An evolutionary conserved early replicating segment on the sex chromosomes of man and the great apes

Replication studies on prometaphase chromosomes of man, the chimpanzee, the pygmy chimpanzee, the gorilla, and the orangutan reveal great interspecific homologies between the autosomes. The early replicating X chromosomes clearly show a high degree of conservation of both the pattern and the time course of replication. An early replicating segment on the short arm of the X chromosomes of man (Xp22.3) which escapes inactivation can be found on the X chromosomes of the great apes as well. Furthermore, the most early replicating segment on the Y chromosomes of all species tested appears to be homologous to this segment on the X chromosomes. Therefore, these early replicating segments in the great apes may correspond to the pseudoautosomal segment proposed to exist in man. From further cytogenetic characterization of the Y chromosomes it is evident that structural alterations have resulted in an extreme divergence in both the euchromatic and heterochromatic parts. It is assumed, therefore, that, in contrast to the X chromosomes, the Y chromosomes have undergone a rapid evolution within the higher primates.     

Dynamics of DNA Methylation in Recent Human and Great Ape Evolution

DNA methylation is an epigenetic modification involved in regulatory processes such as cell differentiation during development, X-chromosome inactivation, genomic imprinting and susceptibility to complex disease. However, the dynamics of DNA methylation changes between humans and their closest relatives are still poorly understood. We performed a comparative analysis of CpG methylation patterns between 9 humans and 23 primate samples including all species of great apes (chimpanzee, bonobo, gorilla and orangutan) using Illumina Methylation450 bead arrays. Our analysis identified ∼800 genes with significantly altered methylation patterns among the great apes, including ∼170 genes with a methylation pattern unique to human. Some of these are known to be involved in developmental and neurological features, suggesting that epigenetic changes have been frequent during recent human and primate evolution. We identified a significant positive relationship between the rate of coding variation and alterations of methylation at the promoter level, indicative of co-occurrence between evolution of protein sequence and gene regulation. In contrast, and supporting the idea that many phenotypic differences between humans and great apes are not due to amino acid differences, our analysis also identified 184 genes that are perfectly conserved at protein level between human and chimpanzee, yet show significant epigenetic differences between these two species. We conclude that epigenetic alterations are an important force during primate evolution and have been under-explored in evolutionary comparative genomics.     

Techniques and Significance of Gamete Collection and Storage in the Great Apes

Rectal probe electroejaculation (RPE) is the most frequently used method for semen recovery in the great apes. Artificial insemination has been successful in the chimpanzee and gorilla. Oocytes can be recovered using laparoscopic techniques similar to those used in human medicine. At this time there has been no successful in vitro fertilization with birth of an infant in the great apes. Semen can be successfully frozen in the apes, as documented by recovery of motility of sperm after thawing. Pregnancies have been initiated in the chimpanzee and gorilla using frozen thawed semen.     

Evolution of subterminal satellite (StSat) repeats in hominids

Subterminal satellite (StSat) repeats, consisting of 32-bp-long AT-rich units (GATATTTCCATGTT(T/C)ATACAGATAGCGGTGTA), were first found in chimpanzee and gorilla (African great apes) as one of the major components of heterochromatic regions located proximal to telomeres of chromosomes. StSat repeats have not been found in orangutan (Asian great ape) or human. This patchy distribution among species suggested that the StSat repeats were present in the common ancestor of African great apes and subsequently lost in the lineage leading to human. An alternative explanation is that the StSat repeats in chimpanzee and gorilla have different origins and the repeats did not occur in human. The purpose of the present study was quantitative evaluation of the above alternative possibilities by analyzing the nucleotide variation contained in the repeats. We collected large numbers of sequences of repeat units from genome sequence databases of chimpanzee and gorilla, and also bonobo (an African great ape phylogenetically closer to chimpanzee). We then compared the base composition of the repeat units among the 3 species, and found statistically significant similarities in the base composition. These results support the view that the StSat repeats had already formed multiple arrays in the common ancestor of African great apes. It is thus suggested that humans lost StSat repeats which had once grown to multiple arrays.     

Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.     

Nucleotide sequences of immunoglobulin-epsilon pseudogenes in man and apes and their phylogenetic relationships

To understand the phylogenetic relationships between hominoids, the nucleotide sequences of immunoglobulin-epsilon processed pseudogenes from chimpanzee, gorilla and orangutan were determined. The basic structures of these processed pseudogenes agreed with their human counterpart. Although the degrees of nucleotide differences between man and the African apes had no statistical significance, all the analytical data examined supported the theory that chimpanzee is the closest relative of man. This result was consistent with that deduced by our recent qualitative study. Studies on the nucleotide sequences of globin genes have suggested that the molecular clock runs more slowly in hominoids than in non-hominoid primates. According to the present data, however, further retardation of the evolutionary rate was not observed in the human lineage. Assuming that orangutan diverged 14 million years ago and that the evolutionary rate between the orangutan lineage and the lineage leading to the other three species is constant, the divergence dates of chimpanzee and gorilla were estimated to be 4.9(+/- 0.9) and 5.9(+/- 0.9) million years ago, respectively.     

