郷 康広

Odor molecules in the environment are detected by olfactory receptors (ORs), being encoded by a large multigene family in mammalian genomes. It is generally thought that primates are vision oriented and dependent weakly on olfaction. Previous studies suggested that Old World monkeys (OWMs) and hominoids lost many functional OR genes after the divergence from New World monkeys (NWMs) due to the acquisition of well-developed trichromatic vision. To examine this hypothesis, here we analyzed OR gene repertoires of five primate species including NWMs, OWMs, and hominoids for which high-coverage genome sequences are available, together with two prosimians and tree shrews with low-coverage genomes. The results showed no significant differences in the number of functional OR genes between NWMs (marmosets) and OWMs/hominoids. Two independent analyses, identification of orthologous genes among the five primates and estimation of the numbers of ancestral genes by the reconciled tree method, did not support a sudden loss of OR genes at the branch of the OWMs/hominoids ancestor but suggested a gradual loss in every lineage. Moreover, we found that humans retain larger numbers of ancestral OR genes that were in the common ancestor of NWMs/OWMs/hominoids than orangutans and macaques and that the OR gene repertoire in humans is more similar to that of marmosets than those of orangutans and macaques. These results suggest that the degeneration of OR genes in primates cannot simply be explained by the acquisition of trichromatic vision, and our sense of smell may not be inferior to other primate species.

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 (similar to 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.

Despite relatively frequent gene or segment duplications, the number of functional loci in the major histocompatibility complex (MHC) is relatively small. The dual function of MHC molecules (triggering the immune system and limiting T-cell receptor repertoires) is likely to balance the number of functional loci. The effect of this dual function on the number of functional MHC loci has been argued mainly in the theoretical and computer simulation studies, but the evidence from empirical data has not been fully examined. Here, we attempt to evaluate this effect based on the analysis of nucleotide sequence data. We hypothesized that due to the dual function, even becoming a pseudogene (pseudogenization) of MHC is advantageous for the organisms. To evaluate this hypothesis, we compared the distribution of the waiting time (T(W)) till pseudogenization for HLA (human MHC) with that of the human olfactory receptor (OR) and bitter taste receptor (T2R) genes. The result shows that T(W) in HLA has a tendency to be relatively shorter as the emergence time (T) of the gene becomes older, while in OR T(W) becomes proportionally longer as T becomes older and in T2R it is almost null irrespective of T. Furthermore, T(W) in HLA is strongly influenced by the extent of functional differentiation in the peptide-binding region. Taken together, these results show that MHC molecules have optimal numbers of functional loci, and these numbers are regulated by the advantageous pseudogenization of duplicated copies.

In 1999, we measured the body mass of 101 wild ring-tailed lemurs (Lemur catta) inhabiting the Berenty Reserve, Madagascar. In addition, we counted the number of ticks [Haemaphysalis (Rhipistoma) lemuris Hoogstraal, 1953] infesting their facial skin and external auditory meatuses. For both males and females, the body mass appeared to increase until the age of 3 years. With the apparent exception of infants, there were no sexual differences in body mass. Within a group, higher-ranked adult males tended to be heavier than lower-ranked males. In contrast, there was no consistent correlation between the body mass of females and their ranks. Among the study groups, there was a small difference in body mass and significant difference in the number of ticks infesting the facial skin and external auditory meatuses. In particular, lemurs of a group who inhabited an area of gallery forest in the study area exhibited the smallest values of body mass and were severely infested with ticks. Such group variations were not consistently correlated with the reproductive parameters of the study groups. In three groups moderately infested with ticks, ticks infested adult males and subadults more heavily than adult females, juveniles, and infants.

The sense of bitter taste plays a critical role in how organisms avoid generally bitter toxic and harmful substances. Previous studies revealed that there were 25 intact bitter taste receptor (T2R) genes in humans and 34 in mice. However, because the recent chicken genome project reported only three T2R genes, it appears that extensive gene expansions occurred in the lineage leading to mammals or extensive gene contractions occurred in the lineage leading to birds. Here, I examined the T2R gene repertoire in placental mammals (dogs, Canis familiaris; and cows, Bos taurus), marsupials (opossums, Monodelphis domestica), amphibians (frogs, Xenopus tropicalis), and fishes (zebrafishes, Danio rerio; and pufferfishes, Takifugu rubripes) to investigate the birth-and-death process of T2R genes throughout vertebrate evolution. I show that (1) the first extensive gene expansions occurred before the divergence of mammals from reptiles/birds but after the divergence of amniotes (reptiles/birds/mammals) from amphibians, (2) subsequent gene expansions continuously took place in the ancestral mammalian lineage and the lineage leading to amphibians, as evidenced by the presence of 15, 18, 26, and 49 intact T2R genes in the dog, cow, opossum, and frog genome, respectively, and (3) contractions of the gene repertoire happened in the lineage leading to chickens. Thus, continuous gene expansions have shaped the T2R repertoire in mammals, but the contractions subsequent to the first round of expansions have made the chicken T2R repertoire narrow. These dramatic changes in the repertoire size might reflect the daily intake of foods from an external environment as a driving force of evolution.

Since the process of becoming dead genes or pseudogenes (pseudogenization) is irreversible and can occur rather rapidly under certain environmental circumstances, it is one plausible determinant for characterizing species specificity. To test this evolutionary hypothesis, we analyzed the tempo and mode of duplication and pseudogenization of bitter taste receptor (T2R) genes in humans as well as in 12 nonhuman primates. The results show that primates have accumulated more pseudogenes than mice after their separation from the common ancestor and that lineage-specific pseudogenization becomes more conspicuous in humans than in nonhuman primates. Although positive selection has operated on some amino acids in extracellular domains, functional constraints against T2R genes are more relaxed in primates than in mice and this trend has culminated in the rapid deterioration of the bitter-tasting capability in humans. Since T2R molecules play an important role in avoiding generally bitter toxic and harmful substances, substantial modification of the T2R gene repertoire is likely to reflect different responses to changes in the environment and to result from species-specific food preference during primate evolution.

