DNA in wheat seeds from European archaeological sites

We have used hybridization analysis to detect ancient DNA in wheat seeds collected from three archaeological sites in Europe and the Middle East. One of these samples, carbonizedT. spelta dated to the first millennium BC, has yielded PCR products after amplification with primers directed at the leader regions of the HMW (high molecular weight) glutenin alleles. Sequences obtained from these products suggest that the DNA present in the Danebury seeds is chemically damaged, as expected for ancient DNA, and also indicate that it should be possible to study the genetic variability of archaeological wheat by ancient DNA analysis. Finally, we describe a PCR-based system that enables tetraploid and hexaploid wheats to be distinguished.     

The biochemistry of ancient DNA in bone

The amount of DNA in ancient bone was determined by ethidium bromide staining after the removal of the potent Taq inhibitor, fulvic acid. A complete decalcification and a perfusion protocol were used to recover DNA from bone. A variety of purification techniques including molecular sieve, hydroxyapatite binding and Magic preparations yielded DNA that spanned from 3.4g/g of bone to below detectable limits. Fulvic acid was shown to interfere with the quantification of DNA derived from ancient human skeletal material one hundred to over seven thousand years old. Scanning UV in the 300 to 230 nm range is a simple and sensitive technique for documenting fulvic acid contamination in ancient bone extracts.     

Ancient DNA sequences reveal unsuspected phylogenetic relationships within New Zealand wrens (Acanthisittidae)

Ancient DNA sequences from preserved specimens are increasingly being used for the investigation of Pacific Island ecosystems prior to the large scale modification and extinction of endemic biota associated with human colonization. However, many difficulties are associated with the use of ancient DNA sequences in studies of genetically close taxa. In this paper, these difficulties are discussed as they relate to a study involving extinct and extant members of an ancient New Zealand avian family, the New Zealand wrens (Acanthisittidae).Sequences of the mitochondrial small ribosomal subunit RNA gene (12S) were obtained from museum specimens of several wren taxa in order to investigate their phylogenetic relationships and the taxonomic status of a rock wren (Xenicus gilviventris) subspecies. Limitations due to sample size and 12S sequence variability as well as the difficulties in authenticating ancient DNA sequences prevent firm conclusions but the data suggest unsuspected phylogenetic relationships exist and raise the possibility that conservation management of rock wren populations is required.     

Ancient DNA from Bronze Age bones of European rabbit (Oryctolagus cuniculus)

The European rabbit (Oryctolagus cuniculus) is now widely distributed throughout the world as a result of transportation by man. The original populations, however, were confined to southern France and Spain. In order to investigate the role of human intervention in determining the genetic diversity of rabbit populations, we are studying the origin of rabbits introduced onto a small Mediterranean island (Zembra) near Tunis over 1400 years ago, by examining ancient DNA extracted from rabbit bones found both on Zembra and on the European mainland. Ancient DNA was successfully extracted from rabbit bones found at two archaeological sites dated to at least the Early Bronze Age (more than 3500 years ago) in south-central France, and compared to that found in modern mainland and island populations using a small variable region of the cytochromeb gene. The results confirm that the Zembra Island population is descended from that present over 1400 years ago. The technical aspects of DNA extraction from bones and the implications of this type of research for determining the origin of introduced rabbit populations are discussed.     

Mitochondrial DNA evidence for the 19th century introduction of African honey bees into the United States

Since the introduction of an African subspecies into Brazil in the mid-1950's¹, descendent Africanized honey bees (Apis mellifera L.) have spread throughout the Neotropics and into temperate North America. Restriction enzyme analysis of 422 feral honey bee colonies collected from non-Africanized areas in the southern United States revealed that over 21% of them had mitochondrial DNA (mtDNA) derived from a European race established in North America by the 17th century, 77% of them had mtDNA common in honey bees maintained by beekeepers and about 1% exhibited African mtDNA. Further analysis revealed that the African mtDNA was derived from a north African subspecies imported to the US in the 19th century.     

Ancient DNA: Methodological challenges

The study of ancient DNA offers the possibility of following genetic change over time. However, the field is plagued by a problem which is unique in molecular biology-the difficulty of verifying results by reproduction. Some of the reasons for this are technical and derive from the low copy number and damaged state of ancient DNA molecules. Other reasons are the unique nature of many of the objects from which DNA is extracted. We describe methodological approaches with which these problems can be alleviated in order to ensure that results are scientific in the sense that they can be reproduced by others.     