Genome size and «C-heterochromatic-DNA» in man and the african apes

The genome sizes and the amounts of DNA after C-banding pretreatments (C-heterochromatic DNA) were measured by quantitative cytochemical methods in man and the African apes,Gorilla gorilla andPan troglodytes. As evaluated by flow cytometry on propidium-iodide-stained lymphocytes, gorilla and chimpanzee have genome sizes larger than man. On the basis of the different resistance of metaphase chromosome DNA to the C-banding procedure, two genome compartments were defined, i.e.,C-heterochromatic-DNA andeuchromatic-DNA. The latter proved to be fairly constant in man and the African apes (as well as in two hylobatid species), whereas the variable amounts ofC-heterochromatic-DNA account well for the interspecific differences of genome size among the hominoid species studied so far. During karyotype diversification, quantitative changes (with either gains or losses) ofC-heterochromatic-DNA seem to have taken place independently in the hylobatid and the man/African ape lineages.     

DA/DAPI fluorescent bands in the chromosomes of Pan paniscus

The fluorochrome pattern produced by DA/DAPI double staining in Pan paniscus chromosomes is reported. The location of DA/DAPI prominent bands differs from that reported for all other hominoid species. However, the pattern in the pygmy chimpanzee is most similar to that seen in Pan troglodytes. Comparison of the DA/DAPI pattern of the other hominoid species allows the construction of a proposed hominoid ancestral karyotype and a preliminary phylogenetic reconstruction of DA/DAPI bands for the great apes and man.     

A Y-chromosomal DNA fragment is conserved in human and chimpanzee.

A human male-specific Y-chromosomal DNA fragment (lambda YH2D6) has been isolated. By deletion-mapping analysis, 2D6 has been localized to the euchromatic portion of the long arm (Yq11) of the human Y chromosome. Among great apes, this fragment was found to be conserved in male chimpanzee but was lacking in male gorilla and male orangutan. No homologous fragments were detected in females of orangutan, gorilla, chimpanzee, or human. Nucleotide sequence analysis indicated the presence of partial-Alu-elements and of sequences similar to the GATA repeats of the snake Bkm sequence.     

Origin of human chromosome 2 revisited

Similarities in chromosome banding patterns and hornologies in DNA sequence between chromosomes of the great apes and humans have suggested that human chromosome 2 originated through the fusion of two ancestral ape chromosomes. A lot of work has been directed at understanding the nature and mechanism of this fusion. The recent availability of the human chrornosome-2-specific alpha satellite DNA probe D2Z and the human chromosome-2p-specific subtelomeric DNA probe D2S445 prompted us to attempt cross-hybridization with chromosomes of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) to search for equivalent locations in the great apes and to comment on the origin of human chromosome 2. The probes gave different results. No hybridization to the chromosome-2-specific alpha satellite DNA probe was observed on the presumed homologous great ape chromosomes using both high-stringency and low-stringency post-hybridization washes, whereas the subtelomeric-DNA probe specific for chromosome 2p hybridized to telomeric sites of the short arm of chromosome 12 of all three great apes. These observations suggest an evolutionary difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes presumably involved in the chromosome fusion. Nevertheless, complete conservation of DNA sequence of the subtelomeric repeat sequence D2S445 in the ape chromosomes is demonstrated.     

A young Alu subfamily amplified independently in human and African great apes lineages.

A variety of Alu subfamilies amplified in primate genomes at different evolutionary time periods. Alu Sb2 belongs to a group of young subfamilies with a characteristic two-nucleotide deletion at positions 65/66. It consists of repeats having a 7-nucleotide duplication of a sequence segment involving positions 246 through 252. The presence of Sb2 inserts was examined in five genomic loci in 120 human DNA samples as well as in DNAs of higher primates. The lack of the insertional polymorphism seen at four human loci and the absence of orthologous inserts in apes indicated that the examined repeats retroposed early in the human lineage, but following the divergence of great apes. On the other hand, similar analysis of the fifth locus (butyrylcholinesterase gene) suggested contemporary retropositional activity of this subfamily. By a semi-quantitative PCR, using a primer pair specific for Sb2 repeats, we estimated their copy number at about 1500 per human haploid genome; the corresponding numbers in chimpanzee and gorilla were two orders of magnitude lower, while in orangutan and gibbon the presence of Sb2 Alu was hardly detectable. Sequence analysis of PCR-amplified Sb2 repeats from human and African great apes is consistent with the model in which the founding of Sb2 subfamily variants occurred independently in chimpanzee, gorilla and human lineages.     

