Gorilla and orangutan c-myc nucleotide sequences: Inference on hominoid phylogeny

The nucleotide sequences of the gorilla and orangutan myc loci have been determined by the dideoxy nucleotide method. As previously observed in the human and chimpanzee sequences, an open reading frame (ORF) of 188 codons overlapping exon 1 could be deduced from the gorilla sequence. However, no such ORF appeared in the orangutan sequence.The two sequences were aligned with those of human and chimpanzee as hominoids and of gibbon and marmoset as outgroups of hominoids. The branching order in the evolution of primates was inferred from these data by different methods: maximum parsimony and neighborjoining.Our results support the view that the gorilla lineage branched off before the human and chimpanzee diverged and strengthen the hypothesis that chimpanzee and gorilla are more related to human than is orangutan. Correspondence to: F. Galibert     

Characterization of a Novel Class of Interspersed LTR Elements in Primate Genomes: Structure, Genomic Distribution, and Evolution

Retrovirus-like sequences and their solitary (solo) long terminal repeats (LTRs) are common repetitive elements in eukaryotic genomes. We reported previously that the tandemly arrayed genes encoding U2 snRNA (the RNU2 locus) in humans and apes contain a solo LTR (U2-LTR) which was presumably generated by homologous recombination between the two LTRs of an ancestral provirus that is retained in the orthologous baboon RNU2 locus. We have now sequenced the orthologous U2-LTRs in human, chimpanzee, gorilla, orangutan, and baboon and examined numerous homologs of the U2-LTR that are dispersed throughout the human genome. Although these U2-LTR homologs have been collectively referred to as LTR13 in the literature, they do not display sequence similarity to any known retroviral LTRs; however, the structure of LTR13 closely resembles that of other retroviral LTRs with a putative promoter, polyadenylation signal, and a tandemly repeated 53-bp enhancer-like element. Genomic blotting indicates that LTR13 is primate-specific; based on sequence analysis, we estimate there are about 2,500 LTR13 elements in the human genome. Comparison of the primate U2-LTR sequences suggests that the homologous recombination event that gave rise to the solo U2-LTR occurred soon after insertion of the ancestral provirus into the ancestral U2 tandem array. Phylogenetic analysis of the LTR13 family confirms that it is diverse, but the orthologous U2-LTRs form a coherent group in which chimpanzee is closest to the humans; orangutan is a clear outgroup of human, chimpanzee, and gorilla; and baboon is a distant relative of human, chimpanzee, gorilla, and orangutan. We compare the LTR13 family with other known LTRs and consider whether these LTRs might play a role in concerted evolution of the primate RNU2 locus. Received: 29 September 1997 / Accepted: 16 January 1998     

A comparison of the small ribosomal RNA genes from the mitochondrial DNA of the great apes and humans: sequence, structure, evolution, and phylogenetic implications

Restriction endonuclease fragments produced by EcoRI/AvaI or KpnI digestion and containing the small (12S) ribosomal RNA (rRNA) genes from the mitochondrial DNAs (mtDNAs) of the common chimpanzee, pygmy chimpanzee, gorilla, and orangutan were inserted into the plasmids pBR322 or pADD1. After species verification the inserted fragments were digested with SauIIIA, subcloned into M13mp7 vectors, and sequenced. The small rRNA gene sequences were compared with each other and with the published human sequence (Anderson et al. 1981). Substitutions were detected at 118 of the 955 nucleotide positions compared. Pairwise, the sequence differences ranged from 1% (between the chimpanzee species) to 9% (comparisons involving the orangutan); the proportion that were transitions ranged from 87% to 100%. Deletions and/or additions were noted at seven locations. With respect to evolutionary sequence lability, kinetic analysis indicated the presence of at least two classes of nucleotide positions; the more labile class occurs in sequences thought to form self-complementary duplexes (stems) in the mature rRNA. The high frequency of compensating substitutions, which maintain base-pairing within these sequences, corroborates their inferred structure. Phylogenetic inferences drawn from the sequence comparisons support the notion of an approximately equidistant relationship among chimpanzees, gorilla, and man, with the orangutan much less closely related. However, inference from a shared deletion suggests that the gorilla and the chimpanzees may be more closely related to one another than they are to man.     

