Recombination in the genesis and evolution of hepatitis B virus genotypes

Hepatitis B virus (HBV) infection is widely distributed in both human and ape populations throughout the world and is a major cause of human morbidity and mortality. HBV variants are currently classified into the human genotypes A to H and species-associated chimpanzee and gibbon/orangutan groups. To examine the role of recombination in the evolution of HBV, large-scale data retrieval and automated phylogenetic analysis (TreeOrder scanning) were carried out on all available published complete genome sequences of HBV. We detected a total of 24 phylogenetically independent potential recombinants (different genotype combinations or distinct breakpoints), eight of which were previously undescribed. Instances of intergenotype recombination were observed in all human and ape HBV variants, including evidence for a novel gibbon/genotype C recombinant among HBV variants from Vietnam. By recording sequence positions in trees generated from sequential fragments across the genome, violations of phylogeny between trees also provided evidence for frequent intragenotype recombination between members of genotypes A, D, F/H, and gibbon variants but not in B, C, or the Asian B/C recombinant group. In many cases, favored positions for both inter- and intragenotype recombination matched positions of phylogenetic reorganization between the human and ape genotypes, such as the end of the surface gene and the core gene, where sequence relationships between genotypes changed in the TreeOrder scan. These findings provide evidence for the occurrence of past, extensive recombination events in the evolutionary history of the currently classified genotypes of HBV and potentially in changes in its global epidemiology and associations with human disease.     

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.     

Species association of hepatitis B virus (HBV) in non-human apes; evidence for recombination between gorilla and chimpanzee variants

Hepatitis B virus (HBV) infections are widely distributed in humans, infecting approximately one third of the world's population. HBV variants have also been detected and genetically characterised from Old World apes; Gorilla gorilla (gorilla), Pan troglodytes (chimpanzee), Pongo pygmaeus (orang-utan), Nomascus nastusus and Hylobates pileatus (gibbons) and from the New World monkey, Lagothrix lagotricha (woolly monkey). To investigate species-specificity and potential for cross species transmission of HBV between sympatric species of apes (such as gorillas and chimpanzees in Central Africa) or between humans and chimpanzees or gorillas, variants of HBV infecting captive wild-born non-human primates were genetically characterised. 9 of 62 chimpanzees (11.3%) and two from 11 gorillas (18%) were HBV-infected (15% combined frequency), while other Old world monkey species were negative. Complete genome sequences were obtained from six of the infected chimpanzee and both gorillas; those from P. t .ellioti grouped with previously characterised variants from this subspecies. However, variants recovered from P. t. troglodytes HBV variants also grouped within this clade, indicative of transmission between sub-species, forming a paraphyletic clade. The two gorilla viruses were phylogenetically distinct from chimpanzee and human variants although one showed evidence for a recombination event with a P.t.e.-derived HBV variant in the partial X and core gene region. Both of these observations provide evidence for circulation of HBV between different species and sub-species of non-human primates, a conclusion that differs from the hypothesis if of strict host specificity of HBV genotypes.     

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.     

Molecular evolution of the psi eta-globin gene locus: gibbon phylogeny and the hominoid slowdown

An 8.4-kb genomic region spanning both the psi eta-globin gene locus and flanking DNA was sequenced from the common gibbon (Hylobates lar). In addition, sequencing of the entire orthologous region from galago (Galago crassicaudatus) was completed. The gibbon and galago sequences, along with published orthologous sequences from 10 other species, were aligned. These noncoding nucleotide sequences represented four human alleles, four apes (chimpanzee, gorilla, organgutan, and gibbon), an Old World monkey (rhesus monkey), two New World monkeys (spider and owl monkeys), tarsier, two strepsirhines (galago and lemur), and goat. Divergence and maximum parsimony analyses of the psi eta genomic region first groups humans and chimpanzees and then, at progressively more ancient branch points, successively joins gorillas, orangutans, gibbons, Old World monkeys, New World monkeys, tarsiers, and strepsirhines (the lemuriform-lorisiform branch of primates). This cladistic pattern supports the taxonomic grouping of all extant hominoids into family Hominidae, the division of Hominidae into subfamilies Hylobatinae (gibbons) and Homininae, the division of Homininae into tribes Pongini (orangutans) and Hominini, and the division of Hominini into subtribes Gorillina (gorillas) and Hominina (chimpanzees and humans). The additional gibbon and galago sequence data provide further support for the occurrence of a graded evolutionary-rate slowdown in the descent of simian primates, with the slowing rate being more pronounced in the great-ape and human lineages than in the gibbon or monkey lineages. A comparison of global versus local molecular clocks reveals that local clock predictions, when focused on a specific number of species within a narrow time frame, provide a more accurate estimate of divergence dates than do those of global clocks.     