A human moderately repeated Y-specific DNA sequence is evolutionarily conserved in the Y chromosome of the great apes.

Evolutionary conservation of the human-derived moderately repeated Y-specific DNA sequence Y-190 (DYZ5) was investigated in the chimpanzee, orangutan, and gorilla. Southern blot analysis showed the presence of the sequence in the Y chromosome of all great apes. Pulsed-field gel electrophoresis and in situ hybridization revealed that the repeat is organized in one major block and confined to a small region of the Y chromosome of the three species. DYZ5 was assigned to the proximal short arm of the Y chromosome of the chimpanzee and orangutan and to the long arm of the Y chromosome of the gorilla. In light of its evolutionary conservation, DYZ5 may have an as yet undetermined structural function in the Y chromosome.     

Mitochondrial DNA evolution in primates: Transition rate has been extremely low in the lemur

Summary Based on mitochondrial DNA (mt-DNA) sequence data from a wide range of primate species, branching order in the evolution of primates was inferred by the maximum likelihood method of Felsenstein without assuming rate constancy among lineages. Bootstrap probabilities for being the maximum likelihood tree topology among alternatives were estimated without performing a maximum likelihood estimation for each resampled data set. Variation in the evolutionary rate among lineages was examined for the maximum likelihood tree by a method developed by Kishino and Hasegawa. From these analyses it appears that the transition rate of mtDNA evolution in the lemur has been extremely low, only about 1/10 that in other primate lines, whereas the transversion rate does not differ significantly from that of other primates. Furthermore, the transition rate in catarrhines, except the gibbon, is higher than those in the tarsier and in platyrrhines, and the transition rate in the gibbon is lower than those in other catarrhines. Branching dates in primate evolution were estimated by a molecular clock analysis of mtDNA, taking into account the rate of variation among different lines, and the results were compared with those estimated from nuclear DNA. Under the most likely model, where the evolutionary rate of mtDNA has been unifrom within a great apes/human calde, human/chimpanzee clustering is preferred to the alternative branching orders among human, chimpanzee, and gorilla.     

The evolution of the azoospermia factor region AZFa in higher primates

Clones of a PAC contig encompassing the human AZFa region in Yq11.21 were comparatively FISH mapped to great ape Y chromosomes. While the orthologous AZFa locus in the chimpanzee, the bonobo and the gorilla maps to the long arm of their Y chromosomes in Yq12.1-->q12.2, Yq13.1-->q13.2 and Yq11.2, respectively, it is found on the short arm of the orang-utan subspecies of Borneo and Sumatra, in Yp12.3 and Yp13.2, respectively. Regarding the order of PAC clones and genes within the AZFa region, no differences could be detected between apes and man, indicating a strong evolutionary stability of this non-recombining region.     

Blood groups of anthropoid apes and their relationship to human blood groups

Affinity between blood groups of man and those of anthropoid apes is reflected not only in similarities or identities of reactions of the red cells with many specific typing reagents, but also in overall structures of some of the main blood group systems defined in man and in apes.Besides specificities of human-type, such as A-B-O, M-N, Rh-Hr, I-i, etc. known to be present on the red cells of various species of apes, specific reagents were produced by iso- or cross-immunization of chimpanzees that detect red cell specificities characteristic for apes only. Some of those specificities were found to be shared by several ape species and to fall into separate blood group systems that are counterparts of the human blood group systems. Recently obtained serological, as well as population data, indicate that the chimpanzee R-C-E-F blood group system is the counterpart of the human Rh-Hr system. Similarly to the Rh-Hr system, it is built around a main antigen, the R^c antigen, to which secondary specificities are attached by means of multiple allelic genes. The R^c is not only the principal factor of the chimpanzee R-C-E-F group system, but also constitutes a direct link with the human Rh-Hr blood group system, since anti-R^c reagents also detect Rh₀ specificity on the human red cells. Another chimpanzee blood group system, the V-A-B-D system, is counterpart of the M-N-S-s system, and is built around the central antigen V^c. the V^c is not only the principal specificity of the chimpanzee V-A-B-D system, but it also constitutes the direct link with the human M-N-S-s system since anti-V^c reagent gives with chimpanzee red cells reactions parralleling those obtained with anti-N lectin (N^v) while in tests with human red cells it detects specificity identical or closely related to the Mi^a specificity.     

