To examine the evolutionary diversities of the polymorphic repetitive region of the neurotransmitter-related genes, such as dopamine receptor D4 (DRD4), the serotonin transporter (5-HTT) and dopamine transporter (DAT1), we amplified and sequenced corresponding region in nonhuman primates.
Human DRD4 is polymorphic in repeat numbers of the 48-bp units located in the third cytoplasmic loop of the receptor. It has been indicated that individuals with long repeats (6 or more) display higher scores in a personality test for novelty seeking than those with shorter repeats. Four or more repeats have been reported in the DRD4 genes of simians. However, we found that most prosimians have 1 or 2 repeat unit(s). This indicates that 1) the ancestral primate may have had one 48-bp unit, 2) duplication occurred at the stage of prosimians, and 3) the repeat number increased after divergence into simian primates.
Tandem repeats consisting of 20-23 -bp units are located in the promoter region of human 5-HTT gene. It has been shown that individuals with 14 repeats in the promoter displayed higher scores in a personality test for anxiety/neuroticism than those with 16 repeats. Eight alleles including 15-23 repeats were observed in apes and polymorphism in repeat numbers was observed except chimpanzees. Allele including 14 repeats was observed only in humanand the frequency of allele with smaller number of repeats appears to increase during the process of hominization.
Tandem repeats consisting of 40-bp units are located in the 3' untranslated region of human DAT1 gene. Allele including 10 repeats is most common and the relationships between repeat numbers and personality (novelty seeking) or neuropsychiatry diseases have been reported. In this gene increase or decrease of the repeat number was not observed in the evolutionary context. Alleles including only 1 or 2 repeat unit(s) were observed in great apes, while DAT1 was highly polymorphic in gibbons with 8 alleles including 5-13 repeats. Tandem repeat structure of DAT1 gene was widely observed in simian primates.

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Human fibroblasts have a limited replicative lifespan and never undergo infinite cell division in vitro. In contrast, rodent fibroblasts spontaneously and highly frequently immortalize in vitro. Therefore, rodent is inappropriate as a model animal to study human aging in vitro. To test effects of macaque monkey as the model system, macaque adherent cells were cultured and passaged in vitro and analyzed cytologically.
Long-tailed macaque (Macaca fascicularis), Japanese macaque (Macaca fuscata), and bonnet monkey (Macaca radiata) were subjected to the study. Adherent cells were isolated from their skin, kidney, and lung. A total of 19 cell cultures were examined until terminating cell division.
Most of the cultures (17/19) exhibited senescence by 7-25 Population Doubling Levels (PDLs), showing enlargement of cell size, decrease of saturation density and extension of doubling interval, and then terminated cell division [Mortality stage 1 (M1)]. The remaining two cell cultures showed distinct pattern. They first exhibited senescent morphology at around 20PDLs, but continued cell division through M1 up to 106 PDLs and then went into crisis stage [Mortality stage 2 (M2)]. In all cell cultures tested, telomerase activity was not detected. Consistently, telomeres appeared to be shortened by every PDLs.
Macaque cells showed an intermediate pattern of in vitro aging between human and rodent cells, whereas, they showed no telomerase activity similarly to human cells. Therefore, the macaques must serve as an excellent animal model to study human cellular aging and provide us with a key to study the mechanism of the transition to critical stages in cellular aging.

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We report a species identification by mitochondrial DNA of a partial macaque skeleton excavated from Shuri Castle, Okinawa, the estimated age of which is around the 16th or 17th century AD. Species identification by gross morphology was not possible because of the following reasons; sufficient parts are not preserved, no natural distribution of non-human primates including macaque is recorded in the Ryukyu islands, and the genus Macaca is highly speciose. DNA amplification of D-loop variable region (ca 200bp) was first unsuccessful with the use of previously devised sets of primers which encompass the whole region, and is accomplished by using newly devised sets of primers, each of which is designed for anterior or posterior portion of the targeted region. Sequenced DNA of the Shuri macaque completely agrees with a sequence of Yaku macaques (Macaca fuscata yakui). For the comparison with or species identification of Shuri macaque, we sequenced the same region of DNA (variable region of D-loop) from four species of macaques (Macaca fuscata, M. cyclopis, M. mulatta and M. fascicularis). Ample variations of substitutions and insertion/deletion mutations are discovered both intra- and inter-specifically. A neighbor-joining tree based on nucleotide substitutions is depicted with bootstrap values (Fig. 4). In this study, a monkey skeletal remain excavated from Okinawa is safely identified as a Yaku macaque by the use of mitochondrial DNA. This suggests a promising future of genetic analyses for archaeological information retrieval. It is also emphasized that, for the proper assessment of the genetic information from archaeological remains, fuller genetic studies of the living animals are critically important.