Major histocompatibility complex genes (Mhc-DQB and Mltc-DRB) were sequenced in seven aye-ayes (Daubentonia madagascariecsis), which is an endemic and endangered species in Madagascar. An aye-aye from a north-eastern population showed genetic relatedness to individuals of a north-western population and had a somewhat different repertoire from another north-eastern individual. These observations suggest that the extent of genetic variation in Mhc genes is not excessively small in the aye-aye in spite of recent rapid destruction of their habitat by human activities. In light of Mhc gene evolution, trans-species and allelic poly-morphisms can be estimated to have been retained for more than 50 Ma (million years) based on the time scale of lemur evolution.

Major histocompatibility complex (MHC) class I genes have complicated and profound evolutionary histories. To reconstruct and better understand their histories, partial class I genes (exon 2-intron 2-exon 3) were sequenced in a sampling of prosimians (Strepsirhini, Primates). In total, we detected 117 different sequences from 36 Malagasy prosimians (lemurs) and 1 non-Malagasy prosimian (galago) representing 4 families, 7 genera, and 13 species. Unlike the MHC class II genes (MHC-DRB), MHC class I genes show a generally genus-specific mode of evolution in lemurs. Additionally, no prosimian class I loci were found to be orthologous to HLA genes, even at highly conserved loci (such as HLA-E, HLA-F). Phylogenetic analysis indicates that nucleotide diversity among loci was very small and the persistence time of the polymorphisms was short, suggesting that the origin of the lemur MHC class I genes detected in this study was relatively recent. The evolutionary mode of these genes is similar to that of classic HLA genes, HLA-A, HLA-B, and HLA-C, in terms of their recent origin and rarity of pseudogenes, and differs from them with respect to the degree of gene duplications. From the viewpoint of MHC genes evolution, some interlocus sequence exchanges were apparently observed in the lemur lineage upon phylogenetic and amino acid motif analyses. This is also in contrast to the evolutionary mode of HLA genes, where intralocus exchanges have certainly occurred but few interlocus exchanges have taken place. Consequently, the gene conversion model for explaining the generation of the MHC diversity among different loci can be thought to play more important roles in the evolution of lemur MHC class I genes than in that of HLA genes.

Partial exon 2 sequences (202 bp) of the lemur Mhc-DRB genes were sequenced. A total of 137 novel sequences were detected in 66 lemurs, representing four out of the five extant families. Trans-species polymorphisms and even identical sequences were observed not only among genera but also among families. Based on the time-scale of lemur evolution, these findings suggest that some identical sequences have been maintained for more than 40 million years. This is in contrast to the evolutionary mode of simian DRB genes, where such identical sequences have been retained for at most several million years. To explore the reasons behind these unexpected findings, the degree of recombination and the synonymous substitution rate in lemurs and simians were examined. We found that (1) little difference existed in the extent of recombination, (2) frequent recombination occurred within the alpha-helix as well as between the beta-pleated sheet and the alpha-helix, and (3) the synonymous substitution rate was significantly reduced in lemur lineages. Upon phylogenetic analysis, lemur DRB genes were clustered by themselves and separated from the other primate DRB genes (simians and non-Malagasy prosimians). This result suggests that the DRB variations in extant lemur populations have been generated after the divergence of the lemurs from the remaining primates. This mode of substitution accumulation is also supported by a pattern of mismatch distribution among lemur DRB genes. These observations correspond with the postulation that a severe bottleneck occurred when the ancestors of lemurs settled into Madagascar from the African continent.

Chromosomal localization of 18S rDNA and telomere sequence was attempted on the chromosomes of the aye-aye (2n = 30) using fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS), respectively. The rDNA was localized at the tip or whole of the short arm of acrocentric chromosomes 13 and 14 in all spreads observed. However, post-FISH silver-nitrate (Ag) staining showed that transcriptional activity of the rRNA genes was variable, particularly in chromosome 14, which was most frequently negative in one homologue carrying the smaller copy number of rDNA. This observation supports, at the molecular cytogenetic level, previous data concerning the relationship between the copy number of rDNA and its trancriptional activity. On the other hand, telomere sequence was localized only at the telomeric region of all chromosomes, the so-called telomere-only pattern, a characteristic similar to that of the greater bushbaby. These data may provide information on the chromosomal evolution of the lemur, because locations of rDNA and telomere sequences frequently offer important clues in reconstruction of karyotype differentiation.

We examined the chromosomal localization of the telomeric sequence, (TTAGGG)(n), in seven species of the lemurs and one greater galago, as an outgroup, using the primed in-situ labeling (PRINS) technique. As expected, the telomeric sequence was identified at both ends of all chromosomes of the eight prosimians. However, six species showed a signal at some pericentromeric regions involving constitutive heterochromatin as well. The pericentromeric region of chromosome 1 of Verreaux's sifaka (Propithecus verreauxi verreauxi) was labeled with a large and intense signal. The range of the signal considerably exceeded the area of DAPI positive heterochromatin. On the other hand, in the five lemurs, a large signal was detected also in the short arm of acrocentric chromosomes. Acquisition of the large block of the telomeric sequence into the acrocentric short arm might be interpretable in terms of the tandem growth of the heterochromatic short arm and the reciprocal translocation between heterochromatic short arms involving the telomeric sequence. Subsequently, it was postulated that meta- or submetacentric chromosomes possessing the telomeric sequence at the pericentromeric region could be formed by centric fusion between such acrocentric chromosomes.