Detection of 400-year-old Yersinia pestis DNA in human dental pulp: An approach to the diagnosis of ancient septicemia

Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague (“plague teeth”) and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human β-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase β-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.

Histone underacetylation is an ancient component of mammalian X chromosome inactivation

Underacetylation of histone H4 is thought to be involved in the molecular mechanism of mammalian X chromosome inactivation, which is an important model system for large-scale genetic control in eukaryotes. However, it has not been established whether histone underacetylation plays a critical role in the multistep inactivation pathway. Here we demonstrate differential histone H4 acetylation between the X chromosomes of a female marsupial, Macropus eugenii. Histone underacetylation is the only molecular aspect of X inactivation known to be shared by marsupial and eutherian mammals. Its strong evolutionary conservation implies that, unlike DNA methylation, histone underacetylation was a feature of dosage compensation in a common mammalian ancestor, and is therefore likely to play a central role in X chromosome inactivation in all mammals.

An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein

The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of ≈50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome ≈30 million years ago.

Carbonic anhydrase is an ancient enzyme widespread in prokaryotes

Carbonic anhydrases catalyze the reversible hydration of CO2 and are ubiquitous in highly evolved eukaryotes. The recent identification of a third class of carbonic anhydrase (γ class) in a methanoarchaeon and our present finding that the β class also extends into thermophilic species from the Archaea domain led us to initiate a systematic search for these enzymes in metabolically and phylogenetically diverse prokaryotes. Here we show that carbonic anhydrase is widespread in the Archaea and Bacteria domains, and is an ancient enzyme. The occurrence in chemolithoautotrophic species occupying deep branches of the universal phylogenetic tree suggests a role for this enzyme in the proposed autotrophic origin of life. The presence of the β and γ classes in metabolically diverse species spanning the Archaea and Bacteria domains demonstrates that carbonic anhydrases have a far more extensive and fundamental role in prokaryotic biology than previously recognized.

Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations

Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer’s disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent ρ° (mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the ρ° cells, demonstrating that these sequences are also present in the human nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then tested for each of the five “AD” missense mutations by restriction endonuclease site variant assays. All five mutations were found in the nuclear CO1 and CO2 PCR products from ρ° cells, but none were found in the PCR products obtained from cells with normal mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products were cloned and sequenced, all five missense mutations were found, as well as a linked synonymous mutation. Unlike the findings in the original report, an additional 32 base substitutions were found, including two in adjacent tRNAs and a two base pair deletion in the CO2 gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences revealed that they diverged from modern human mtDNAs early in hominid evolution about 770,000 years before present. These data would be consistent with the interpretation that the missense mutations proposed to cause AD may be the product of ancient mtDNA variants preserved as nuclear pseudogenes.

Intron positions correlate with module boundaries in ancient proteins

We analyze the three-dimensional structure of proteins by a computer program that finds regions of sequence that contain module boundaries, defining a module as a segment of polypeptide chain bounded in space by a specific given distance. The program defines a set of “linker regions” that have the property that if an intron were to be placed into each linker region, the protein would be dissected into a set of modules all less than the specified diameter. We test a set of 32 proteins, all of ancient origin, and a corresponding set of 570 intron positions, to ask if there is a statistically significant excess of intron positions within the linker regions. For 28-Å modules, a standard size used historically, we find such an excess, with P < 0.003. This correlation is neither due to a compositional or sequence bias in the linker regions nor to a surface bias in intron positions. Furthermore, a subset of 20 introns, which can be putatively identified as old, lies even more explicitly within the linker regions, with P < 0.0003. Thus, there is a strong correlation between intron positions and three-dimensional structural elements of ancient proteins as expected by the introns-early approach. We then study a range of module diameters and show that, as the diameter varies, significant peaks of correlation appear for module diameters centered at 21.7, 27.6, and 32.9 Å. These preferred module diameters roughly correspond to predicted exon sizes of 15, 22, and 30 residues. Thus, there are significant correlations between introns, modules, and a quantized pattern of the lengths of polypeptide chains, which is the prediction of the “Exon Theory of Genes.”