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Comparative sequence analysis of primate genes is a powerful tool for studying human evolution. However, current public databases have little entries of nonhuman primate mRNA sequences and expressed sequence tags (ESTs). In an attempt to establish a database of nonhuman primate ESTs, we have constructed full length-enriched cDNA libraries for two species, cynomolgus monkey (Macaca fascicularis) and chimpanzee (Pan troglodytes), using the Oligo-Capping method. In the cynomolgus monkey, cDNA libraries were constructed from brain, liver, heart and kidney. Those from brain consisted of eight region-specific cDNA libraries (frontal lobe, temporal lobe, medulla oblongata, cerebellum cortex, hypophysis, mesencephalon, parietal lobe and brain stem). The ESTs were obtained by single-pass sequencing from the 5'-ends of randomly selected cDNA clones. A total of, 13, 738 ESTs were obtained from these cDNA libraries. In the chimpanzee, a skin cDNA library was constructed and 650 SSTs were obtained. Sequence similarity test against GenBank (database: nr and dbEST) indicated that 92.2% of ESTs in the cynomolgus monkey and 86.8% of ESTs in the chimpanzee matched with registered sequences in the public databases. In order to investigate the degree of identity in the 5'-end regions of mRNAs that include both 5'-untranslated regions (UTRs) and partial coding sequences (CDSs), 68 cynomolgus monkey ESTs and 49 chimpanzee ESTs were aligned with human homologous sequences. Sequence identities between cynomolgus monkey and human were 94.3% in the 5'-UTRs and 97.8% in the 5'-CDSs. Those between chimpanzee and human were 97.2% in 5'UTRs and 99.3% in the 5'-CDSs. These results show that the 5'-end regions of the mRNAs are highly conserved in primate species. There were, however, several exceptional genes that showed significant sequence differences in the 5'-end regions.

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Most New World (NW) monkeys exhibit extensive intra-specific variations in color vision, where males are dichromatic (red-green colorblind) and females are either dichromatic or trichromatic. This is explained by a model that the red-green visual pigment genes of NW monkeys exist as a single-locus gene with three allelic variations residing on X-chromosome (“tri-allelic single-locus X-chromosome model”). In this study we investigated genomic organization of red-green visual pigment genes of common marmoset (Callithrix jacchus) and owl monkey (Aotus trivirgatus). By fluorescence in situ hybridization (FISH), we directly localized the pigment gene at a locus on X-chromosome of common marmoset. The study on the marmoset provided direct and conclusive evidences for the “tri-allelic single-locus X-chromosome model” on the genetic basis of marmoset color vision.
By using in vitro pigment regeneration method, we determined exact peak absorption spectrum (λmax) of all three allelic pigments, red, yellow, and green, of the marmoset, to be 561, 553, and 539nm, respectively, and green pigment of owl monkey to be 539nm, which were precise to within ±1nm. These values were virtually identical to predicted values based on amino acid sequence at five critical sites: 180, 197, 277, 285, 308.
Interestingly, the nocturnal owl monkey was found to possess multiple green pseudogenes. The phylogenetic analysis indicated that duplications of the green genes occurred in owl monkey lineage and were independent from that in the howler monkey, the only known NW monkey having red and green genes on separate loci. By Southern hybridization, we found one male capuchin monkey (Cebus apella) that appeared to have two red-green-like visual pigment genes. The results obtained from studies on owl monkey and capuchin suggest that the evolution of color vision in NW monkey is more complicated than ever considered.

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Primates are known to be basically herbivorous, eating leaves and buds of trees, fruits, etc. However, the diversified food habits are appreciable in several primates such as humans, chimpanzees, and common marmosets who actively eat animal flesh. Not only morphological adaptations but also molecular adaptations of digestive enzymes might be necessary to adapt new food habits. We discuss the molecular adaptations of pepsinogen, the gastric digestive proteinase, during primate evolution. Pepsinogen levels in primate stomachs are found to be the highest among all mammals examined so far. This high level might be due to the tact that primates are basically herbivorous, and need to digest plant foods efficiently. Although the occurrence of type-A and C pepsinogens is known in mammals, the multiplicity of type-A pepsinogen is found to be extreme in primates. The exception to this being, New World monkeys, for which the multiplicity of pepsinogen A and C is not found. Instead, New World monkeys express prochymosin at adult stage, a known neonate-specific pepsinogen in other mammals. Three types of pepsinogen work for digestion in New World monkeys. Old World monkeys have 3-4 types of pepsinogen A. Gene duplication generates the multiple forms bearing different enzymatic functions in protein digestion. In hominoids, the occurrence of 7-15 pepsinogen A isozymogens is remarkable. They can be classified into different groups acconling to their amino acid composition and enzymatic properties. The presence of multiple pepsinogens in Old World monkeys and hominoids might be advantageous in the efficient digestion of a variety of foods and may be correlated with the development of the central nervous system of these primates.