Comparative studies on an antigenicity of plasma proteins from humans and apes by ELISA: a close relationship of chimpanzee and human

1. Antigenic differences between human and ape plasma proteins were quantitatively investigated by enzyme-linked immunosorbent assay (ELISA) using antisera against human and chimpanzee plasmas. 2. With anti-human plasma serum, both the chimpanzee and gorilla were very close to the human, although the chimpanzee was slightly closer to the human than to the gorilla; relative immunological distance (relative ID) of the chimpanzee was 71, while that of the gorilla was 74. 3. With anti-chimpanzee plasma serum, the chimpanzee was found to be closely related to the human; relative ID of the chimpanzee was 58, while that of the gorilla was 75. 4. From these a molecular phylogeny for humans and apes was deduced; among living apes, the chimpanzee is the most closely related species to the human.     

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。     

Serum cholinesterase activities and inhibition profiles of nonhuman primates

Serum cholinesterase activities and inhibition profiles of 169 chimpanzees, 15 gorillas, 26 orangutans, seven gibbons, and 12 rhesus monkeys were determined. Mean values of activities against benzoylcholine (μmols/min/ml) and dibucaine, fluoride, and Ro 2-0683 numbers (percentage inhibition of benzoylcholine hydrolysis) are: chimpanzee, 2.276, 80, 64, and 97; gorilla, 9.403, 82, 71, and 96; orangutan, 0.747, 94, 6, and 98; gibbon, 0.071, 89, 7, and 94; and rhesus monkey, 0.859, 95, 10, and 99, respectively. Sernylan numbers were determined of the last 100 chimpanzee serums collected and of each of the gorilla, orangutan, gibbon, and rhesus monkey serums. Mean values of Sernylan numbers are: chimpanzee, 80; gorilla, 81; orangutan, 95; gibbon, 94; and rhesus monkey, 96. The chimpanzee and the gorilla have dibucaine, fluoride, Ro 2-0683, and Sernylan numbers within the range found in men who are homozygotes for the usual cholinesterase (genotype E₁^uE₁^u). No cholinesterase variant was found in any chimpanzee or gorilla. The orangutan, gibbon, and rhesus monkey have inhibition profiles that resemble one another, with higher dibucaine and Sernylan numbers and much lower fluoride numbers than the chimpanzee or the gorilla. The results of the inhibition tests suggest that the African apes, chimpanzee and gorilla, are related more closely to man than are the Asian apes, orangutan and gibbon.     

Molecular evolution of IgG subclass among nonhuman primates: implication of differences in antigenic determinants among Apes

The cross-reactivity of five different rabbit polyclonal antibodies to human IgG and IgG subclass (IgG1, IgG2, IgG3, and IgG4) was determined by competitive ELISA with nine nonhuman primate species including five apes, three Old World monkeys, and one New World monkey. As similar to those previously reported, the reactivity of anti-human IgG antibody with plasma from different primate species was closely related with phylogenic distance from human. Every anti-human IgG subclass antibody showed low cross-reactivity with plasma from Old World and New World monkeys. The plasma from all apes except for gibbons (Hylobates spp.) showed 60 to 100% of cross-reactivity with anti-human IgG2 and IgG3 antibodies. On the other hand, chimpanzee (Pan troglodytes andPan paniscus) and orangutan (Pongo pygmaeus) plasma showed 100% cross-reactivity with anti-human IgG1 antibody, but gorilla (Gorilla gorilla) and gibbon plasma showed no cross-reactivity. The chimpanzee and gorilla plasma cross-reacted with anti-human IgG4 antibody at different reactivity, 100% in chimpanzee and 50% in gorilla, but no cross-reactivity was observed in orangutan and gibbon plasma. These results suggest the possibilities that the divergence of “human-type” IgG subclasses might occur at the time of divergence ofHomo sapience fromHylobatidae, and that the molecular evolution of IgG1 as well as IgG4 is different from that of IgG2 and IgG3 in great apes, this is probably caused by different in development of immune function in apes during the course of evolution.     

Nucleotide sequence comparison of a chromosome rearrangement on human chromosome 12 and the corresponding ape chromosomes

Chromosome rearrangement has been considered to be important in the evolutionary process. Here, we demonstrate the evolutionary relationship of the rearranged human chromosome 12 and the corresponding chromosome XII in apes (chimpanzee, bonobo, gorilla, orangutan, and gibbon) by examining PCR products derived from the breakpoints of inversions and by conducting shotgun sequencing of a gorilla fosmid clone containing the breakpoint and a "duplicated segment" (duplicon). We confirmed that a pair of 23-kb duplicons flank the breakpoints of inversions on the long and short arms of chimpanzee chromosome XII. Although only the 23-kb duplicon on the long arm of chimpanzee chromosome XII and its telomeric flanking sequence are found to be conserved among the hominoids (human, great apes, and gibbons), the duplicon on the short arm of chimpanzee chromosome XII is suggested to be the result of a duplication from that on the long arm. Furthermore, the shotgun sequencing of a gorilla fosmid indicated that the breakpoint on the long arm of the gorilla is located at a different position 1.9 kb from that of chimpanzee. The region is flanked by a sequence homologous to that of human chromosome 6q22. Our findings and sequence analysis suggest a close relationship between segmental duplication and chromosome rearrangement (or breakpoint of inversion) in Hominoidea. The role of the chromosome rearrangement in speciation is also discussed based on our new results.     

