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Molecular Biology Section, Dept. of Cellular and Molecular Biology, Primate Research Institute, Kyoto University

Hirohisa HIRAI

Hirohisa HIRAI

Molecular cytogenetics and Chromosome evolution in primates

Professor, Director (Concurrent)

Hirohisa HIRAI

Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan
Tel: +81-568-63-0528, Fax: +81-0568-63-0085,

Academic Experience

1979-1992 Research Associate, Kumamoto University, Medical School, Japan
1987-1989 Post-doctoral Fellow and Research Assistant Professor, State University of New York at Buffalo, U.S.A.
1992-1999 Instructor, Primate Research Institute, Kyoto University, Japan
2000-2005 Associate Professor, Primate Research Institute, Kyoto University, Japan
2005-present Professor, Primate Research Institute, Kyoto University, Japan

Research interest

Molecular cytogenetics and Chromosome evolution in primates

New topics

A most distant intergeneric hybrid offspring (Larcon) of lesser apes, Nomascus leucogenys and Hylobates lar.:
Unlike humans, which are the sole remaining representatives of a once larger group of bipedal apes (hominins), the "lesser apes" (hylobatids) are a diverse radiation with numerous extant species. Consequently, the lesser apes can provide a valuable evolutionary window onto the possible interactions (e.g. interbreeding) of hominin lineages coexisting in the same time and place. In the present work, we employ chromosomal analyses to verify the hybrid ancestry of an individual (Larcon) produced by two of the most distant genera of lesser apes, Hylobates (lar-group gibbons) and Nomascus (concolor-group gibbons). In addition to a mixed pelage pattern, the hybrid animal carries a 48-chromosome karyotype that consists of the haploid complements of each parental species: Hylobates lar (n=22) and Nomascus leucogenys leucogenys (n=26). Studies of this animal's karyotype shed light onto the processes of speciation and genus-level divergence in the lesser apes and, by extension, across the Hominoidea.

Chromosomes of Primates

Japanese macaque(2n = 42, centromeres detected by alpha satelitte DNA)

Aye-aye(2n = 30, conventional stain)

Blue Sykes monkey(2n = 70, C-band)

Current main researchs

(1) Differentiation of genome wasteland and evolution of organisms

Humans and chimpanzees share very similar cytogenetic karyotypes. Significant chromosomal structural differences include a number of peri- and paracentric inversions, and the fusion of chimpanzee chromosomes 12 and 13 to form human chromosome 2 (Yunis et al. 1980). Additionally, more than half the 48 chimpanzee chromosome arms have large terminal blocks of constitutive heterochromatin (C-bands) (Marks 1985; Yunis et al. 1980), subterminal satellite DNA (Royle et al. 1994), and subterminal and interstitial telomeric sequence arrays (Hirai 2001), which are absent from humans. On the other hand, a human chimpanzee comparative genome analysis revealed that the difference between the chimpanzee and human genomes at the nucleotide level was 1.23% (Fujiyama et al. 2002). In fact, however, the phenotypes of these two species are distinct from each other. What, besides gene differences, makes us human? Are any of the phenotypic differences between humans and chimpanzees due to chromosomal differences between these two species? These questions are not easily answered, but need to be investigated in the post-genome era using new insights. The human genome includes 45% repetitive arrays derived from transposable elements. Segmental duplications are also a notable feature of the human genome (International Human Genome Sequencing Consortium 2001). The human genome sequencing project suggested that it might be intriguing to investigate whether such genomic upheavals caused by repetitive sequences and duplications have a role in speciation events. This report presents some qualitative evidence that the genomic wasteland seems to influence chromosome configuration and pairing and chiasma formation in male meiosis of chimpanzees, and postulates that it may bring about gene silencing and a bias in gene shuffling.