In vitro reconstitution of homologous recombination reactions

The proteins essential to homologous recombination inE. coli have been purified and their individual activities have been identified, permitting biochemical reconstitution of steps that comprise the cellular recombination process. This review focuses on the biochemical events responsible for the initiation and homologous pairing steps of genetic recombination. The properties of an in vitro recombination reaction that requires the concerted action of recA, recBCD, and SSB proteins and that is stimulated by the recombination hotspot, Chi(), are described. The recBCD enzyme serves as the initiator of this reaction; its DNA helicase activity produces single-stranded DNA that is used by the recA protein to promote homologous pairing and DNA strand invasion of supercoiled (recipient) DNA. The SSB protein acts to trap the single-stranded DNA produced by recBCD enzyme and to facilitate pairing by the recA protein. The regulatory sequence acts incis by attenuating the nuclease, but not the helicase, activity of recBCD enzyme. This attenuation assures the preservation of ssDNA produced by the DNA helicase activity and is responsible for the simulation in vitro and, presumably, in vivo. The attenuation of nuclease activity by results in the loss or functional inactivation of the recD subunit.     

Quantitative genetic models of sexual selection

Summary Quantitative genetic models of sexual selection have disporven some of the central tenets of both the handicap mechanism and the sexy son hypothesis. These results suggest that the good genes approach to sexual selection may generally lead to erroneous results.Runaway sexual selection seems possible under a wide variety of circumstances. Quantittive genetic models have revealed runaway processes for sexually selected attributes expressed in both sexes and for attributes of parental care. Furthermore, the runaway could occur simultaneously in a series of populations that straddle an environmental gradient. While the models support the feasibility of runaway processes, empirical studies are needed to evaluate whether runaways actually happen. Estimates of critical genetic parameters are particularly needed, as well as measures of natural and sexual selection acting on the same population.The models also show that sexual selection has tremendous potential to produce population differentiation, particularly in epigamic traits. Differentiation is promoted by indeterminancy of evolutionary outcome, transient differences among populations during the final slow approach to equilibrium, sampling drift among equilibrium populations, and the tendency of sexual selection to amplify geographic variation arising from spatial differences in natural selection. Recent work with two- and three-locus models of sexual selection has produced results that parallel the results of the polygenic models^36–38,58. Thus the feature of indeterminate equilibria (outcome dependent on initial conditions) is common to both types of model.     

Molecular paleontology

Molecular paleontology, i.e., the recovery of DNA from ancient human, animal, and plant remains is an innovative research field that has received progressively more attention from the scientific community since the 1980s. In the last decade, the field was punctuated by claims which aroused great interest but eventually turned out to be fakes - the most famous being the sequence of dinosaur DNA later shown to be of human origin. At present, the discipline is characterized by some certainties and many doubts. We know, for example, that we have reasonable chances to recover authentic DNA from a mammoth carcass, while our chances are negligible (or nonexistent) in the case of a dynastic mummy from Egypt. On the other hand, though we are developing convincing models of DNA decay in bone, we are not yet able to predict whether a certain paleontological or archeological site will yield material amenable to DNA analysis. This article reviews some of the most important and promising investigations using molecular paleontology approaches, such as studies on the conservation of DNA in human bone, the quest for ancient DNA in permafrost-frozen fauna, the Tyrolean iceman, and the Neandertals. Received 5 April 2001; received after revision 5 July 2001; accepted 5 July 2001     

Mitochondrial DNA mutators

In this article we review our current knowledge of the mechanisms by which point mutations arise in the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae and discuss to what extent these mechanisms operate in human mtDNA mutagenesis. The 3–5 exonuclease proofreading activity of Pol ensures accuracy of mtDNA replication in both yeast and humans, while the role of base excision repair in mtDNA error avoidance remains debated. The mitochondrial mismatch repair Msh1 protein, which removes transitions in yeast, is absent in humans, a particularity that might cause accumulation of transitions, while the most frequent substitution in yeast mtDNA is A:T to T:A transversion. Proofreading-deficient mutator human cell lines and knockin mice have been created. They will be useful for studying the mechanisms by which mtDNA mutations accumulate in diseases, ageing, malignancy and drug therapy.Received 25 May 2004; received after revision 21 June 2004; accepted 7 July 2004     