Evolution of four human Y chromosomal unique sequences

Four cloned unique sequences from the human Y chromosome, two of which are found only on the Y chromosome and two of which are on both the X and Y chromosomes, were hybridized to restriction enzyme-treated DNA samples of a male and a female chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and pig-tailed macaque (Macaca nemestrina); and a male orangutan (Pongo pygmaeus) and gibbon (Hylobates lar). One of the human Y-specific probes hybridized only to male DNA among the humans and great apes, and thus its Y linkage and sequence similarities are conserved. The other human Y-specific clone hybridized to male and female DNA from the humans, great apes, and gibbon, indicating its presence on the X chromosome or autosomes. Two human sequences present on both the X and Y chromosomes also demonstrated conservation as indicated by hybridization to genomic DNAs of distantly related species and by partial conservation of restriction enzyme sites. Although conservation of Y linkage can only be demonstrated for one of these four sequences, these results suggest that Y-chromosomal unique sequence genes do not diverge markedly more rapidly than unique sequences located on other chromosomes. However, this sequence conservation may in part be due to evolution while part of other chromosomes.     

Phylogenetic isolation of a human alu flounder gene: Drift to new subfamily identity

A severe bottleneck in the size of the PV Alu subfamily in the common ancestor of human and gorilla has been used to isolate an Alu source gene. The human PV Alu subfamily consists of about one thousand members which are absent in gorilla and chimpanzee DNA. Exhaustive library screening shows that there are as few as two PV Alus in the gorilla genome. One is gorilla-specific, i.e., absent in the orthologous loci in both human and chimpanzee, suggesting the independent retrotranspositional activity of the PV subfamily in the gorilla lineage. The second of these two gorilla PV Alus is present in both human and chimpanzee DNAs and is the single PV Alu known to precede the radiation of these three species. The orthologous Alu in gibbon DNA resembles the next older Alu subfamily. Thus, this Alu locus is originally templated by a non-PV source gene and acquired characteristic PV sequence variants by mutational drift in situ, consequently becoming the first member and presumptive founder of this PV subfamily. Correspondence to: C.W. Schmid     

人与大猩猩,黑猩猩和猩猩亲缘关系的探讨

有关人锆超科的系统发育仍然存在刍议。争论焦点在与大猩猩和黑猩猩哪 个关系更近一点。酪氨酸酶是黑色素合成中的关键酶,酪氨酶基因的突变将导致白化病。测定了人猿科中大猩猩,黑猩猩、猩猩和长臂锆产基因全部５个外显子的ＤＮＡ序列。     

Anti-human red cell monoclonal antibodies produced by macaque-mouse heterohybridomas: their reactivity with human and nonhuman primate erythrocytes.

Eighteen monoclonal antibodies (Mabs) against human red blood cells (RBCs) produced by macaque mouse heterobybridomas gave uniformly positive reactions with all human samples except for some with particular null phenotypes. Based on reactions with latter cells, the human antigenic targets of 11 antibodies could be identified: six were specific for glycophorin-related antigens (Wr(b), En(a), Ge4), and each of the live remaining antibodies showed one of the following specificities: CD55, CD44, CD59, Kell, and Rh proteins. Four Mabs recognized the Vc antigen of the chimpanzee V-A-B-D system. Six macaque Mabs detected polymorphisms in chimpanzee, gorilla, orangutan, and gibbon that did not correspond to any known blood group in these animals, while other Mabs gave monomorphic reactions with ape RBCs. The reagents produced by macaque hybridomas are useful tools not only for human blood grouping tests, but also for tracing the relationships among blood group antigens of man and anthropoid apes.     

Prevalence of hepatitis B virus infections in nonhuman primates

The aim of this study was to determine the prevalence of hepatitis B virus (HBV) infection in nonhuman primates. Serum samples from Europe, Thailand and Vietnam were analyzed. Sera obtained from 262 apes and 454 monkeys were tested for HBV infection serologically and for HBV DNA using nested PCR (nPCR). A total number of 198 ape sera and all but one (Cercopithecus aethiops) of the 4543 monkey sera had no serological signs of HBV infection. Among the 64 of 262 (24.4%) seropositive ape sera, we found, as in humans, different stages of HBV infection: very early HBV infection, active infection with high level of infectivity, virus carriers with low infectivity, and passed HBV infection. In the cases with passed infection, 47.8% harbored HBV DNA in the presence of protective antibodies to the HBV surface antigen (HBsAb). This indicates HBV persistence in apes despite immune control. In contrast to apes, in monkeys HBV infection is a very rare event.     

