Birth of 'human-specific' genes during primate evolution

Humans and other Anthropoids share very similar chromosome structure and genomic sequence as seen in the 98.5% homology at the DNA level between us and Great Apes. However, anatomical and behavioral traits distinguish Homo sapiens from his closest relatives. I review here several recent studies that address the issue by using different approaches: large-scale sequence comparison (first release) between human and chimpanzee, characterization of recent segmental duplications in the human genome and analysis of exemplary gene families. As a major breakthrough in the field, the heretical concept of human-specific genes has recently received some supporting data. In addition, specific chromosomal regions have been mapped that display all the features of gene nurseries and could have played a major role in gene innovation and speciation during primate evolution. A model is proposed that integrates all known molecular mechanisms that can create new genes in the human lineage.     

Assignment of 118 novel cDNAs of cynomolgus monkey brain to human chromosomes

In order to isolate genes that may not be represented in current human brain cDNA libraries, we have sequenced about 20,000 sequence tags of cDNA clones derived from cerebellum and parietal lobe of cynomolgus monkeys (Macaca fascicularis). We determined the entire cDNA sequence of approximately 700 clones whose 5'-terminal sequences showed no homology to annotated putative genes or expressed sequence tags in current databases of genetic information. From this, 118 clones with sequences encoding novel open reading frames of more than 100 amino acid residues were selected for further analysis. To localize the genes corresponding to these 118 newly identified cDNA clones on human chromosomes, we performed a homology search using the human genome sequence and fluorescent in situ hybridization. In total, 108 of 118 clones were successfully assigned to specific regions of human chromosomes. This result demonstrates that genes expressed in cynomolgus monkey are highly conserved throughout primate evolution, and that virtually all had human homologs. Furthermore, we will be able to discover novel human genes in the human genome using monkey homologs as probes.     

A scan for positively selected genes in the genomes of humans and chimpanzees

Since the divergence of humans and chimpanzees about 5 million years ago, these species have undergone a remarkable evolution with drastic divergence in anatomy and cognitive abilities. At the molecular level, despite the small overall magnitude of DNA sequence divergence, we might expect such evolutionary changes to leave a noticeable signature throughout the genome. We here compare 13,731 annotated genes from humans to their chimpanzee orthologs to identify genes that show evidence of positive selection. Many of the genes that present a signature of positive selection tend to be involved in sensory perception or immune defenses. However, the group of genes that show the strongest evidence for positive selection also includes a surprising number of genes involved in tumor suppression and apoptosis, and of genes involved in spermatogenesis. We hypothesize that positive selection in some of these genes may be driven by genomic conflict due to apoptosis during spermatogenesis. Genes with maximal expression in the brain show little or no evidence for positive selection, while genes with maximal expression in the testis tend to be enriched with positively selected genes. Genes on the X chromosome also tend to show an elevated tendency for positive selection. We also present polymorphism data from 20 Caucasian Americans and 19 African Americans for the 50 annotated genes showing the strongest evidence for positive selection. The polymorphism analysis further supports the presence of positive selection in these genes by showing an excess of high-frequency derived nonsynonymous mutations.     

The chimpanzee and cynomolgus monkey erythrocyte immune adherence receptors are encoded by CR1-like genes