Evolutionary conservation of 5' upstream sequence of nine genes between human and great apes

Nucleotide sequences of nine 5' upstream gene regions for human, chimpanzee, gorilla, and orangutan were determined. We estimated nucleotide differences (d) for each region between human and great apes. The overall d was 0.027 (ranged from 0.004 to 0.052). Rates of nucleotide substitution were estimated by using d and divergence times of human, chimpanzee, gorilla, and orangutan. The overall rate of nucleotide substitution between human and other hominoids was estimated to be 0.52-0.85 x 10(-9). This rate in 5' upstream regions was lower than that of synonymous sites, suggesting that 5' upstream regions have evolved under some functional constraints. Because lower rates have been reported for coding sequences in primates compared to rodents, we also estimated the rate (1.17-1.76 x 10(-9)) of nucleotide substitutions for the corresponding 5' upstream regions in rodents (mouse/rat comparison). Thus the primate rate was lower than rodent rate also for the 5' upstream regions.     

Lineage-Specific Homogenization of the Polyubiquitin Gene Among Human and Great Apes

Ubiquitin is a highly conserved protein, and is encoded by a multigene family among eukaryote species. The polyubiquitin genes, UbB and UbC, comprise tandem multiple ubiquitin coding units without a spacer region or intron. We determined nucleotide sequences for the UbB and UbC of human, chimpanzee, gorilla, and orangutan. The ubiquitin repeat number of UbB was constant (3) in human and great apes, while that of UbC varied: 6 to 11 for human, 10 to 12 for chimpanzee, 8 for gorilla, and 10 for orangutan. The heterogeneity of the repeat number within closely related hominoid species suggests that a lineage-specific unequal crossover and/or gene duplication occurred. A marked homogenization of UbC occurred in gorilla with a low level of synonymous difference (p_s). The homogenization of UbC also occurred in chimpanzee and less strikingly in human. The first and last ubiquitin coding units of UbC were clustered independently between species, and less affected by homogenization during the hominoid evolution. Therefore, the homogenization of ubiquitin coding units is likely due to an unequal crossing-over inside the ubiquitin units. The lineage-specific homogenization of UbC among closely related species suggests that concerted evolution has a key role in the short-term evolution of UbC.     

Mass spectral analyses of the two major apolipoproteins of great ape high density lipoproteins

The two major apolipoproteins associated with human and chimpanzee (Pan troglodytes) high density lipoproteins (HDL) are apoA-I and dimeric apoA-II. Although humans are closely related to great apes, apolipoprotein data do not exist for bonobos (Pan paniscus), western lowland gorillas (Gorilla gorilla gorilla) and the Sumatran orangutans (Pongo abelii). In the absence of any data, other great apes simply have been assumed to have dimeric apoA-II while other primates and most other mammals have been shown to have monomeric apoA-II. Using mass spectrometry, we have measured the molecular masses of apoA-I and apoA-II associated with the HDL of these great apes. Each was observed to have dimeric apoA-II. Being phylogenetically related, one would anticipate these apolipoproteins having a high percentage of invariant sequences when compared with human apolipoproteins. However, the orangutan, which diverged from the human lineage between 16 and 21 million years ago, had an apoA-II with the lowest monomeric mass, 8031.3 Da and the highest apoA-I value, 28,311.7 Da, currently reported for various mammals. Interestingly, the gorilla that diverged from the lineage leading to the human–chimpanzee branch after the orangutan had almost identical mass values to those reported for human apoA-I and apoA-II. But chimpanzee and the bonobo that diverged more recently had identical apoA-II mass values that were slightly larger than reported for the human apolipoprotein. The chimpanzee A-I mass values were very close to those of humans; however, the bonobo had values intermediate to the molecular masses of orangutan and the other great apes. With the already existing genomic data for chimpanzee and the recent entries for the orangutan and gorilla, we were able to demonstrate a close agreement between our mass spectral data and the calculated molecular weights determined from the predicted primary sequences of the respective apolipoproteins. Post-translational modification of these apolipoproteins, involving truncation and oxidation of methionine, are also reported.     