Molecular ecology of a Neolithic meadow: The DNA of the grass remains from the archaeological site of the Tyrolean Iceman

The paper reports on the molecular analysis of samples of approximately 5,300-year-old grass found at the alpine archaeological site where the so-called Tyrolean Iceman was discovered. The grass comes from a cloak made of long grass blades and/or the stuffing of the snow footwear worn by the Iceman. The results show that while the largest fraction of the DNA extractable from the grass is of foreign origin, a much smaller part belongs to the original genetic material of the grass itself, and can be used as a valuable taxonomic clue to the plant species utilized by neolithic men to manufacture their equipment. On the other hand, the foreign DNA, or at least a portion of it, comes from microorganisms-mainly filamentous fungi and unicellular algae-which seem to have been associated with the grass since the time the grass was harvested.     

The use of museum specimens to reconstruct the genetic variability and relationships of extinct populations

In this review, we discuss the use of DNA from museum specimens to address conservation genetic questions. We provide four examples from our previous studies of the northern hairy-nosed wombat, African wild dog, Ethiopian wolf and red wolf. These species were genetically surveyed using two molecular approaches: first, analysis of short sequences in the mitochondrial genome using species-specific primers, and second, analysis of hypervariable microsatellite loci. The studies demonstrate that museum-derived DNA adds an important dimension to the genetic study of extant populations. Inaccessible populations can be studied, and both the loss of genetic variation and its distribution over space and time can be better understood. Finally, analysis of additional museum material provides definitive evidence for a hybrid origin of the red wolf.     

PCR jumping in clones of 30-million-year-old DNA fragments from amber preserved termites (Mastotermes electrodominicus)

DNA from 30-million-year-old amber preserved termites (Mastotermes electrodominicus) was PCR amplified with nuclear ribosomal RNA small subunit primers and cloned into the TA vector (INVITROGEN). We obtained several classes of recombinant clones as a result. AuthenticMastotermes electrodominicus clones were identified. The source of other classes of clones was identified as contaminants of the ancient DNA template. Several of the clones appeared to be chimeric in structure with half of the clone identical to the termite sequence and the other half identical to contaminant sequences. The phenomenon of PCR jumping was identified as a possible source for the chimeric clones.     

Some trends in medical populations genetics

Summary Five topics concerning medical population genetics have been selected for discussion: in the field of population cytogenetics, the frequency of chromosomal aberrations and the roles of mutation and selection in the maintenance of balanced rearrangements are studied; the long term genetic effects of treatment and prevention of genetic diseases are reviewed; the relationships between malaria and the sickle-cell trait are discussed; some recent works concerning human DNA polymorphisms in the field of population, genetics are presented, and finally, some methods of genetic epidemiology are described.     

Biochemical aspects of radiation biology

Summary In order to analyze the mechanisms of biological radiation effects, the events after radiation energy absorption in irradiated organisms have to be studied by physico-chemical and biochemical methods. The radiation effects in vitro on biomolecules, especially DNA, are described, as well as their alterations in irradiated cells. Whereas in vitro, in aqueous solution, predominantly OH radicals are effective and lead to damage in single moieties of the DNA, in vivo the direct absorption of radiation energy leads to locally multiply-damaged sites, which produce DNA double-strand breaks and locally denatured regions. DNA damage will be repaired in irradiated cells. Error free repair leads to the original nucleotide sequence in the genome by excision or by recombination. Error prone repair (mutagenic repair), leads to mutation. However, the biochemistry of these processes, regulated by a number of genes, is poorly understood. In addition, more complex reactions, such as gene amplification and transposition of mobile gene elements, are responsible for mutation or malignant transformation.     

