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.

A genetic record of population isolation in pocket gophers during Holocene climatic change

A long-standing question in Quaternary paleontology is whether climate-induced, population-level phenotypic change is a result of large-scale migration or evolution in isolation. To directly measure genetic variation through time, ancient DNA and morphologic variation was measured over 2,400 years in a Holocene sequence of pocket gophers (Thomomys talpoides) from Lamar Cave, Yellowstone National Park, Wyoming. Ancient specimens and modern samples collected near Lamar Cave share mitochondrial cytochrome b sequences that are absent from adjacent localities, suggesting that the population was isolated for the entire period. In contrast, diastemal length, a morphologic character correlated with body size and nutritional level, changed predictably in response to climatic change. Our results demonstrate that small mammal populations can experience the long-term isolation assumed by many theoretical models of microevolutionary change.

A molecular analysis of dietary diversity for three archaic Native Americans

DNA was extracted from three fecal samples, more than 2,000 years old, from Hinds Cave, Texas. Amplification of human mtDNA sequences showed their affiliation with contemporary Native Americans, while sequences from pronghorn antelope, bighorn sheep, and cottontail rabbit allowed these animals to be identified as part of the diet of these individuals. Furthermore, amplification of chloroplast DNA sequences identified eight different plants as dietary elements. These archaic humans consumed 2–4 different animal species and 4–8 different plant species during a short time period. The success rate for retrieval of DNA from paleofeces is in strong contrast to that from skeletal remains where the success rate is generally low. Thus, human paleofecal remains represent a source of ancient DNA that significantly complements and may in some cases be superior to that from skeletal tissue.

Thermostable archaeal O6-alkylguanine-DNA alkyltransferases

Archaea represent some of the most ancient organisms on earth, and they have relatively uncharacterized DNA repair processes. We now show, using an in vitro assay, that extracts of two Crenarchaeota (Sulfolobus acidocaldarius and Pyrobaculum islandicum) and two Euryarchaeota (Pyrococcus furiosus and Thermococcus litoralis) contain the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase). The ATase activities found in the archaea were extremely thermostable, with half-lives at 80°C ranging from 0.5 hr (S. acidocaldarius) to 13 hr (T. litoralis). The temperature optima of the four proteins ranged from ≈75 to ≈100°C, although activity was seen at 37°C, the temperature optimum of the Escherichia coli and human ATases. In all cases, preincubaton of extracts with a short oligonucleotide containing a single O6-methylguanine residue caused essentially complete loss of ATase activity, suggesting that the alkylphosphotriester-DNA alkyltransferase activity seen in some prokaryotes is not present in Archaea. The ATase from Pyrobaculum islandicum had an apparent molecular mass of 15 kDa, making it the smallest of these proteins so far described. In higher organisms, ATase is responsible for the repair of toxic and mutagenic O6-alkylguanine lesions in alkylated DNA. The presence of ATase in these primitive organisms therefore suggests that endogenous or exogenous exposure to agents that generate appropriate substrates in DNA may be an early event in evolution.

Typing of urinary JC virus DNA offers a novel means of tracing human migrations

Although polyomavirus JC (JCV) is the proven pathogen of progressive multifocal leukoencephalopathy, the fatal demyelinating disease, this virus is ubiquitous as a usually harmless symbiote among human beings. JCV propagates in the adult kidney and excretes its progeny in urine, from which JCV DNA can readily be recovered. The main mode of transmission of JCV is from parents to children through long cohabitation. In this study, we collected a substantial number of urine samples from native inhabitants of 34 countries in Europe, Africa, and Asia. A 610-bp segment of JCV DNA was amplified from each urine sample, and its DNA sequence was determined. A worldwide phylogenetic tree subsequently constructed revealed the presence of nine subtypes including minor ones. Five subtypes (EU, Af2, B1, SC, and CY) occupied rather large territories that overlapped with each other at their boundaries. The entire Europe, northern Africa, and western Asia were the domain of EU, whereas the domain of Af2 included nearly all of Africa and southwestern Asia all the way to the northeastern edge of India. Partially overlapping domains in Asia were occupied by subtypes B1, SC, and CY. Of particular interest was the recovery of JCV subtypes in a pocket or pockets that were separated by great geographic distances from the main domains of those subtypes. Certain of these pockets can readily be explained by recent migrations of human populations carrying these subtypes. Overall, it appears that JCV genotyping promises to reveal previously unknown human migration routes: ancient as well as recent.

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.

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.