人猿超科和旧大陆猴7种高等灵长类FKN全基因序列的测定及进化分析

测定人猿超科(人、黑猩猩、大猩猩、红毛猩猩和长臂猿)和旧大陆猴(猕猴和叶猴)7种高等灵长类FKN全基因序列, 探讨其系统进化分析。用简并引物PCR(Degenerated PCR)法分别扩增FKN的3个外显子, 其产物经琼脂糖凝胶回收、纯化后测序, 然后用BioEdit软件剪切拼接FKN基因全序列, 用DNAStar比对后比较基因和氨基酸序列同源性, Mega软件重构FKN基因进化树, 应用Datamonkey分析FKN的负选择位点。序列分析发现人猿超科较旧大陆猴FKN基因除了有散在的点突变外, 还有一明显的30 bp的核苷酸缺失突变; 人FKN基因序列与黑猩猩、大猩猩、红毛猩猩、长臂猿、猕猴和叶猴的同源性分别是99.2%、98.4%、98.1%、96.5%、95.9%和93.8%, 由此推导的氨基酸序列同源性分别是98.5%、98.0%、97.7%、94.7%、93.7%和90.5%; FKN基因进化树表明人与黑猩猩关系更近, FKN基因进化和通常认为的物种进化一致; Datamonkey分析结果显示FKN存在3个负选择位点53Q、84D、239N。成功获得人、黑猩猩、大猩猩、红毛猩猩、长臂猿、猕猴和叶猴7种高等灵长类物种FKN全基因序列, 为后续探讨FKN在高等灵长类物种进化过程中免疫学功能演变及其结构与功能的关系奠定基础。     

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.     

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.     

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Evolution of the X-linked Zinc Finger Gene and the Y-linked Zinc Finger Gene in Primates

We have sequenced the partial exon of the zinc finger genes (ZFX and ZFY) in 5 hominoids, 2 Old World monkeys, 1 New World monkey, and 1 prosimian. Among these primate species, the percentage similarities of the nucleotide sequence of the ZFX gene were 96-100% and 91.2-99.7% for the ZFY gene. Of 397 sites in the ZFX and ZFY gene sequences, 20 for ZFX gene and 42 for ZFY gene were found to be variable. Substitution causes 1 amino acid change in ZFX, and 5 in ZFY, among 132 amino acids. The numbers of synonymous substitutions per site (Ks) between human and the chimpanzee, gorilla and orangutan for ZFY gene were 0.026, 0.033, and 0.085, respectively. In contrast, the Ks value between human and hominoid primates for the ZFX gene was 0.008 for each comparison. Comparison of the ZFX and ZFY genes revealed that the synonymous substitution levels were higher in hominoids than in other primates. The rates of synonymous substitution per site per year were higher in the ZFY exon than in the SRY exon, and higher in the ZFY exon than in the ZFY intron, in hominoid primates.     

Fluorescent (F) bodies in the spermatozoa of man and the great apes

Mature spermatozoa of the chimpanzee (Pan troglodytes), the gorilla (Gorilla gorilla), and the orangutan (Pongo pygmaeus) were stained with quinacrine dihydrochloride. Fluorescent (F) bodies were visualized in the spermatozoa of the chimpanzee and gorilla but were absent in the orangutan, in which there is no brilliant fluorescence in any chromosome. The F bodies appeared to be randomly located in the sperm heads of these two species, as they usually are in human spermatozoa. However, the proportion of sperm showing one or more F bodies in the chimpanzee and gorilla was not comparable to what is usually found in man. The F bodies in the chimpanzee presumably represent brilliant regions in the autosomes, since the Y chromosome has no brilliant fluorescence in this species. This is contrary to man, in which the F body is an useful indicator of the Y chromosome. In the gorilla, the F bodies probably correspond to both the Y chromosome and to some brilliant regions in the autosomes.     

Concerted evolution of alpha satellite DNA: Evidence for species specificity and a general lack of sequence conservation among alphoid sequences of higher primates

We investigated relationships among alpha satellite DNA families in the human, gorilla, chimpanzee, and orangutan genomes by filter hybridization with cloned probes which correspond to chromosome-specific alpha satellite DNAs from at least 12 different human chromosomes. These include representatives of both the dimer-based and pentamer-based subfamilies, the two major subfamilies of human alpha satellite. In addition, we evaluated several high-copy dimer-based probes isolated from gorilla genomic DNA. Under low stringency conditions, all human probes tested hybridized extensively with gorilla and chimpanzee alpha satellite sequences. However, only pentameric and other non-dimeric human alphoid probes hybridized with orangutan alpha satellite sequences; probes belonging to the dimer subfamily did not cross-hybridize detectably with orangutan DNA. Moreover, under high stringency conditions, each of the human probes hybridized extensively only with human genomic DNA; none of the probes cross-hybridized effectively with other primate DNAs. Dimer-based gorilla alpha satellite probes hybridized with human and chimpanzee, but not orangutan, sequences under low stringency hybridization conditions, yet were specific for gorilla DNA under high stringency conditions. These results indicate that the alpha satellite DNA family has evolved in a concerted manner, such that considerable sequence divergence is now evident among the alphoid sequences of closely related primate species.     

