanzees, but the OR gene repertoires are quite different between them. This difference might be responsible for the species-specific ability of odor perception.     

Comparative genomic analysis of human and chimpanzee proteases

Proteolytic enzymes are implicated in multiple physiological and pathological processes. The availability of the sequence of the chimpanzee genome has allowed us to determine that the chimpanzee degradome-the repertoire of protease genes from this organism-is composed of at least 559 protease and protease-like genes and is virtually identical to that of human, containing 561 genes. Despite the high degree of conservation between both genomes, we have identified important differences that vary from deletion of whole genes to small insertion/deletion events or single nucleotide changes that lead to the specific gene inactivation in one species, mostly affecting immune system genes. For example, the genes encoding PRSS33/EOS, a macrophage serine protease conserved in most mammals, and GGTLA1 are absent in chimpanzee, while the gene for metalloprotease MMP23A, located in chromosome 1p36, has been specifically duplicated in the human genome together with its neighbor gene CDC2L1. Other differences arise from single nucleotide changes in protease genes, such as NAPSB and CASP12, resulting in the presence of functional genes in chimpanzee and pseudogenes in human. Finally, we have confirmed that the Trypanosoma lytic factor HPR is inactive in chimpanzee, likely contributing to the susceptibility of chimpanzees to T. brucei infection. This study provides the first analysis of the chimpanzee degradome and might contribute to the understanding of the molecular bases underlying variations in host defense mechanisms between human and chimpanzee.     

A comparative analysis of regulatory regions of the transthyretin gene in the mouse, human, and chimpanzee genomes

A full genome analysis of differences between the gene expression in the human and chimpanzee brains revealed that the gene for transthyretin, the carrier of thyroid hormones, is differently transcribed in the cerebella of these species. A 7-kbp DNA fragment of chimpanzee was sequenced to identify possible regulatory sequences responsible for the differences in expression. One hundred and thirteen substitutions were found in the chimpanzee sequence in comparison with the human sequence. About 40% of the substitutions were revealed within the repeating elements of the genome; their location and sizes did not differ from those in the corresponding fragments of the human genome, and the nucleotide sequences had a high degree of identity. A comparison of nucleotide sequences of the transthyretin region of human, chimpanzee, and mouse genes revealed substantial differences in the distribution of G + C content along the examined fragment in the human (chimpanzee) and mouse genes and allowed us to localize three sequence tracts with a higher degree of identity in the three species. One of these tracts is located in the promoter region of the gene, and the other two probably determine the specificity of transthyretin gene expression in the liver and brain. One of the conserved tracts of the chimpanzee genome was found to have a single and a triple nucleotide substitution. The triple substitution distinguishes chimpanzees from humans and mice, which have identical sequences of this site. It is likely that these substitutions are responsible for the differences in the expression levels of the transthyretin gene in the human and chimpanzee brains.     

A Comparative Analysis of Regulatory Regions of the Transthyretin Gene in the Mouse, Human, and Chimpanzee Genomes

A full genome analysis of differences between the gene expression in the human and chimpanzee brains revealed that the gene for transthyretin, the carrier of thyroid hormones, is differently transcribed in the cerebella of these species. A 7-kbp DNA fragment of chimpanzee was sequenced to identify possible regulatory sequences responsible for the differences in expression. One hundred and thirteen substitutions were found in the chimpanzee sequence in comparison with the human sequence. About 40% of the substitutions were revealed within the repeating elements of the genome; their location and sizes did not differ from those in the corresponding fragments of the human genome, and the nucleotide sequences had a high degree of identity. A comparison of nucleotide sequences of the transthyretin region of human, chimpanzee, and mouse genes revealed substantial differences in the distribution of G + C content along the examined fragment in the human (chimpanzee) and mouse genes and allowed us to localize three sequence tracts with a higher degree of identity in the three species. One of these tracts was located in the promoter region of the gene, and the other two probably determine the specificity of transthyretin gene expression in the liver and brain. One of the conserved tracts of the chimpanzee genome was found to have a single and a triple nucleotide substitution. The triple substitution distinguishes chimpanzees from humans and mice, which have identical sequences of this site. It is likely that these substitutions are responsible for the differences in the expression levels of the transthyretin gene in the human and chimpanzee brains.