Concerted evolution of the primate immunoglobulin alpha-gene through gene conversion.

We determined four nucleotide sequences of the hominoid immunoglobulin alpha (C alpha) genes (chimpanzee C alpha 2, gorilla C alpha 2, and gibbon C alpha 1 and C alpha 2 genes), which made possible the examination of gene conversions in all hominoid C alpha genes. The following three methods were used to detect gene conversions: 1) phenetic tree construction; 2) detection of a DNA segment with extremely low variability between duplicated C alpha genes; and 3) a site by site search of shared nucleotide changes between duplicated C alpha genes. Results obtained from method 1 indicated a concerted evolution of the duplicated C alpha genes in the human, chimpanzee, gorilla, and gibbon lineages, while results obtained from method 2 suggested gene conversions in the human, gorilla, and gibbon C alpha genes. With method 3 we identified clusters of shared nucleotide changes between duplicated C alpha genes in human, chimpanzee, gorilla, and gibbon lineages, and in their hypothetical ancestors. In the present study converted regions were identified over the entire C alpha gene region excluding a few sites in the coding region which have escaped from gene conversion. This indicates that gene conversion is a general phenomenon in evolution, that can be clearly observed in non-functional regions.     

Repetitive sequences originating from the centromere constitute large-scale heterochromatin in the telomere region in the siamang, a small ape

Chromosomes of the siamang Symphalangus syndactylus (a small ape) carry large-scale heterochromatic structures at their ends. These structures look similar, by chromosome C-banding, to chromosome-end heterochromatin found in chimpanzee, bonobo and gorilla (African great apes), of which a major component is tandem repeats of 32-bp-long, AT-rich units. In the present study, we identified repetitive sequences that are a major component of the siamang heterochromatin. Their repeat units are 171 bp in length, and exhibit sequence similarity to alpha satellite DNA, a major component of the centromeres in primates. Thus, the large-scale heterochromatic structures have different origins between the great apes and the small ape. The presence of alpha satellite DNA in the telomere region has previously been reported in the white-cheeked gibbon Nomascus leucogenys, another small ape species. There is, however, a difference in the size of the telomere-region alpha satellite DNA, which is far larger in the siamang. It is not known whether the sequences of these two species (of different genera) have a common origin because the phylogenetic relationship of genera within the small ape family is still not clear. Possible evolutionary scenarios are discussed.     

The role of behavioral research in the conservation of chimpanzees and gorillas

Chimpanzees and gorillas are among man's closest living relatives, sharing most of the human genetic code and having many similarities to humans in anatomy, physiology, and behavior. Like humans, these apes make and use tools and have strong family bonds. Chimpanzees even show population-specific behaviors similar to those of human cultures. However, chimpanzee and gorilla populations are in dramatic decline due to bushmeat hunting, habitat loss, and the varied risks of small, isolated populations. The first step in conserving the world's ape populations in the wild is to recognize and understand the complexities of these threats. Mitigating the risks takes a deeper understanding of ape behavior. This article provides examples of how gorilla and chimpanzee behavioral studies intersect with, and are critical to, conservation efforts.     

Mitochondrial 16S rRNA sequence diversity of hominoids

We determined nucleotide sequences of the 16S rRNA gene of mitochondrial DNA (mtDNA) (about 1.6 kb) for 35 chimpanzee, 13 bonobo, 10 gorilla, 16 orangutan, and 23 gibbon individuals. We compared those data with published sequences and estimated nucleotide diversity for each species. All the ape species showed higher diversity than human. We also constructed phylogenetic trees and networks. The two orangutan subspecies were clearly separated from each other, and Sumatran orangutans showed much higher nucleotide diversity than Bornean orangutans. Some gibbon species did not form monophyletic clusters, and variation within species was not much different from that among species in the subgenus Hylobates.     

The Role of Behavioral Research in the Conservation of Chimpanzees and Gorillas

Chimpanzees and gorillas are among man's closest living relatives, sharing most of the human genetic code and having many similarities to humans in anatomy, physiology, and behavior. Like humans, these apes make and use tools and have strong family bonds. Chimpanzees even show population-specific behaviors similar to those of human cultures. However, chimpanzee and gorilla populations are in dramatic decline due to bushmeat hunting, habitat loss, and the varied risks of small, isolated populations. The first step in conserving the world's ape populations in the wild is to recognize and understand the complexities of these threats. Mitigating the risks takes a deeper understanding of ape behavior. This article provides examples of how gorilla and chimpanzee behavioral studies intersect with, and are critical to, conservation efforts.