Analysis of GC-rich repetitive nucleotide sequences in great apes

The genomes of four primate species, belonging to the families Pongidae (chimpanzee, gorilla, and orangutan) and Hylobatidae (gibbons), have been analyzed for the presence and organization of two human GC-rich heterochromatic repetitive sequences: Satellite (Sat) and LongSau (LSau) repeats. By Southern blot hybridization and PCR, both families of repeats were detected in all the analyzed species, thus indicating their origin in an ape ancestor. In the chimpanzee and gorilla, as in man, Sat sequences showed a 68-bp Sau3A periodicity and were preferentially organized in large clusters, whereas in the orangutan, they were organized in DNA fragments of 550 bp, which did not seem to be characterized by a tandem organization. On the contrary, in each of the analyzed species, the bulk of LSau sequences showed a longer Sau3A periodicity than that observed in man (450–550 bp). Furthermore, only in the chimpanzee genome some of LSau repeats seemed to be interspersed within blocks of Sat sequences. This sequence organization, which also characterizes the human genome, is probably absent in the gorilla. In fact, the analysis of a gorilla genomic library suggested that LSau repeats are not preferentially in linkage with Sat sequences. Moreover, LSau sequences were found in a genomic sector characterized by the simultaneous presence of L1 and (CA) repeats, as well as of anonymous sequences and known genes. In spite of the different sequence organization, the nucleotide differences between complete human and gorilla LSau repeats were very few, whereas one gorilla LSau repeat, interrupted by a probably-truncated L1 transposon, showed a higher degree of divergence. Besides the gorilla, this unusual sequence organization was detected in man, and, to a lesser extent, in the chimpanzee. Correspondence to: R. Meneveri     

Fast adaptive coevolution of nuclear and mitochondrial subunits of ATP synthetase in orangutan

Nuclear and mitochondrial genomes have to work in concert to generate a functional oxidative phosphorylation (OXPHOS) system. We have previously shown that we could restore partial OXPHOS function when chimpanzee or gorilla mitochondrial DNA (mtDNA) were introduced into human cells lacking mtDNA. However, we were unable to maintain orangutan mitochondrial DNA in a human cell. We have now produced chimpanzee, gorilla, orangutan, and baboon cells lacking mtDNA and attempted to introduce mtDNA from different apes into them. Surprisingly, we were able to maintain human mtDNA in an orangutan nuclear background, even though these cells showed severe OXPHOS abnormalities, including a complete absence of assembled ATP synthetase. Phylogenetic analysis of complex V mtDNA-encoded subunits showed that they are among the most evolutionarily divergent components of the mitochondrial genome between orangutan and the other apes. Our studies showed that adaptive coevolution of nuclear and mitochondrial components in apes can be fast and accelerate in recent branches of anthropoid primates.     

Mitochondrial DNA sequences of primates: Tempo and mode of evolution

Summary We cloned and sequenced a segment of mitochondrial DNA from human, chimpanzee, gorilla, orangutan, and gibbon. This segment is 896 bp in length, contains the genes for three transfer RNAs and parts of two proteins, and is homologous in all 5 primates. The 5 sequences differ from one another by base substitutions at 283 positions and by a deletion of one base pair. The sequence differences range from 9 to 19% among species, in agreement with estimates from cleavage map comparisons, thus confirming that the rate of mtDNA evolution in primates is 5 to 10 times higher than in nuclear DNA. The most striking new finding to emerge from these comparisons is that transitions greatly outnumber transversions. Ninety-two percent of the differences among the most closely related species (human, chimpanzee, and gorilla) are transitions. For pairs of species with longer divergence times, the observed percentage of transitions falls until, in the case of comparisons between primates and non-primates, it reaches a value of 45. The time dependence is probably due to obliteration of the record of transitions by multiple substitutions at the same nucleotide site. This finding illustrates the importance of choosing closely related species for analysis of the evolutionary process. The remarkable bias toward transitions in mtDNA evolution necessitates the revision of equations that correct for multiple substitutions at the same site. With revised equations, we calculated the incidence of silent and replacement substitutions in the two protein-coding genes. The silent substitution rate is 4 to 6 times higher than the replacement rate, indicating strong functional constraints at replacement sites. Moreover, the silent rate for these two genes is about 10% per million years, a value 10 times higher than the silent rate for the nuclear genes studied so far. In addition, the mean substitution rate in the three mitochondrial tRNA genes is at least 100 times higher than in nuclear tRNA genes. Finally, genealogical analysis of the sequence differences supports the view that the human lineage branched off only slightly before the gorilla and chimpanzee lineages diverged and strengthens the hypothesis that humans are more related to gorillas and chimpanzees than is the orangutan.Abbreviations mtDNA mitochondrial DNA - bp base pair - URF unidentified reading frame     