The human erythrocyte immune adherence (IA) receptor is the Mr 220,000 type one complement receptor, or CR1. Nonhuman primate IA receptors are comprised of a family of smaller erythrocyte complement receptors (E-CRs) of unknown origin. Recently, the Mr 65,000 baboon E-CR was identified as a glycophosphatidylinositol (GPI)-linked protein encoded by a partially duplicated CR1 gene termed CR1-like. The purpose of this study was to determine the genetic origin of the Mr 75,000 chimpanzee E-CR. Two previously identified cDNAs, an alternative splice product of CR1 termed CR1a and a chimpanzee form of CR1-like, were synthesized and amplified from chimpanzee bone marrow RNA, and transiently expressed in COS-7 cells. By SDS-PAGE, the CR1a protein had a relative mobility slightly greater than chimpanzee E-CR, whereas that of the CR1-like protein was slightly less. Affinity chromatography demonstrated that little chimpanzee CR1a bound to human C3i linked to activated thiol-Sepharose (C3i-ATS), while over 50% of both chimpanzee CR1-like and chimpanzee E-CR bound to C3i-ATS. Treatment with phosphatidylinositol-specific phospholipase C (PIPLC) to assess GPI linkage released E-CR from chimpanzee erythrocytes, and E-CR from cynomolgus monkey erythrocytes. Based on size, ligand-binding specificity, and PIPLC sensitivity, we conclude that the chimpanzee E-CR is encoded by the CR1-like gene. Furthermore, based on PIPLC sensitivity, the cynomolgus monkey E-CR is also likely encoded by a CR1-like sequence. Thus, CR1-like, which is a genetic element of unknown significance in humans, is the gene that encodes the erythrocyte IA receptor of many nonhuman primates.     

Rapid evolution of prostatic protein PSP94 suggested by sequence divergence between rhesus monkey and human cDNAs

Comparison of the protein coding region of mRNA for the prostatic secretory protein PSP94 in human (hPSP94) with that in rhesus monkey (rmPSP94) indicates that, for the most part, its sequence has evolved with few constraints and at a relatively fast rate. Interestingly, half of the 22 residue differences between the two species involve charge changes, reflected by the acidic pI (5.4) of hPSP94 and the basic pI (10.6) of rmPSP94. However, the 10 cysteines and 5 of the 6 prolines of PSP94 were unaffected, suggesting that the three-dimensional conformations of the human and the monkey proteins may be similar. Rapid evolution of this gene might explain the apparent absence in nonprimates of homologous sequences detectable by hybridization.     

Remodeling of the involucrin gene during primate evolution

The protein involucrin is a product of terminal differentiation in the epidermal cell and related cell types. By comparing the nucleotide sequence of the involucrin gene of the lemur with that of the human, it is clear that the gene has undergone unusual evolution in the primates. The coding region of the gene contains an ancestral segment, most of which is common to the lemur and the human, and a species-specific segment of repeats derived from the ancestral segment. Instead of the modern segment of repeats found in the human gene, the lemur gene possesses repeats derived from another sequence at a different location in the ancestral segment. The two kinds of segments of repeats probably represent alternative ways of creating a repeat structure in the involucrin molecule. The modern segment of repeats must have been created after divergence of the higher primates from the prosimians.     

Plasticity of human chromosome 3 during primate evolution

Comparative mapping of more than 100 region-specific clones from human chromosome 3 in Bornean and Sumatran orangutans, siamang gibbon, and Old and New World monkeys allowed us to reconstruct ancestral simian and hominoid chromosomes. A single paracentric inversion derives chromosome 1 of the Old World monkey Presbytis cristata from the simian ancestor. In the New World monkey Callithrix geoffroyi and siamang, the ancestor diverged on multiple chromosomes, through utilizing different breakpoints. One shared and two independent inversions derive Bornean orangutan 2 and human 3, implying that neither Bornean orangutans nor humans have conserved the ancestral chromosome form. The inversions, fissions, and translocations in the five species analyzed involve at least 14 different evolutionary breakpoints along the entire length of human 3; however, particular regions appear to be more susceptible to chromosome reshuffling. The ancestral pericentromeric region has promoted both large-scale and micro-rearrangements. Small segments homologous to human 3q11.2 and 3q21.2 were repositioned intrachromosomally independent of the surrounding markers in the orangutan lineage. Breakage and rearrangement of the human 3p12.3 region were associated with extensive intragenomic duplications at multiple orangutan and gibbon subtelomeric sites. We propose that new chromosomes and genomes arise through large-scale rearrangements of evolutionarily conserved genomic building blocks and additional duplication, amplification, and/or repositioning of inherently unstable smaller DNA segments contained within them.     

