A novel Australian flying-fox retrovirus shares an evolutionary ancestor with Koala, Gibbon and Melomys gamma-retroviruses.

A novel gamma-retroviral sequence (7912 bp), inclusive of both partial 5' and 3' long terminal repeat regions, was identified from the brain of a black flying-fox (Pteropus alecto), Queensland, Australia. The sequence was distinct from other retroviral sequences identified in bats and showed greater identity to Koala, Gibbon ape leukaemia, Melomys burtoni and Woolly monkey retroviruses, forming their own phylogenetic clade. This finding suggests that these retroviruses may have an unknown common ancestor and that further investigation into the diversity of gamma-retroviruses in Australian Pteropus species may elucidate their evolutionary origins.

Transcriptional activation of a chimeric retrogene PIPSL in a hominoid ancestor.

Retrogenes are a class of functional genes derived from the mRNA of various intron-containing genes. PIPSL was created through a unique mechanism, whereby distinct genes were assembled at the RNA level, and the resulting chimera was then reverse transcribed and integrated into the genome by the L1 retrotransposon. Expression of PIPSL RNA via its transcription start sites (TSSs) has been confirmed in the testes of humans and chimpanzee. Here, we demonstrated that PIPSL RNA is expressed in the testis of the white-handed gibbon. The 5'-end positions of gibbon RNAs were confined to a narrow range upstream of the PIPSL start codon and overlapped with those of orangutan and human, suggesting that PIPSL TSSs are similar among hominoid species. Reporter assays using a luciferase gene and the flanking sequences of human PIPSL showed that an upstream sequence exhibits weak promoter activity in human cells. Our findings suggest that PIPSL might have acquired a promoter at an early stage of hominoid evolution before the divergence of gibbons and ultimately retained similar TSSs in all of the lineages. Moreover, the upstream sequence derived from the phosphatidylinositol-4-phosphate 5-kinase, type I, alpha 5' untranslated region and/or neighboring repetitive sequences in the genome possibly exhibits promoter activity. Furthermore, we observed that a TATA-box-like sequence has emerged by nucleotide substitution in a lineage leading to humans, with this possibly responsible for a broader distribution of the human PIPSL TSSs.

A new look at the origins of gibbon ape leukemia virus.

Is the origin of gibbon ape leukemia virus (GALV) human after all? When GALV was discovered and found to cause neoplastic disease in gibbons, it stimulated a great deal of research including investigations into the origins of this virus. A number of publications have suggested that the GALV progenitor was a retrovirus present in one of several species of South East Asian rodents that had close contact with captive gibbons. However, there are no published retroviral sequences from any South East Asian species to support this view. Here we present an alternative hypothesis that the origin of GALV is a virus closely related to Melomys burtoni retrovirus, and that this virus infected human patients in Papua New Guinea from whom biological material was obtained or in some way contaminated these samples. This material we propose contained infectious MbRV-related virus that was then unwittingly introduced into gibbons which subsequently developed GALV infections.

Novel variable number of tandem repeats of gibbon MAOA gene and its evolutionary significance.

Variable number of tandem repeats (VNTRs) are scattered throughout the primate genome, and genetic variation of these VNTRs have been accumulated during primate radiation. Here, we analyzed VNTRs upstream of the monoamine oxidase A (MAOA) gene in 11 different gibbon species. An abundance of truncated VNTR sequences and copy number differences were observed compared to those of human VNTR sequences. To better understand the biological role of these VNTRs, a luciferase activity assay was conducted and results indicated that selected VNTR sequences of the MAOA gene from human and three different gibbon species (Hylobates klossii, Hylobates lar, and Nomascus concolor) showed silencing ability. Together, these data could be useful for understanding the evolutionary history and functional significance of MAOA VNTR sequences in gibbon species.

Unresolved molecular phylogenies of gibbons and siamangs (Family: Hylobatidae) based on mitochondrial, Y-linked, and X-linked loci indicate a rapid Miocene radiation or sudden vicariance event.