Panels of somatic cell hybrids specific for chimpanzee, gorilla, orangutan, and baboon

The generation of panels of somatic cell hybrids specific for chimpanzee, gorilla, orangutan, and olive baboon is reported. The chromosome content of each hybrid clone was characterized using reverse painting on human normal metaphases and by the use of appropriate sequence tag sites (STSs), one for each chromosome arm. These resources can be advantageously exploited in the characterization of chromosome architecture of different primate species, with special reference to the discrimination of inter- and intra-chromosomal arrangement of segmental duplications.     

Molecular evolution of intergenic DNA in higher primates: pattern of DNA changes, molecular clock, and evolution of repetitive sequences

A 3.1-kb intergenic DNA fragment located between the psi beta-globin and delta-globin genes in the beta-globin gene cluster was cloned from gorilla, orangutan, rhesus monkey, and spider monkey, and the nucleotide sequence of each fragment was determined. The phylogeny of these four sequences, together with two previously published allelic sequences from humans and one from chimpanzee, was constructed, and the accumulation of mutations in the region was analyzed. The sites of base substitutions are not evenly distributed within the region: two Alu repeats have accumulated 0.21 + 0.02 substitutions/site with 0.15 + 0.008 substitutions/site in the remainder of the fragment. The occurrence of substitutions at neighboring sites is more frequent than would be expected if they were independent. The observed excesses disappear when ancestral -CG- dinucleotide sites are excluded. The phylogenetic relationships of the sequences indicate that the human sequence shares a most recent coancestor with the chimpanzee sequence. The data also show that great apes have accumulated fewer mutations in this part of the genome than has the rhesus monkey. The relative rates of accumulation of 12 kinds of nucleotide substitution in the region during primate evolution are asymmetric in the DNA strands. From these rates of accumulation, the origin of a simple stretch of sequence near the 3' end of the 3.1-kb fragment was deduced to be a sequence comprising 50% T and 50% C on one strand. The two oppositely oriented Alu sequences in the 3.1-kb region were inserted at their present positions before the divergence of the New-World monkeys from other lineages. Our analysis shows that the nucleotide sequences of the two Alu repeats in spider monkey are unexpectedly similar both to each other and to the deduced ancestral sequence of Alu repeats. The data suggest that there has been some type of recombinational event between the spider monkey Alu repeats but that it was not a simple gene conversion.      

Regional localization of human M-BCR gene to chromosome 23 band q11 in the great apes

We hybridized a human M-BCR DNA probe to the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilld) and orangutan (Pongo pygmaeus) by FISH-technique. The human M-BCR gene was localized to chromosome 23 band q11 (23q11), which is equivalent to the human chromosome 22 band q11 in all three species. The conservation of M-BCR gene in higher primates at the corresponding human chromosome locus provides phylogenetic clues concerning the evolution of genes.     

Examination of hominid evolution by DNA sequence homology

Hominoid phylogeny was investigated in terms of unique DNA sequence homologies. In comparisons from the human standpoint the ΔTe₅₀ DNA values were Man 0, chimpanzee 0·7, gorilla 1·4, gibbon 2·7, orangutan 2·9, and African green monkey 5·7. In comparisons from the orangutan standpoint the ΔTe₅₀ DNA values were orangutan 0, chimpanzee 1·8, Man 1·9, gorilla 2·3, gibbon 2·4 and African green monkey 4·3. These results indicate that chimpanzee and gorilla are cladistically closer to Man than to orangutan and other primates, and that gorilla DNA may have diverged slightly more from the ancestral state than chimpanzee or human DNA. Comparisons from chimpanzee and gorilla DNA standpoints are needed to achieve a more definitive picture of hominoid phylogeny.