From barcodes to genomes: extending the concept of DNA barcoding

DNA barcoding has had a major impact on biodiversity science. The elegant simplicity of establishing massive scale databases for a few barcode loci is continuing to change our understanding of species diversity patterns, and continues to enhance human abilities to distinguish among species. Capitalizing on the developments of next generation sequencing technologies and decreasing costs of genome sequencing, there is now the opportunity for the DNA barcoding concept to be extended to new kinds of genomic data. We illustrate the benefits and capacity to do this, and also note the constraints and barriers to overcome before it is truly scalable. We advocate a twin track approach: (i) continuation and acceleration of global efforts to build the DNA barcode reference library of life on earth using standard DNA barcodes and (ii) active development and application of extended DNA barcodes using genome skimming to augment the standard barcoding approach.     

DNA Gyrase: Structure and Function

Abstract

DNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. The mechanism by which gyrase is able to influence the topological state of DNA molecules is of inherent interest from an enzymological standpoint. In addition, much attention has been focused on DNA gyrase as the intracellular target of a number of antibacterial agents and as a paradigm for other DNA topoisomerases. In this review we summarize the current knowledge concerning DNA gyrase by addressing a wide range of aspects of the study of this enzyme.     

Forensic DNA analysis for animal protection and biodiversity conservation: A review

The use of DNA analysis in forensic investigations into animal persecution and biodiversity conservation is now commonplace and crimes such as illegal collection/smuggling, poaching, and illegal trade of protected species are increasingly being investigated using DNA based evidence in many countries. Using DNA analysis, it is possible to identify the species and geographical origin (i.e. population) of a forensic sample, and to also individualise the sample with high levels of probability. Despite extensive literature in animal species, there is unfortunately a serious lack of information on plant species, with only a handful of recent studies. In this review, I detail the applications and diverse forensic investigations that have been carried out to date whilst also highlighting recent developmental studies which offer forensic potential for many species in the future.     

Mechanisms of replication fork protection: a safeguard for genome stability

During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.     

Single cell gel electrophoresis assay: methodology and applications

The single cell gel electrophoresis or Comet assay is a sensitive, reliable, and rapid method for DNA double- and single-strand breaks, alkali-labile sites and delayed repair site detection, in eukariotic individual cells. Given its overall characteristics, this method has been widely used over the past few years in several different areas. In this paper we review the studies published to date about the principles, the basic methodology with currently used variations. We also explore the applications of this assay in: genotoxicology, clinical area, DNA repair studies, environmental biomonitoring and human monitoring.     

Ribonucleotides in DNA: Origins,repair and consequences

While primordial life is thought to have been RNA-based (Cech, Cold Spring Harbor Perspect. Biol. 4 (2012) a006742), all living organisms store genetic information in DNA, which is chemically more stable. Distinctions between the RNA and DNA worlds and our views of “DNA” synthesis continue to evolve as new details emerge on the incorporation, repair and biological effects of ribonucleotides in DNA genomes of organisms from bacteria through humans.     

Pulsed field gel electrophoresis: Theory,instruments and application

Pulsed field gel electrophoresis (PFGE) is a technique for the fractionation of high-molecular-weight DNA ranging from 10 kb to 10 Mb by electrophoresis in agarose gel with an electric field that alternates (pulsates) in two directions. This technology plays a key role in modern genomics, as it allows manipulations with DNA of whole chromosomes or their large fragments. In this review, we discuss (1) the theory behind PFGE; (2) different instruments based on the principle of pulsed field, as well as their advantages and limitations; (3) factors affecting the DNA mobility in PFGE gel; and (4) practical applications of the technique.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: Cellular Roles of DNA Polymerase Beta

Since its discovery and purification in 1971, DNA polymerase ß (Pol ß) is one of the most well-studied DNA polymerases. Pol ß is a key enzyme in the base excision repair (BER) pathway that functions in gap filling DNA synthesis subsequent to the excision of damaged DNA bases. A major focus of our studies is on the cellular roles of Pol ß. We have shown that germline and tumor-associated variants of Pol ß catalyze aberrant BER that leads to genomic instability and cellular transformation. Our studies suggest that Pol ß is critical for the maintenance of genomic stability and that it is a tumor suppressor. We have also shown that Pol ß functions during Prophase I of meiosis. Pol ß localizes to the synaptonemal complex and is critical for removal of the Spo11 complex from the 5’ ends of double-strand breaks. Studies with Pol ß mutant mice are currently being undertaken to more clearly understand the function of Pol ß during meiosis. In this review, we will highlight our contributions from our studies of Pol ß germline and cancer-associated variants.     

CpG DNA:一种新型免疫佐剂

美国依阿华大学医学院Ｋｒｉｅｇ［１］等报道,一种含有胞嘧啶鸟嘌呤二核苷酸（ＣｐＧ）的ＤＮＡ片段是一种强烈的非特异性免疫刺激剂。这种ＣｐＧＤＮＡ可作用于多种免疫细胞。用含ＣｐＧ序列的细菌ＤＮＡ可诱导小鼠９５％的Ｂ细胞进入细胞增殖周期并分泌ＩｇＭ、ＩＬ－...     