According to recent taxonomic reclassification, the primate family Hylobatidae contains four genera (Hoolock, Nomascus, Symphalangus, and Hylobates) and between 14 and 18 species, making it by far the most species-rich group of extant hominoids. Known as the "small apes", these small arboreal primates are distributed throughout Southeast, South and East Asia. Considerable uncertainty surrounds the phylogeny of extant hylobatids, particularly the relationships among the genera and the species within the Hylobates genus. In this paper we use parsimony, likelihood, and Bayesian methods to analyze a dataset containing nearly 14 kilobase pairs, which includes newly collected sequences from X-linked, Y-linked, and mitochondrial loci together with data from previous mitochondrial studies. Parsimony, likelihood, and Bayesian analyses largely failed to find a significant difference among phylogenies with any of the four genera as the most basal taxon. All analyses, however, support a tree with Hylobates and Symphalangus as most closely related genera. One strongly supported phylogenetic result within the Hylobates genus is that Hylobates pileatus is the most basal taxon. Multiple analyses failed to find significant support for any singular genus-level phylogeny. While it is natural to suspect that there might not be sufficient data for phylogenetic resolution (whenever that situation occurs), an alternative hypothesis relating to the nature of gibbon speciation exists. This lack of resolution may be the result of a rapid radiation or a sudden vicariance event of the hylobatid genera, and it is likely that a similarly rapid radiation occurred within the Hylobates genus. Additional molecular and paleontological evidence are necessary to better test among these, and other, hypotheses of hylobatid evolution.

Hominoid seminal protein evolution and ancestral mating behavior.

Hominoid mating systems show extensive variation among species. The degree of sexual dimorphism in body size and canine size varies among primates in accordance with their mating system, as does the testes size and the consistency of ejaculated semen, in response to differing levels of sperm competition. To investigate patterns of evolution at hominoid seminal proteins and to make inferences regarding the mating systems of extinct taxa, we sequenced the entire coding region of the prostate-specific transglutaminase (TGM4) gene in human, chimpanzee, bonobo, western lowland gorilla, eastern lowland gorilla, orangutan, and siamang, including multiple humans, chimps, and gorillas. Partial DNA sequence of the coding regions was also obtained for one eastern lowland gorilla at the semenogelin genes (SEMG1 and SEMG2), which code for the predominant proteins in semen. Patterns of nucleotide variation and inferred protein sequence change were evaluated within and between species. Combining the present data with previous studies demonstrates a high rate of amino acid substitutions, and low intraspecific variation, at seminal proteins in Pan, presumably driven by strong sperm competition. Both gorilla species apparently possess nonfunctional TGM4, SEMG1, and SEMG2 genes, suggesting that gorillas have had low sperm competition, and therefore their current polygynous mating system, for a long time before their divergence. Similarly, orangutans show longstanding stasis at TGM4, which may be interpreted as evidence for an unchanging mating system for most of their evolution after their divergence from African apes. In contrast to the great apes, the data from humans could be interpreted as evidence of fluctuations between different mating systems or alternatively as a relaxed functional constraint in these proteins. It is our hope that this study is a first step toward developing a model to predict ancestral mating systems from extant molecular data to complement interpretations from the fossil record.

Primate TNF promoters reveal markers of phylogeny and evolution of innate immunity.

BACKGROUND: Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry. METHODOLOGY/PRINCIPAL FINDINGS: Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages. CONCLUSIONS/SIGNIFICANCE: Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF.

Fluorescence in situ hybridization to chromosomes as a tool to understand human and primate genome evolution.

For the last 15 years molecular cytogenetic techniques have been extensively used to study primate evolution. Molecular probes were helpful to distinguish mammalian chromosomes and chromosome segments on the basis of their DNA content rather than solely on morphological features such as banding patterns. Various landmark rearrangements have been identified for most of the nodes in primate phylogeny while chromosome banding still provides helpful reference maps. Fluorescence in situ hybridization (FISH) techniques were used with probes of different complexity including chromosome painting probes, probes derived from chromosome sub-regions and in the size of a single gene. Since more recently, in silico techniques have been applied to trace down evolutionarily derived chromosome rearrangements by searching the human and mouse genome sequence databases. More detailed breakpoint analyses of chromosome rearrangements that occurred during higher primate evolution also gave some insights into the molecular changes in chromosome rearrangements that occurred in evolution. Hardly any "fusion genes" as known from chromosome rearrangements in cancer cells or dramatic "position effects" of genes transferred to new sites in primate genomes have been reported yet. Most breakpoint regions have been identified within gene poor areas rich in repetitive elements and/or low copy repeats (segmental duplications). The progress in various molecular and molecular-cytogenetic approaches including the recently launched chimpanzee genome project suggests that these new tools will have a significant impact on the further understanding of human genome evolution.