Chiasma suppression by position effect of genomic wasteland

Abstract 1: The terminal C-bands that are a specific feature of chimpanzee chromosomes were dissected using a molecular cytogenetic technique, PRINS, with primers for telomeric sequences, subterminal satellite, and retrotransposable elements (HERV-K and -W). These DNA elements jointly formed a large block of retrotransposable compound repeat DNA organization (RCRO) at the terminal C-band regions of 30 chromosomes, and are also located at the centromeric regions of some chromosomes. Additionally, a block consisting of all members of the RCRO has transposed to the middle (q31.1) of the long arm of chromosome 6, and three members, the subterminal satellite and the two HERVs, have integrated into the proximal region (q14.4) of the long arm of chromosome 14. Terminal RCROs seem to induce and prolong the bouquet stage in meiotic prophase, and to affect chiasma formation, together with interstitial RCROs. It is also postulated that RCROs may cause a position effect to gene expression, resulting in gene silencing and/or late replication. (Hirai et al. Cytogenetic and Genome Research 108: 248-254, 2005)

Gene silencing of rDNA by position effect of genomic wasteland. Green, rDNA. Orange, genomic wasteland. Pink, position effect. Blue, gene expression. Red, gene silencing.

Abstract 2: Polymorphisms related to transcriptional inactivation of nucleolus organizer regions (NORs) have long been described in many animals, particularly humans. However, the precise aetiology of such variations is not always clear. We conducted analyses to investigate the repression mechanisms in humans and chimpanzees using FISH (fluorescence in situ hybridisation) with 18S rDNA, Ag-NOR (silver nitrate) staining, C-banding, and the in situ nick translation technique with the HpaII restriction enzyme. Examination of 48 humans and 46 chimpanzees suggested that there are at least three different mechanisms that produce inactivation of NORs. These include: (1) elimination of rDNA; (2) DNA methylation; (3) gene silencing due to position effects induced by heterochromatin (C-bands) and/or telomeres. (Guillen et al. Chromosome Research 12: 225-237, 2004)

If these speculations were true, such biases of gene silencing and gene shuffling by position effects of RCROs may have worked to produce divergence of two species with similar genes during five million years, resulting in the large phenotypic differentiation between chimpanzees and humans.

(2) Phylobiogeography of small apes

Silver gibbon

Kloss gibbon

Small apes (Hylobatidae) are included in Appendix I (threatened species) of CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) as endangered species, together with great apes and other monkeys (Soehartono & Mardiastuti 2002). Forest clearance has reduced their habitat, which is primary forest with tall trees. If this continues in the future, it may cause the extinction of small apes. To prevent such an undesirable outcome, conservation programs start in several places. As part of these programs, conservation genetics could be important in reducing current rates of extinction and to preserve biodiversity (reviewed in Frankham et al. 2002). That is, genetic factors that affect extinction risk can be determined, and the genetic management schemes required to minimize these risks can be put in place. As basic major genetic issues for starting conservation biology, evolutionary, population and quantitative genetics and taxonomy are probably the most important for the precise understanding of the biodiversity of these species.

In gibbons, hitherto, there have been problems with the identification of species or subspecies in zoological institutions. Most such captive gibbons are of unknown origin, so that identification is very difficult and is sometimes confused due to morphological similarity. Such confusion results in strange molecular phylogenetic relationships. Phylogenetic trees have also been produced showing topology such as メtrans-species polymorphismsモ of mitochondrial DNA sequences in investigations using samples obtained from zoological institutions (unpublished data), as a result of misidentification or interspecific hybridization in captivity. Such indefinite data lead to confusion in genetic monitoring to obtain standard taxonomic information in conservation programs. To avoid such problems, samples of known origin are required as an initial step.