Fish and mammals in the economy of an ancient Peruvian kingdom

Fish and mammal bones from the coastal site of Cerro Azul, Peru shed light on economic specialization just before the Inca conquest of A.D. 1470. The site devoted itself to procuring anchovies and sardines in quantity for shipment to agricultural communities. These small fish were dried, stored, and eventually transported inland via caravans of pack llamas. Cerro Azul itself did not raise llamas but obtained charqui (or dried meat) as well as occasional whole adult animals from the caravans. Guinea pigs were locally raised. Some 20 species of larger fish were caught by using nets; the more prestigious varieties of these show up mainly in residential compounds occupied by elite families.

Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes

Isopentenyl diphosphate (IPP) is the central intermediate in the biosynthesis of isoprenoids, the most ancient and diverse class of natural products. Two distinct routes of IPP biosynthesis occur in nature: the mevalonate pathway and the recently discovered deoxyxylulose 5-phosphate (DXP) pathway. The evolutionary history of the enzymes involved in both routes and the phylogenetic distribution of their genes across genomes suggest that the mevalonate pathway is germane to archaebacteria, that the DXP pathway is germane to eubacteria, and that eukaryotes have inherited their genes for IPP biosynthesis from prokaryotes. The occurrence of genes specific to the DXP pathway is restricted to plastid-bearing eukaryotes, indicating that these genes were acquired from the cyanobacterial ancestor of plastids. However, the individual phylogenies of these genes, with only one exception, do not provide evidence for a specific affinity between the plant genes and their cyanobacterial homologues. The results suggest that lateral gene transfer between eubacteria subsequent to the origin of plastids has played a major role in the evolution of this pathway.

Anthropoid humeri from the late Eocene of Egypt

A number of recent studies have, by necessity, placed a great deal of emphasis on the dental evidence for Paleogene anthropoid interrelationships, but cladistic analyses of these data have led to the erection of phylogenetic hypotheses that appear to be at odds with biogeographic and stratigraphic considerations. Additional morphological data from the cranium and postcranium of certain poorly understood Paleogene primates are clearly needed to help test whether such hypotheses are tenable. Here we describe humeri attributable to Proteopithecus sylviae and Catopithecus browni, two anthropoids from late Eocene sediments of the Fayum Depression in Egypt. Qualitative and morphometric analyses of these elements indicate that humeri of the oligopithecine Catopithecus are more similar to early Oligocene propliopithecines than they are to any other Paleogene anthropoid taxon, and that Proteopithecus exhibits humeral similarities to parapithecids that may be symplesiomorphies of extant (or “crown”) Anthropoidea. The humeral morphology of Catopithecus is consistent with certain narrowly distributed dental apomorphies—such as the loss of the upper and lower second premolar and the development of a honing blade for the upper canine on the lower third premolar—which suggest that oligopithecines constitute the sister group of a clade containing propliopithecines and Miocene-Recent catarrhines and are not most closely related to Proteopithecus as has recently been proposed.

Dentition of Proteopithecus sylviae, an archaic anthropoid from the Fayum, Egypt

Proteopithecus sylviae is an archaic anthropoid from the late Eocene quarry L-41, Fayum Province, Egypt. The dentition of Proteopithecus is very primitive and does not closely resemble that of other, better known, primates from the Fayum (e.g., parapithecids and propliopithecids). The dental morphology, much of which is described herein, shows a platyrrhine-like level of organization, suggesting that P. sylviae may occupy a position near the base of the modern anthropoid radiation.

Ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene Pto

We have investigated the origin of the Pto disease resistance (R) gene that was previously identified in the wild tomato species Lycopersicon pimpinellifolium and isolated by map-based cloning. Pto encodes a serine-threonine protein kinase that specifically recognizes strains of Pseudomonas syringae pv. tomato (Pst) that express the avirulence gene avrPto. We examined an accession of the distantly related wild species Lycopersicon hirsutum var. glabratum that exhibits avrPto-specific resistance to Pst. The Pst resistance of L. hirsutum was introgressed into a susceptible Lycopersicon esculentum background to create the near-isogenic line 96T133-3. Resistance to Pst(avrPto) in 96T133-3 was inherited as a single dominant locus and cosegregated with a restriction fragment length polymorphism detected by the Pto gene. This observation suggested that a member of the Pto gene family confers Pst(avrPto) resistance in this L. hirsutum line. Here we report the cloning and characterization of four members of the Pto family from 96T133-3. One gene (LhirPto) is 97% identical to Pto and encodes a catalytically active protein kinase that elicits a hypersensitive response when coexpressed with avrPto in leaves of Nicotiana benthamiana. In common with the Pto kinase, the LhirPto protein physically interacts with AvrPto and downstream members of the Pto signaling pathway. Our studies indicate that R genes of the protein kinase class may not evolve rapidly in response to pathogen pressure and rather that their ability to recognize specific Avr proteins can be highly conserved.

Ancient Maya documents concerning the movements of Mars

A large part of the pre-Columbian Maya book known as the Dresden Codex is concerned with an exploration of commensurate relationships among celestial cycles and their relationship to other, nonastronomical cycles of cultural interest. As has long been known, pages 43b–45b of the Codex are concerned with the synodic cycle of Mars. New work reported here with another part of the Codex, a complex table on pages 69–74, reveals a concern on the part of the ancient Maya astronomers with the sidereal motion of Mars as well as with its synodic cycle. Two kinds of empiric sidereal intervals of Mars were used, a long one (702 days) that included a retrograde loop and a short one that did not. The use of these intervals, which is indicated by the documents in the Dresden Codex, permitted the tracking of Mars across the zodiac and the relating of its movements to the terrestrial seasons and to the 260-day sacred calendar. While Kepler solved the sidereal problem of Mars by proposing an elliptical heliocentric orbit, anonymous but equally ingenious Maya astronomers discovered a pair of time cycles that not only accurately described the planet's motion, but also related it to other cosmic and terrestrial concerns.

The Sm domain is an ancient RNA-binding motif with oligo(U) specificity

Sm and Sm-like proteins are members of a family of small proteins that is widespread throughout eukaryotic kingdoms. These proteins form heteromers with one another and bind, as heteromeric complexes, to various RNAs, recognizing primarily short U-rich stretches. Interestingly, completion of several genome projects revealed that archaea also contain genes that may encode Sm-like proteins. Herein, we studied the properties of one Sm-like protein derived from the archaebacterium Archaeoglobus fulgidus and overexpressed in Escherichia coli. This single small protein closely reflects the properties of an Sm or Sm-like protein heteromer. It binds to RNA with a high specificity for oligo(U), and assembles onto the RNA to form a complex that exhibits, as judged by electron microscopy, a ring-like structure similar to the ones observed with the Sm core ribonucleoprotein and the like Sm (LSm) protein heteromer. Importantly, multivariate statistical analysis of negative-stain electron-microscopic images revealed a sevenfold symmetry for the observed ring structure, indicating that the proteins form a homoheptamer. These results support the structural model of the Sm proteins derived from crystallographic studies on Sm heterodimers and demonstrate that the Sm protein family evolved from a single ancestor that was present before the eukaryotic and archaeal kingdoms separated.

Global biodiversity and the ancient carbon cycle

Paleontological data for the diversity of marine animals and land plants are shown to correlate significantly with a concurrent measure of stable carbon isotope fractionation for approximately the last 400 million years. The correlations can be deduced from the assumption that increasing plant diversity led to increasing chemical weathering of rocks and therefore an increasing flux of carbon from the atmosphere to rocks, and nutrients from the continents to the oceans. The CO2 concentration dependence of photosynthetic carbon isotope fractionation then indicates that the diversification of land plants led to decreasing CO2 levels, while the diversification of marine animals derived from increasing nutrient availability. Under the explicit assumption that global biodiversity grows with global biomass, the conservation of carbon shows that the long-term fluctuations of CO2 levels were dominated by complementary changes in the biological and fluid reservoirs of carbon, while the much larger geological reservoir remained relatively constant in size. As a consequence, the paleontological record of biodiversity provides an indirect estimate of the fluctuations of ancient CO2 levels.