Phylogenetic shadowing of primate sequences to find functional regions of the human genome.

Nonhuman primates represent the most relevant model organisms to understand the biology of Homo sapiens. The recent divergence and associated overall sequence conservation between individual members of this taxon have nonetheless largely precluded the use of primates in comparative sequence studies. We used sequence comparisons of an extensive set of Old World and New World monkeys and hominoids to identify functional regions in the human genome. Analysis of these data enabled the discovery of primate-specific gene regulatory elements and the demarcation of the exons of multiple genes. Much of the information content of the comprehensive primate sequence comparisons could be captured with a small subset of phylogenetically close primates. These results demonstrate the utility of intraprimate sequence comparisons to discover common mammalian as well as primate-specific functional elements in the human genome, which are unattainable through the evaluation of more evolutionarily distant species.

Mutations in the factor IX gene (F9) during the past 150 years have relative rates similar to ancient mutations.

Pollutants and dietary mutagens have been associated with somatic mutation and cancer, but the extent of their influence on germline mutation is not clear. Since deleterious germline mutations can be transmitted for thousands of years, any influence on germline mutation from the vast increase in man-made chemicals of the past 150 years would be an important public health issue. Observed disease causing mutations in the X-linked factor IX gene (F9) of hemophilia B patients originated predominantly in the past 150 years, since the half-life of these mutations in human populations had been about two generations before effective treatment became available about a generation ago. Recent changes in germline mutational processes may be detected by comparison of the observed hemophilia B causing mutation pattern in F9 with the pattern of neutral polymorphisms which occurred over a much longer period of time. By scanning a total of 1.5 megabases of deep intronic regions of F9 in the genomic DNA from 84 individuals, 42 neutral polymorphisms were found in 23 haplotypes that differed by at least 11 mutations from the ancestral primate haplotype. By sequencing F9 in seven non-human primates, 39 of these polymorphisms were characterized as ancient mutations relative to a unanimous ancestral primate allele. This ancient mutation pattern was compared to the recent pattern of hemophilia B causing mutations. Remarkably, no significant difference was found (P=0.5), suggesting that the vast increase in man-made chemicals during the past 150 years has not had a major impact on the pattern of human germline mutation. This result is consistent with the hypothesis that endogenous processes dominate germline mutation.

A classification efficiency test of spectral karyotyping and multiplex fluorescence in situ hybridization: identification of chromosome homologies between Homo sapiens and Hylobates leucogenys.

Two digital fluorescence microscopy systems, spectral karyotyping (SKY) and multiplex fluorescence in situ hybridisation (M-FISH), are used with multicolour probe sets to assist in the detection of chromosome aberrations. We have compared the resolution of the two methods in their ability to identify karyotype rearrangements, which have occurred during the divergence of Homo sapiens and Hylobates leucogenys in evolution. A 24-color human paint kit distinguishes 74 conserved autosomal segments in H. leucogenys, some of which are difficult to resolve. We examined the extent to which the SKY and M-FISH techniques are able to detect the smallest of these bands. We have found this to be a rigorous test of multicolour chromosome classification systems. We conclude from our results that both systems are able invariably to classify the majority of conserved segments but differ in the efficiency of detection of small inserts.

Characterization of chromosomal aberrations in lung cancer cell lines by cross-species color banding.

Using cross-species color banding (RxFISH) and chromosome painting techniques, chromosomal aberrations were investigated in six lung cancer cell lines (NCI-H524, H865, H522, H1373, H358, A549). Each cell line had a variable number of numerical and structural cytogenetic aberrations. While NCI-H524, -H865, and -H522 had near diploidy, NCI-H358, -H1373, and A549 had near triploidy. The origins of the marker chromosomes were further identified by RxFISH and chromosome painting: Nonrandom chromosomal rearrangements were seen on 1p, 3q, 5p10-p15, 6q13-q21, 7q22-q31, 9p32, 15q22-qter, 17p, 17q21-q25, and 21. These abnormal cytogenetic findings indicate that multiple genetic lesions are associated with the development of lung cancer, and thus, these might be possible candidate regions for the abnormal genes involved in lung cancer.