Topological measurement of an A-tract bend angle: effect of magnesium

Sequences of four to six adenine residues, termed A-tracts, have been shown to produce curvature in the DNA double helix. A-tracts have been used extensively as reference standards to quantify bending induced by other sequences as well as by DNA binding proteins when they bind to their sites. However, the ability of an A-tract to serve as such a standard is hampered by the wide variation of values reported for the amount of bend conferred by an A-tract. One experimental condition that differs in these studies is the presence of divalent cation. To evaluate this effect, a new application of a topological method, termed rotational variant analysis, is used here to measure for the first time the effect of the presence of magnesium ion on the bend angle conferred by an A-tract. This method, which has the unique ability to measure a bend angle in the presence or absence of magnesium ion, demonstrates that magnesium ion markedly increases the bend angle. For example, when measured in a commonly used gel electrophoretic buffer, the bend angle conferred by a tract of six adenine residues increases from about 7 degrees in the absence of magnesium ion to 19 degrees in the presence of 3.9 mM magnesium ion. This quantitative demonstration of substantial magnesium ion dependence has several important implications. First, it explains discrepancies among bend values reported in various previous studies, particularly those employing gel electrophoretic versus other solution methods. In addition, these findings necessitate substantial revisions of the conclusions in a large number of studies that have used A-tract DNA as the bend angle reference standard in comparison measurements. Finally, any such future studies employing this comparison methodology will need to use the same sequence analyzed in the original measurements as well as replicate the original measurement conditions (e.g. ionic composition and temperature).     

染色质免疫沉淀技术——一种研究基因表达调控的工具

染色质结构在基因表达调节中起着重要的调节作用。利用甲醛固定活细胞中的DNA与蛋白质,通过免疫沉淀分离复合物的染色质免疫沉淀法是研究体内DNA和蛋白质相互作用的一种新方法,它不仅可用来研究体内反式因子与DNA的相互作用,也可以用来研究组蛋白修饰与基因表达的关系,从而分析蛋白质及其体内的DNA结合序列。目前,该方法在染色质结构研究中获得了广泛的应用。     

DNA甲基转移酶的表达调控及主要生物学功能

DNA甲基化是表观遗传学的重要部分, 同组蛋白修饰相互作用, 通过改变染色质结构, 调控基因表达。在哺乳类细胞或人体细胞中, DNA甲基化与细胞的增殖、衰老、癌变等生命现象有着重大关系。对催化DNA甲基化的DNA甲基转移酶(DNA methyltransferase, Dnmt)的研究可以揭示DNA甲基化对基因表达调控的机制, 从而研究与之相关的重要生命活动。文章以DNA甲基转移酶作为切入点, 探讨DNA甲基转移酶在基因表达调控中发挥的作用及其主要生物学功能。     

Technical note: Bone DNA extraction and purification using silica-coated paramagnetic beads

The goal of this study was to develop a simple method to improve DNA recovery from challenging bone samples. To this end, an optimized procedure was developed that combined the demineralization and DNA extraction into a single step, followed by DNA purification using an automated silica-coated paramagnetic bead procedure. This method replaced a previous silica-membrane-based procedure, which was able to recover sufficient DNA to obtain full autosomal and Y chromosome STR profiles from greater than 90% of the samples, including samples greater than 20 years old. The development process began with the evaluation of buffer and demineralization systems to determine the best reagent combination. During the developmental process, we observed that the addition of EDTA and DTT affected silica-based DNA purification methods by raising the pH of the digest buffer. The protocols with buffer ATL, PK, EDTA, and DTT followed by lowering the pH with sodium acetate just before purification resulted in the best yields. The method reduced the extraction volume from 10 to 1.5 ml and used commercially available reagents already being utilized in forensic DNA casework. Because of the simplicity and small volume needed for the procedure, many steps where contamination could be introduced have been eliminated or minimized. This study demonstrated a new method of recovering DNA from bone samples capable of extracting trace quantities of DNA, removing potential inhibitors, and minimizing the potential for exogenous DNA contamination.     

Phylogenetic and biogeographical relationships of the Sander pikeperches (Percidae: Perciformes): patterns across North America and Eurasia

North America and Eurasia share several closely related taxa that diverged either from the breakup of the Laurasian supercontinent or later closures of land bridges. Their modern population structures were shaped in Pleistocene glacial refugia and via later expansion patterns, which are continuing. The pikeperch genus Sander contains five species – two in North America (S. canadensis and S. vitreus) and three in Eurasia (S. lucioperca, S. marinus, and S. volgensis) – whose evolutionary relationships and relative genetic diversities were previously unresolved, despite their fishery importance. This is the first analysis to include the enigmatic and rare sea pikeperch S. marinus, nuclear DNA sequences, and multiple mitochondrial DNA regions. Bayesian and maximum‐likelihood trees from three mitochondrial and three nuclear gene regions support the hypothesis that Sander diverged from its sister group Romanichthys/Zingel ～24.6 Mya. North American and Eurasian Sander then differentiated ～20.8 Mya, with the former diverging ～15.4 Mya, congruent with North American fossils dating to ～16.3–13.6 Mya. Modern Eurasian species date to ～13.8 Mya, with S. volgensis being basal and comprising the sister group to S. lucioperca and S. marinus, which diverged ～9.1 Mya. Genetic diversities of the North American species are higher than those in Eurasia, suggesting fewer Pleistocene glaciation bottlenecks. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 156–179.     

Torque-induced deformations of charged elastic DNA rods: thin helices,loops, and precursors of DNA supercoiling

We study the deformations of charged elastic rods under applied end forces and torques. For neutral filaments, we analyze the energetics of initial helical deformations and loop formation. We supplement this elastic approach with electrostatic energies of bent filaments and find critical conditions for buckling depending on the ionic strength of the solution. We also study force-induced loop opening, for parameters relevant for DNA. Finally, some applications of this nano-mechanical DNA model to salt-dependent onset of the DNA supercoiling are discussed.