Chromosomes of small apes are highly differentiated from those of other apes in spite of belonging to the same hominoid group. First, the genome of each subgenus of small apes is very intricately rearranged compared with humans and great apes due to numerous translocations (Jauch et al. 1992; Koehler et al. 1995; Arnold et al. 1996; Nie et al. 2001; Muller et al. 2003). These rearrangements are more drastic than those that distinguish human and Old World monkeys, and are a puzzle of primate chromosome evolution. However, the chromosome number is an important feature for separating the four subgenera of small apes, that is, Bunopithecus (2n = 38), Hylobates (2n = 44), Symphalangus (2n = 50), and Nomascus (2n = 52) (reviewed in Geissmann 1995). Additionally, molecular cytogenetic data indicated that the subgenus Bunopithecus is the most basal group of the family Hylobatidae, followed by Hylobates, with Symphalangus and Nomascus as the last to diverge (Muller et al. 2003). This result conflicts with the view from molecular phylogenetic studies with DNA sequences that depicts Nomascus as the most basal group of the Hylobatidae, followed by Symphalangus, with Bunopithecus and Hylobates as the last to diverge (Ross & Geissmann 2001). This discrepancy might occur because speciation of all extant gibbons occurred within a relatively short evolutionary time, resulting in poor correlations between genetic (DNA sequence), cytogenetic (chromosome differentiation), and morphological divergence (Muller et al. 2003). This can also be deduced from the fact that all six species of the subgenus Hylobates share the same three chromosome inversions (e.g., Stanyon et al. 1987; Van Tuinen et al. 1999). In general, chromosomal rearrangements can produce changes that are considered as unique landmarks at the divergence nodes (Muller et al. 2003), because chromosome changes generate reproductive isolation between populations by reducing fitness (White 1973; 1978; King 1993). Furthermore, in new models, chromosomal rearrangements reduce gene flow by suppressing genetic recombination and extending the effects of linked isolating genes (e.g., Rieseberg 2001; Navarro & Barton 2003a), and accelerate protein evolution (Navarro & Barton 2003b). These new and old models may all lead to chromosomal speciation. Since the small ape lineage has passed through a burst of translocations, processes such as meiotic drive, recombination reduction, and molecular divergence in rearranged chromosomes might have operated as valid evolutionary processes in gibbons. Therefore, chromosome markers could be very useful tools for evolutionary and/or conservation studies of gibbons.

Recently, a whole arm translocation (actually an almost whole arm translocation) between chromosomes 8 and 9 was found in the agile gibbon (Hylobates agilis) (Van Tuinen et al. 1999). This translocation (named WAT8/9) seemed to be apparently restricted to Sumatran agile gibbons (Hirai et al. 2003), though data were not adequate because of samples of unknown origin. We thus conducted a research project to determine the situation in individuals of known origin, because if our postulation were correct, such information would be very useful for identifying individuals whose specific or subspecific status was uncertain from purely morphological data. This would be important for conservation programs. This report describes their chromosome differentiation and the population structure of the variation, together with morphological identification, clustering of TSPY (testis-specific protein, Y encoded) gene sequences, and genetic constitution of microsatellite DNA, and discusses mechanisms of formation of WAT8/9 using chromosome-painting data, features of the heterozygote, migration of the species group, and suggestions for conservation programs.

Baby of agile gibbon

Whole arm translocation between chromosomes 8 and 9 occurred in Sumatran agile gibbons.

Phylobiogeographic relationship between Sumatran and Bornean gibbons.

Abstract: Gibbons, like orangutans, are a group of threatened Asian apes, so that genetic monitoring of each species or subspecies is a pressing need for conservation programs. We conducted a project to take, as far as possible, samples of known origin from wild-born animals from Sumatra and Borneo (Central Kalimantan) for genetic monitoring of agile gibbons. As a result, we found a whole arm translocation between chromosomes 8 and 9 (WAT8/9) specific to Sumatran agile gibbons. Furthermore, population surveys suggested that the form with the WAT8/9 seems to be incompatible with an ancestral form, suggesting that the former might have extinguished the latter from Sumatran populations by competition. In any case, this translocation is a useful chromosomal marker for identifying Sumatran agile gibbons. Population genetic analyses with DNA showed that the molecular genetic distance between Sumatran and Bornean agile gibbons is the smallest, although the chromosomal difference is the largest. Thus, it is postulated that WAT8/9 occurred and fixed in a small population of Sumatra after migration and geographical isolation at the last glacial period, and afterwards dispersed rapidly to other populations in Sumatra as a result of the bottleneck effect and a chromosomal isolating mechanism. (Hirai et al. Chromosome Research 13: 123-133, 2005)