Activity of individual ERCC1 and XPF subunits in DNA nucleotide excision repair

ERCC1–XPF is a structure-specific nuclease with two subunits, ERCC1 and XPF. The enzyme cuts DNA at junctions where a single strand moves 5′ to 3′ away from a branch point with duplex DNA. This activity has a central role in nucleotide excision repair (NER), DNA cross-link repair and recombination. To dissect the activities of the nuclease it is necessary to investigate the subunits individually, as studies of the enzyme so far have only used the heterodimeric complex. We produced recombinant ERCC1 and XPF separately in Escherichia coli as soluble proteins. Activity was monitored by a sensitive dual incision assay for NER by complementation of cell extracts. XPF and ERCC1 are unstable in mammalian cells in the absence of their partners but we found, surprisingly, that ERCC1 alone could confer some repair to extracts from ERCC1-defective cells. A version of ERCC1 lacking the first 88 non-conserved amino acids was also functional. This indicated that a small amount of active XPF was present in ERCC1 extracts, and immunoassays showed this to be the case. Some repair in XPF-defective extracts could be achieved by adding ERCC1 and XPF proteins together, but not by adding only XPF. The results show for the first time that functional ERCC1–XPF can be formed from separately produced subunits. Protein sequence comparison revealed similarity between the ERCC1 family and the C-terminal region of the XPF family, including the regions of both proteins that are necessary for the ERCC1–XPF heterodimeric interaction. This suggests that the ERCC1 and XPF families are related via an ancient duplication.

DNA methyltransferases of the cyanobacterium Anabaena PCC 7120

From the characterization of enzyme activities and the analysis of genomic sequences, the complement of DNA methyltransferases (MTases) possessed by the cyanobacterium Anabaena PCC 7120 has been deduced. Anabaena has nine DNA MTases. Four are associated with Type II restriction enzymes (AvaI, AvaII, AvaIII and the newly recognized inactive AvaIV), and five are not. Of the latter, four may be classified as solitary MTases, those whose function lies outside of a restriction/modification system. The group is defined here based on biochemical and genetic characteristics. The four solitary MTases, DmtA/M.AvaVI, DmtB/M.AvaVII, DmtC/M.AvaVIII and DmtD/M.AvaIX, methylate at GATC, GGCC, CGATCG and rCCGGy, respectively. DmtB methylates cytosines at the N4 position, but its sequence is more similar to N6-adenine MTases than to cytosine-specific enzymes, indicating that it may have evolved from the former. The solitary MTases, appear to be of ancient origin within cyanobacteria, while the restriction MTases appear to have arrived by recent horizontal transfer as did five now inactive Type I restriction systems. One Mtase, M.AvaV, cannot reliably be classified as either a solitary or restriction MTase. It is structurally unusual and along with a few proteins of prokaryotic and eukaryotic origin defines a structural class of MTases distinct from all previously described.

Testing ancient RNA–protein interactions

The past decade in molecular biology has seen remarkable advances in the study of the origin and early evolution of life. The mathematical tools for analyzing DNA and protein sequences, coupled with the availability of complete microbial genome sequences, provide insight almost as far back as the age of the nucleic acids themselves. Experimental evolution in the laboratory and especially in vitro evolution of RNA provide insight into a hypothetical world where RNA, or a close relative, may have debuted as a primary functional and informational molecule. The ability to isolate new functional RNAs from random sequences now ultimately makes the world of possible primitive chemical interactions accessible even when the molecules or reactions are no longer present in modern species. Thus we can at last form direct experimental tests of specific models for the origin of RNA–protein associations, such as those that influenced the genetic code. This marks a turning point for probing the origin and early history of life at the molecular level.

Ancient single origin for Malagasy primates.

We report new evidence that bears decisively on a long-standing controversy in primate systematics. DNA sequence data for the complete cytochrome b gene, combined with an expanded morphological data set, confirm the results of a previous study and again indicate that all extant Malagasy lemurs originated from a single common ancestor. These results, as well as those from other genetic studies, call for a revision of primate classifications in which the dwarf and mouse lemurs are placed within the Afro-Asian lorisiforms. The phylogenetic results, in agreement with paleocontinental data, indicate an African origin for the common ancestor of lemurs and lorises (the Strepsirrhini). The molecular data further suggest the surprising conclusion that lemurs began evolving independently by the early Eocene at the latest. This indicates that the Malagasy primate lineage is more ancient than generally thought and places the split between the two strepsirrhine lineages well before the appearance of known Eocene fossil primates. We conclude that primate origins were marked by rapid speciation and diversification sometime before the late Paleocene.

Tiggers and DNA transposon fossils in the human genome.

We report several classes of human interspersed repeats that resemble fossils of DNA transposons, elements that move by excision and reintegration in the genome, whereas previously characterized mammalian repeats all appear to have accumulated by retrotransposition, which involves an RNA intermediate. The human genome contains at least 14 families and > 100,000 degenerate copies of short (180-1200 bp) elements that have 14- to 25-bp terminal inverted repeats and are flanked by either 8 bp or TA target site duplications. We describe two ancient 2.5-kb elements with coding capacity, Tigger1 and -2, that closely resemble pogo, a DNA transposon in Drosophila, and probably were responsible for the distribution of some of the short elements. The deduced pogo and Tigger proteins are related to products of five DNA transposons found in fungi and nematodes, and more distantly, to the Tc1 and mariner transposases. They also are very similar to the major mammalian centromere protein CENP-B, suggesting that this may have a transposase origin. We further identified relatively low-copy-number mariner elements in both human and sheep DNA. These belong to two subfamilies previously identified in insect genomes, suggesting lateral transfer between diverse species.