A Salmonella typhimurium strain genetically engineered to secrete effectively a bioactive human interleukin (hIL)-6 via the Escherichia coli hemolysin secretion apparatus.

Human interleukin-6 (hIL-6) cDNA was genetically fused with the Escherichia coli hemolysin secretorial signal (hlyA[S]) sequence in a plasmid vector. Recombinant E. coli XL-1 Blue and attenuated Salmonella typhimurium secreted a 30 kDa hIL-6-HlyA(S) fusion protein, with an additional form of higher apparent molecular mass produced by S. typhimurium. In S. typhimurium cultures hIL-6-HlyA(S) concentrations entered a plateau at 500 to 600 ng ml(-1) culture supernatant. In contrast to E. coli XL-1 Blue, in S. typhimurium culture supernatants hIL-6-HlyA(S) was accumulated faster reaching three-fold higher maximal concentrations. The cell proliferating activity of hIL-6-HlyA(S) fusion protein(s) was equivalent to that of mature recombinant hIL-6. Furthermore. hIL-6-secreting S. typhimurium were less invasive than the attenuated control strain. Therefore, the bulky hemolysin secretorial peptide at the C-terminus of the fusion protein does not markably affect hIL-6 activity, suggesting that the hemolysin secretion apparatus provides an excellent system to study immunomodulatory effects of in situ synthesized IL-6 in Salmonella vaccine strains.

Multicolor spectral karyotyping of human chromosomes.

The simultaneous and unequivocal discernment of all human chromosomes in different colors would be of significant clinical and biologic importance. Whole-genome scanning by spectral karyotyping allowed instantaneous visualization of defined emission spectra for each human chromosome after fluorescence in situ hybridization. By means of computer separation (classification) of spectra, spectrally overlapping chromosome-specific DNA probes could be resolved, and all human chromosomes were simultaneously identified.

Polymorphism, monomorphism, and sequences in conserved microsatellites in primate species.

Dimeric short tandem repeats are a source of highly polymorphic markers in the mammalian genome. Genetic variation at these hypervariable loci is extensively used for linkage analysis, for the identification of individuals, and may be useful for interpopulation and interspecies studies. In this paper, we analyze the variability and the sequences of a segment including three microsatellites, first described in man, in several species of primates (chimpanzee, orangutan, gibbon, and macaque) using the heterologous primers (man primers). This region is located on the human chromosome 6p, near the tumor necrosis factor genes, in the major histocompatibility complex. The fact that these primers work in all species studied indicates that they are conserved throughout the different lineages of the two superfamilies, the Hominoidea and the Cercopithecidea, represented by the macaques. However, the intervening sequence displays intraspecific and interspecific variability. The sites of base substitutions and the insertion/deletion events are not evenly distributed within this region. The data suggest that it is necessary to have a minimal number of repeats to increase the rate of mutation sufficiently to allow the development of polymorphism. In some species, the microsatellites present single base variations which reduce the number of contiguous repeats, thus apparently slowing the rate of additional slippage events. Species with such variations or a low number of repeats are monomorphic. These microsatellite sequences are informative in the comparison of closely related species and reflect the phylogeny of the Old World monkeys, apes, and man.

Pvu II polymorphism in the primate homologue of the mouse B144 (LST-1). A novel marker gene within the tumor necrosis factor region.

A Pvu II RFLP was mapped within the LST-1 gene, the human homologue of the mouse B144 sequence, establishing LST-1 as a new marker gene within the TNF region. We investigated the distribution of this Pvu II RFLP in 274 unrelated individuals, 132 additional HLA-DR7-positive individuals, 86 homozygous lymphoblastoid cell lines, and in four families. Seventeen of 274 individuals (6.2%) were heterozygous for the Pvu II restriction site (ADB1 = lack and ADB2 = presence of the Pvu II restriction site). In our study population the polymorphism has a much wider distribution than that previously reported in an analysis of selected haplotypes. Besides a strong association of ADB1 with HLA-B14, -DR7, we found a further association with HLA-B35. These results were also validated by family segregation studies and analyses of homozygous cell lines. In addition, five of 17 individuals carrying the ADB1 allele had HLA types other than B14 or B35, emphasizing that the presence of ADB1 is not limited to the HLA-B14, DR7 haplotype. LST-1 and its polymorphism may be used as an additional marker of the TNF region, where genes responsible for autoimmune diseases are suspected to be localized.