Evidence of similar organization of the chromosomes carrying the major histocompatibility complex in man and other primates

The chromosome localization and gene synteny of the major histocompatibility complex (MHC) of the great apes and rhesus monkey were investigated using somatic cell hybrids. The presence of the MHC antigens was determined either with a microadsorption technique employing primate alloantisera, or with a radioimmune assay. The enzymes phosphoglucomutase 3 (PGM3), glyoxalase 1 (GLO1), mitochondrial superoxide dismutase (SOD2), and soluble maleic enzyme (ME1) were assayed in those hybrids where electrophoretic separations could be achieved. A chromosome homologous to the human No. 6 was found in the chimpanzee, gorilla, orangutan and rhesus monkey, and its genomic organization is similar to that of man.     

Improved method for artificial insemination in the great apes

Artificial insemination in the great apes has not achieved its potential as a tool in maintenance of the endangered captive population. Three factors can influence the success rate of artificial insemination: sperm preparation, site of insemination, and timing of insemination. We have tried to optimize methods regarding these three steps. A modified method for insemination is described which has resulted in a 21% success rate (six term pregnancies from 29 inseminations) in the chimpanzee and which has successfully initiated a pregnancy in a gorilla.     

Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data,and the branching order in hominoidea

Summary A maximum likelihood method for inferring evolutionary trees from DNA sequence data was developed by Felsenstein (1981). In evaluating the extent to which the maximum likelihood tree is a significantly better representation of the true tree, it is important to estimate the variance of the difference between log likelihood of different tree topologies. Bootstrap resampling can be used for this purpose (Hasegawa et al. 1988; Hasegawa and Kishino 1989), but it imposes a great computation burden. To overcome this difficulty, we developed a new method for estimating the variance by expressing it explicitly.The method was applied to DNA sequence data from primates in order to evaluate the maximum likelihood branching order among Hominoidea. It was shown that, although the orangutan is convincingly placed as an outgroup of a human and African apes clade, the branching order among human, chimpanzee, and gorilla cannot be determined confidently from the DNA sequence data presently available when the evolutionary rate constancy is not assumed.     

Fluorescent heterochromatin staining in primate chromosomes

Recently, in addition to quinacrine staining, fluorochrome techniques have been developed which brilliantly stain other heterochromatic regions. Two of these staining techniques are Distamycin/DAPI (DA/DAPI) and D287/170. We stained the chromosomes of all species of great apes and 14 species of primates (48 individuals) using these three fluorochrome techniques. Only african apes and man show brilliant quinacrine staining while, man and all the great apes show brilliant DA/DAPI staining and only species belonging to the hominoidea (including the siamang) showed bright D287/170 staining. In the lower primates a medium level of DA/DAPI fluorescence was found in some species with large amount of pericentromeric heterochromatin. Brilliant DA/DAPI staining could represent a derived trait linking all great apes and humans, while D287/170 may link all hominoidea. Fluorochrome staining is believed to be correlated with some satellite DNA sequences. However, data available on the chromosome location of satellite DNAs in non-human primates were derived from buoyant density fractions resulting in cross hybridization and now are not considered reliable. Before making any correlation between fluorochrome staining and satellite DNAs in non human primates there is need of data onin situ hybridization with cloned DNA sequences on primate chromosomes. These data would help clarify the evolution and relationship of satellite DNAs and heterochromatin in primates.     

Conservation of intronic minisatellite polymorphisms in the SCK1/SHC2 gene of Hominidae

The neuronally expressed Shc adaptor homolog SCK1/SHC2 gene contains an unusually high number of minisatellites. In humans, twelve different minisatellite sequences are located in introns of SCK1/SHC2 and ten of them are highly polymorphic. Here we used primers developed for humans to screen ten intronic loci of SCK1/SHC2 in chimpanzee and gorilla, and undertook a comprehensive analysis of the genomic sequence to address the evolutionary events driving these variable repeats. All ten loci amplified in chimpanzee and gorilla contained hypervariable and low-variability minisatellites. The human polymorphic locus TR1 was monomorphic in chimpanzee and gorilla, but we detected polymorphic alleles in these apes for the human monomorphic TR7 locus. When we examined the repeat size among these hominoids, there was no consistent variation by length from humans to great apes. In spite of the inconsistent evolutionary dynamics in repeat length variation, exon 16 was highly conserved between humans and great apes. These results suggest that non-coding intronic minisatellites do not show a consistent evolutionary paradigm but evolved with different patterns among each minisatellite locus. These findings provide important insight for minisatellite conservation during hominoid evolution.