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The sex was identified by the polymerase chain reaction (PCR) using DNA extracted from noninvasive samples, such as urine, feces, food wadges, and hair samples, of Pan troglodytes, Pan paniscus, and Macaca fuscata. The Amelogenin gene was successfully amplified by PCR, and X- and Y-specific DNA regions had different lengths. The ZFX and ZFY genes were also amplified by PCR, and restriction fragment length polymorphism (RFLP) depicted different patterns between the sexes. Sex identification using DNA from noninvasive samples obtained from unidentified wild animals will help to study the range and social structure of these species.

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Microsatellites, which involve a repetitive sequence motif (2-6bp), are evenly interspersed throughout eukaryotic genomes. Because of the variety in the numbers of repeat, they are useful genetic markers in the fields of human genetics, forensic sciences, and so on. Recently, phylogenetic studies using microsatellites have been performed in various species. Microsatellites in intergenic sequences, which evolve faster than coding sequences, are informative in the comparison between closely related species. Many of nonhuman primate species share the same microsatellite loci as human, although repeat numbers or nucleotide sequences vary species to species. We introduce some interesting studies and topics in which the interspecies difference of properties of microsatellites and the birth or death of them has been discussed. We also report the nucleotide sequence analysis of a microsatellite D14S299 locus in primates. The nucleotide sequence analyses of microsatellite alleles in primates provide informative data to study on primate evolution or dynamic processes in microsatellite loci.

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Retroposons become remarkable DNA markers in the study of the evolution of DNA in the recent decade. Retroposons are formed by reverse transcriptase from the RNA and inserted in the random position of the genomic DNA. If it has the promoter region of RNA polymerase, the retroposon will be expressed again and increase copy numbers. The active retroposons such as phosphoglycerate kinase (McCarry and Thomas 1984), BC200, one of the Alu element (Cheng et al. 1997), amylase gene (Samuelson et al. 1990) and others were summarized.
A novel processed gene was found in the intergenic α-globin gene region in macaques, which had an open reading frame of 117 amino acids, poly (A) signal, poly (A) and a flanking direct repeat. It was named P117 gene. The frequency of insertion of this retrosequence P117 (P117^R) was different from locality in the long-tailed macaques, while, the P117^R could not be found in 49 Japanese macaques from three places in the central part of Japan. Also, hominoid genome, so far as examined, do not have this retroposon. The parental P117 (P117^P) gene in the macaque has the length by 3kb, which was longer than that in human by 1kb. The P117^P was consisted from four exons and three introns.
The sequence of P117^R from the long-tailed and Formosan macaques, and capchin were determined. The comparison of the synonymous (k_S) and the non-synonymous substitution number (k_A) between P117^P and P117^R, and the frequency of insertion in macaques, suggest that the insertion occurred after the divergence of the macaques from the hominoid lineage but just before the divergence of the macaque species.
In the case of capchin, the P117^R was assumed to be inserted at around the divergence time between the New World and the Old World monkeys.

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There are three common alleles (A, B, and O) at the human ABO blood group locus which codes glycosyltransferase. The polymorphisms of the ABO blood group are also observed in wide variety of primates. The difference of the glycosyltransferase activity between human A and B enzymes is due to the two amino acid differences. The same amino acid differences are observed for A and B alleles in non-human primates. We determined 19 sequences of chimpanzee, 8 sequences of bonobo, and 2 sequences of Japanese macaque ABO blood group gene for exon 3 and intron 6 (ca 1.7kb). We also determined 3 sequences of Japanese macaque ABO for exon 7 (ca. 0.5kb). We compared those data with published sequences of other hominoids and Old World monkeys. It was suggested that the type changes between A and B occurred independently in the both lineages of the hominoids and Old world monkeys. The alleles A and B appeared to be polymorphic in the ancestral species of macaques, while the different B type allele evolved independently in baboon lineage.

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Nucleotide difference between human and chimpanzee, the closest living organism to human, is about 1.4%. Therefore, half (0.7%) of this difference accumulated after the human lineage diverged from the chimpanzee lineage. All the genetic changes responsible for “humanness” must reside in those differences.
Human genome consists of about 3 billion nucleotides, thus the 0.7% difference is tantamount to 21 million nucleotide changes. Although 95% of the human genome can be considered as junk DNA, we still have 1 million nucleotide changes located in nonjunk DNA. How many changes are really responsible for creating humanness in those changes? Our very rough guess is only 10, 000.
Draft human genome sequences will be completed within a year or so. It is now the time to sequence ape genomes so as compare them with human sequences nucleotide by nucleotide. Because we are interested in the changes which occurred in the human lineage, we should compare not only chimpanzee but also gorilla and orangutan. We can pinpoint human-specific changes after multiplyaligned those ape and human sequences by applying maximum parsimony principle.
We just started this kind of sequencing “Ape genome” (Ag=silver) by picking up interesting part of the human genome sequences. PCR primers are made based on those sequences, and corresponding orthologous DNAs are amplified for apes, followed by direct sequencing. The determined nucleotide sequences were submitted to the DDBJ/EMBL/GenBank International Nucleotide Sequence Database, and are also be available through web site of this project, as follows: http://sayer.lab.nig.ac.jp/silver/.

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