Secretion of pre-beta-migrating apoA-I by cynomolgus monkey hepatocytes in culture

Cynomolgus monkey hepatocytes that had been stored frozen were thawed, established in culture, and used to study apoA-I secretion. Protein synthetic activity was low at first, but increased with time, approaching what appeared to be the constitutive levels of the intact liver by day 7. During the first week, cellular RNA levels increased from 5.3 +/- 0.3 to 18.6 +/- 1.0 micrograms/10(6) cells; albumin secretion rates increased from undetectable to 55.4 micrograms/10(6) cells per day; apoA-I mRNA levels increased from 174 +/- 12 to 564 +/- 145 ng/10(6) cells; and apoA-I secretion rates increased from undetectable to 2.11 +/- 0.27 micrograms/10(6) cells per day. Analysis of day 7-conditioned media by agarose electrophoresis, gradient gel electrophoresis-immunoblotting, and column chromatography, showed that the apoA-I produced by the cells was present in three distinct forms. One had an apparent molecular mass greater than 1 million Da, migrated pre-beta, and accounted for 11 +/- 3 (mean +/- SD)% of the total; one had an apparent molecular mass of 104 kDa, had alpha migration, and accounted for 27 +/- 2% of the total; and one had an apparent molecular mass of 50 kDa, migrated pre-beta, and accounted for 46 +/- 9% of the total. These data support the proposition that the pre-beta-migrating, 50 kDa, apoA-I-containing particles identified in the plasma of cynomolgus monkeys are nascent hepatic HDL.     

Thyroxine 5'-deiodinase in brown adipose tissue of the cynomolgus monkey Macaca fascicularis

Brown adipose tissue was identified in axillary, interscapular, subscapular, and cervical fat deposits of male and female cynomolgus monkeys (Macaca fascicularis) by histological and immunological techniques. Histology included staining of mitochondria with a Novelli stain and identification of mitochondria-rich multilocular cells. Immunological detection involved separation of homogenate proteins by sodium dodecyl sulphate--polyacrylamide gel chromatography, blotting on to nitrocellulose membranes, and identification of the specific uncoupling protein, unique to brown adipose tissue, with an antiserum to purified hamster uncoupling protein followed by detection with 125I-labelled protein A. The activity of thyroxine 5'-deiodinase in monkey brown adipose tissue homogenates was much higher than that seen previously in brown adipose tissue of rats, mice, and hamsters. This is the first demonstration of the presence of this enzyme in brown adipose tissue of a primate species.     

Ancient and recent adaptive evolution of primate non-homologous end joining genes

In human cells, DNA double-strand breaks are repaired primarily by the non-homologous end joining (NHEJ) pathway. Given their critical nature, we expected NHEJ proteins to be evolutionarily conserved, with relatively little sequence change over time. Here, we report that while critical domains of these proteins are conserved as expected, the sequence of NHEJ proteins has also been shaped by recurrent positive selection, leading to rapid sequence evolution in other protein domains. In order to characterize the molecular evolution of the human NHEJ pathway, we generated large simian primate sequence datasets for NHEJ genes. Codon-based models of gene evolution yielded statistical support for the recurrent positive selection of five NHEJ genes during primate evolution: XRCC4, NBS1, Artemis, POLλ, and CtIP. Analysis of human polymorphism data using the composite of multiple signals (CMS) test revealed that XRCC4 has also been subjected to positive selection in modern humans. Crystal structures are available for XRCC4, Nbs1, and Polλ; and residues under positive selection fall exclusively on the surfaces of these proteins. Despite the positive selection of such residues, biochemical experiments with variants of one positively selected site in Nbs1 confirm that functions necessary for DNA repair and checkpoint signaling have been conserved. However, many viruses interact with the proteins of the NHEJ pathway as part of their infectious lifecycle. We propose that an ongoing evolutionary arms race between viruses and NHEJ genes may be driving the surprisingly rapid evolution of these critical genes.