Evolution of hominoid mitochondrial DNA with special reference to the silent substitution rate over the genome.

Focusing on the synonymous substitution rate, we carried out detailed sequence analyses of hominoid mitochondrial (mt) DNAs of ca. 5-kb length. Owing to the outnumbered transitions and strong biases in the base compositions, synonymous substitutions in mtDNA reach rapidly a rather low saturation level. The extent of the compositional biases differs from gene to gene. Such changes in base compositions, even if small, can bring about considerable variation in observed synonymous differences and may result in the region-dependent estimate of the synonymous substitution rate. We demonstrate that such a region dependency is due to a failure to take proper account of heterogeneous compositional biases from gene to gene but that the actual synonymous substitution rate is rather uniform. The synonymous substitution rate thus estimated is 2.37 +/- 0.11 x 10(-8) per site per year and comparable to the overall rate for the noncoding region. On the other hand, the rate of nonsynonymous substitutions differs considerably from gene to gene, as expected under the neutral theory of molecular evolution. The lowest rate is 0.8 x 10(-9) per site per year for COI and the highest rate is 4.5 x 10(-9) for ATPase 8, the degree of functional constraints (measured by the ratio of the nonsynonymous to the synonymous substitution rate) being 0.03 and 0.19, respectively. Transfer RNA (tRNA) genes also show variability in the base contents and thus in the nucleotide differences. The average rate for 11 tRNAs contained in the 5-kb region is 3.9 x 10(-9) per site per year. The nucleotide substitutions in the genome suggest that the transition rate is about 17 times faster than the transversion rate.

Gibbon and marmoset c-myc nucleotide sequences.

The nucleotide sequences of the gibbon and marmoset myc loci have been determined by the dideoxy ribomethod. The number of mutations which occurred during evolution and the branches affected were deduced according to the principle of maximum parsimony, from a comparison with known mammal sequences. As previously observed for the human and chimpanzee myc genes, an Alu repeat belonging to subclass III was observed in the second intron of the gibbon myc gene. In contrast, no such element was found in the marmoset gene. Alignment of the Myc amino acid (aa) sequences provided clues for detecting which aa or which protein regions have been more heavily mutated. Conversely, some regions remained free of mutations and remained unchanged from mouse to human, most probably in connection with some important embedded property(ies). An intriguing feature of the human Myc protein is duplication of 50 aa out of 439. Strikingly, most of these aa remain unchanged in mouse, rat, cat, marmoset, gibbon, chimpanzee and human.

Molecular history of gene conversions in the primate fetal gamma-globin genes. Nucleotide sequences from the common gibbon, Hylobates lar.

Comparative and phylogenetic analyses of homologous sequences from closely related species reveal genetic events which have happened in the past and thus provide considerable insight into molecular genetic processes. One such process which has been especially important in the evolution of multigene families is gene conversion. The fetal gamma 1 and gamma 2-globin genes of catarrhine primates (humans, apes, and Old World monkeys) underwent numerous gene conversion events after they arose from a gene duplication event 25-35 million years ago. By including the gamma 1- and gamma 2-globin gene sequences from the common gibbon, Hylobates lar, the present work expands the gamma-globin data set to represent all major groups of hominoid primates. A computer-assisted algorithm is introduced which reveals converted DNA segments and provides results very similar to those obtained by site-by-site evolutionary reconstruction. Both methods provide strong evidence for at least 14 different converted stretches in catarrhine primates as well as five conversions in ancestral lineages. Features of gene conversions generalized from this molecular history are 1) conversions are restricted to regions maintaining high degrees of sequence similarity, 2) one gene may dominate in converting another gene, 3) sequences involved in conversions may accumulate changes more rapidly than expected, and 4) certain elements, such as polypurine/polypyrimidine [Y)n) and (TG)n elements, appear to be hotspots for initiating or terminating conversion events.