Ancient DNA analysis reveals divergence of the cave bear, Ursus spelaeus, and brown bear, Ursus arctos, lineages.

The cave bear, Ursus spelaeus, represents one of the most frequently found paleontological remains from the Pleistocene in Europe. The species has always been confined to Europe and was contemporary with the brown bear, Ursus arctos. Relationships between the cave bear and the two lineages of brown bears defined in Europe, as well as the origins of the two species, remain controversial, mainly due to the wide morphological diversity of the fossil remains, which makes interpretation difficult [1, 2]. Sequence analysis of ancient DNA is a useful tool for resolving such problems because it provides an independent source of data [3]. We previously amplified a short DNA fragment of the mitochondrial DNA control region (mt control region) of a 40,000-year-old Ursus spelaeus sample [4]. In this paper, we describe the DNA analysis of two mtDNA regions, the control region and the cytochrome b gene. Control region sequences were obtained from ten samples of cave bears ranging from 130,000 to 20,000 years BP, and one particularly well-conserved sample gave a complete cyt b sequence. Our data demonstrate that cave bears split largely before the lineages of brown bears around 1.2 million years ago. Given its abundance, its wide distribution in space and time, and its large morphological diversity, the cave bear is a promising model for direct observation of the evolution of sequences throughout time, extinction periods, and the differentiation of populations shaped by climatic fluctuations during the Pleistocene.

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.

Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites.

On the Middle-Age site of Namur (Belgium) the analysis of coprolites revealed the presence of many well-preserved Ascaris eggs. Following rehydratation of the coprolite samples, 104 eggs were collected and extracted with an ultrasonication and phenol-chloroform based method. Three overlapping fragments of the 18S rRNA gene and one fragment of the cytochrome b gene have been reproducibly amplified, cloned and sequenced. The analysis of these sequences confirms the identification of the eggs as coming from Ascaris. Our study reveals that coprolites can be an interesting source of parasites that can be readily identified using molecular approaches. The study of ancient DNA from helminth parasites is of interest as it may answer long-standing questions in the history of infectious diseases and gives a possibility to compare these ancient sequences with those of modern populations.

Trinucleotide repeat elongation in the huntingtin gene in Huntington's disease patients from 85 French families. The French HD Research Group.

IT15 is a new gene encoding a protein named huntingtin; a polymorphic CAG repeat in the proposed open reading frame of IT15 has been characterized, and an elongation of this repeat has been correlated to Huntington's disease (HD). We have investigated the CAG repeat in the huntingtin gene in 85 unrelated French families with Huntington's disease. In 79 patients (from 60 families, where at least one HD DNA was available) we found repeat lengths of 37 to 100 units, in contrast to 11 to 35 CAG's on normal chromosomes. Comparison of repeat length and age at onset of disease symptoms in 71 individuals confirms an inverse correlation (r = -0.51 for p < 10(-4)) between the age at onset and the number of CAG repeat units.

Diagnosing post-mortem treatments which inhibit DNA amplification from US MIAs buried at the Punchbowl.

The US military is committed to recovering and identifying the remains of unknown military service members. Casualties of the Korean War were exhumed from the National Memorial Cemetery of the Pacific, or Punchbowl, and submitted to the Armed Forces DNA Identification Laboratory (AFDIL) for mtDNA sequencing. Contrary to AFDIL's experience on other samples from this era, most failed to yield amplifiable DNA. Suspicion fell on mortuary practices that may have been applied to the remains, evidenced by a white powder found with the bones, and general records suggesting the use of formaldehyde-based stablizing agents. To improve the chances of successful identification of the unknown individuals, we looked for the causes underlying this failure. We did this by examining the state of the collagen, the most abundant biomolecule in bone, by using differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). The DSC analyses showed collagens with a range of different thermal stabilities. When these results were compared with the DNA amplification results, a clear correlation between elevated thermal stability and amplification failure was evident. TEM analysis revealed that fibril integrity was maintained after thermal and acid treatments in the samples which failed amplification. Together these two approaches implicate a stabilization agent as the cause of problems with DNA analysis, presumably due to excessive cross-linking. Following the initial study, the ability of DSC to rapidly identify problem samples was tested in a blind study of 14 samples, the method successfully identifying all the problematic samples from Punchbowl. Within this unusual context, DSC analysis is a useful method to assess the likelihood of successful DNA extraction and amplification.

[History of the rabbit and ancient DNA]. / Histoire des lapins et ADN ancien.

Present populations of Rabbits (Oryctolagus cuniculus) are organized into two well defined groups A and B according to their mitochondrial DNA sequences. Group A is restricted to the South Western part of the Iberian Peninsula while group B is found everywhere else. Domestic breeds belong to the latter. As evidenced from data on ancient bones (up to 12,000 years BP) the mitochondrial type B1, predominant in domestic animals, originated from Spain. B1 animals were introduced in France by man between late Roman times and Middle Ages.