Ancestry of modern Europeans: contributions of ancient DNA

Understanding the peopling history of Europe is crucial to comprehend the origins of modern populations. Of course, the analysis of current genetic data offers several explanations about human migration patterns which occurred on this continent, but it fails to explain precisely the impact of each demographic event. In this context, direct access to the DNA of ancient specimens allows the overcoming of recent demographic phenomena, which probably highly modified the constitution of the current European gene pool. In recent years, several DNA studies have been successfully conducted from ancient human remains thanks to the improvement of molecular techniques. They have brought new fundamental information on the peopling of Europe and allowed us to refine our understanding of European prehistory. In this review, we will detail all the ancient DNA studies performed to date on ancient European DNA from the Middle Paleolithic to the beginning of the protohistoric period.     

The first known use of vermillion

Vermillion has been shown to be useful in preserving human bones from 5000 years ago. Remarkably well-preserved human bones have been found in the dolmenic burial La Velilla in Osorno (Palencia, Spain), carefully covered by pulverized cinnabar (vermillion) which ensured their preservation even in non-favorable climatic conditions. We believe the red powder was deliberately deposited for preservative use because no cinnabar mine is to be found within 160 km, because of the large amount (hundreds of kilograms) used, and because its composition, red mercuric sulphide, is similar to that of preparations used in technical embalming. This finding pushes back the data of the use of mercury ore for preservation by four millennia in South America, and by at least one millennium in the Old World. Chemical and thermal analyses of vermillion in La Velilla have demonstrated its great purity and shown that the cinnabar was pulverized and washed (but not heated), producing a bright red-orange tone.     

The role of repair in radiobiology

Summary Apart from cancer and mutation induction, radiobiological effects on mammals are mostly attributable to cell death, defined as loss of proliferative capacity. Survival curves relate retention of that capacity to radiation dose, and often manifest a quasi-threshold (shoulder). The shoulder is attributable to an initial mechanism of repair (Q-repair) which is gradually depleted as dose increases. Another form of repair, which is not depleted (P-repair), increases the dose required to deliver an average of one lethal event per cell (dose D₀). Neither form of repair can unambiguously be linked with repair of defects in isolated DNA. An important initial lesion may well be disruption of the complex structural relationship between the DNA, nuclear membrane and associated proteins. One form of P-repair may be restoration of that structural relationship.     

Genetic variation in the New World: Ancient teeth,bone, and tissue as sources of DNA

Examination of ancient and contemporary Native American mtDNA variation via diagnostic restriction sites and the 9-pb Region V deletion suggests a single wave of migration into the New World. This is in contrast to data from Torroni et al.³⁴ which suggested two waves of migration into the New World (the NaDene and Amerind). All four founding lineage types are present in populations in North, Central, and South America suggesting that all four lineages came over together and spead throughout the New World. Ancient Native American DNA shows that all four lineages were present before European contact in North America, and at least two were present in South America. The presence of all four lineages in the NaDene and the Amerinds argues against separate migrations founding these two groups, although admixture between the groups is still a viable explanation for the presence of all four types in the NaDene.     

Alternariol,a dibenzopyrone mycotoxin ofAlternaria spp., is a new photosensitizing and DNA cross-linking agent

Summary The mycotoxin alternariol (3,4,5-trihydroxy-6-methyldibenzo [a] pyrone) but not alternariol monomethyl ether (3,4-dihydroxy-5-methoxy-6-methyldibenzo [a] pyrone) is phototoxic toEscherichia coli in the presence of near UV light (320–400 nm). The phototoxicity bioassays with a DNA repair-deficient mutant ofE. coli suggested that DNA may be the molecular target for photo-induced toxicity of alternariol. Interactions between alternariol and double-stranded, supercoiled DNA suggest that alternariol interacts with DNA by intercalation. No DNA breakage was detected in this system; however, alternariol forms a complex and cross-links double-stranded DNA in near UV light. These results suggest that alternariol is a new phototoxic, DNA-intercalating agent and is a DNA cross-linking mycotoxin in near UV light.Acknowledgment. Dr Albert Stoessl (Agriculture Canada, London, Ontario, Canada) generously provided a mixture of alternariol and alternariol monomethyl ether, and made many helpful suggestions. Dr Ashwood-Smith (University of Victoria, Victoria, British Columbia, Canada) kindly supplied the microorganisms through Dr G.H.N. Towers (University of British Columbia, Vancouver). We gratefully acknowledge the gifts. It is a pleasure to acknowledge the able assistance of Mr S. Tallevi.