Comparative Genomic Study Reveals a Transition from TA Richness in Invertebrates to GC Richness in Vertebrates at CpG Flanking Sites： An Indication for Context-Dependent Mutagenicity of Methylated CpG Sites

Vertebrate genomes are characterized with CpG deficiency, particularly for GCpoor regions. The GC content-related CpG deficiency is probably caused by context-dependent deamination of methylated CpG sites. This hypothesis was examined in this study by comparing nucleotide frequencies at CpG flanking positions among invertebrate and vertebrate genomes. The finding is a transition of nucleotide preference of 5＇ T to 5＇ A at the invertebrate-vertebrate boundary, indicating that a large number of CpG sites with 5＇ Ts were depleted because of global DNA methylation developed in vertebrates. At genome level, we investigated CpG observed/expected （obs/exp） values in 500 bp fragments, and found that higher CpG obs/exp value is shown in GC-poor regions of invertebrate genomes （except sea urchin） but in GC-rich sequences of vertebrate genomes. We next compared GC content at CpG flanking positions with genomic average, showing that the GC content is lower than the average in invertebrate genomes, but higher than that in vertebrate genomes. These results indicate that although 5＇ T and 5＇ A are different in inducing deamination of methylated CpG sites, GC content is even more important in affecting the deamination rate. In all the tests, the results of sea urchin are similar to vertebrates perhaps due to its fractional DNA methylation. CpG deficiency is therefore suggested to be mainly a result of high mutation rates of methylated CpG sites in GC-poor regions.     

Mechanism of DNA Methylation: The Double Role of DNA as a Substrate and as a Cofactor

Methylation of cytosine residues in the DNA is one of the most important epigenetic marks central to the control of differential expression of genes. We perform quantum mechanical calculations to investigate the catalytic mechanism of the bacterial HhaI DNA methyltransferase. We find that the enzyme nucleophile, Cys81, can attack C6 of cytosine only after it is deprotonated by the DNA phosphate group, a reaction facilitated by a bridging water molecule. This finding, which indicates that the DNA acts as both the substrate and the cofactor, can explain the total loss of activity observed in an analogous enzyme, thymidylate synthase, when the phosphate group of the substrate was removed. Furthermore, our results displaying the inability of the phosphate group to deprotonate the side chain of serine is in agreement with the total, or the large extent of, inactivity observed for the C81S mutant. In contrast to results from previous calculations, we find that the active site conserved residues, Glu119, Arg163, and Arg165, are crucial for catalysis. In addition, the enzyme-DNA adduct formation and the methyl transfer from the cofactor S-adenosyl-l-methionine are not concerted but proceed via stepwise mechanism. In many of the different steps of this methylation reaction, the transfer of a proton is found to be necessary. To render these processes possible, we find that several water molecules, found in the crystal structure, play an important role, acting as a bridge between the donating and accepting proton groups.     

Reproducible Alterations of DNA Methylation at a Specific Population of CpG Islands during Blast Formation of Peripheral Blood Lymphocytes

We investigated the changes in the methylation patterns of CpGislands associated with blast formation of human peripheralblood lymphocytes activated by anti-CD3 and interleukin-2 (IL-2),using restriction landmark genomic scanning with a methylation-sensitiverestriction enzyme (RLGS-M) system. Of about 2,100 Not I spot/lociwhich were analyzed, only 10 showed changes, whereas drasticchanges have been observed in cases of malignant and SV40 transformation.These changes were highly reproducible for samples from boththe same and different individuals. Even the timing of the changesafter cultivation was the same. Thus, we concluded that at leastthe genomic DNA methylation state in vivo was essentially retainedin T blast cells activated in vitro by induction with IL-2 andanti-CD3, which are commonly used in biological experimentsas well as clinical diagnosis and therapy.     

Fish disease as a monitor for marine pollution: the case of the North Sea

Summary The use of fish diseases to monitor marine pollution is reviewed and evaluated, with particular reference to the North Sea and associated waters. Criteria for epidemiological surveys are outlined, an international overview of research is given, and recent studies in the North Sea area are described and evaluated.The basic approach is to identify spatial and temporal patterns of disease prevalence, which can be related to pollution. A major obstacle is to distinguish effects of pollution from those of other variables, especially as most diseases appear to have a multifactorial aetiology. Field studies can be evaluated against a number of criteria: these include the accuracy and precision of prevalence estimates, the extent to which possible causal factors other than pollution are taken into account, and whether or not exposure of the study population to pollution is measured directly.A distinction can be made between intensive, thorough studies, which frequently use a histopathological approach, and the more extensive surveys of large numbers of fish for grossly observable lesions. Broadly speaking, North American research has emphasized the former approach, and research in the North Sea the latter. Nevertheless, although the most comprehensive evidence for a causal relationship between disease and pollution has been gathered in North America, there are also good examples from the North Sea area, particularly in local areas with distinct sources of pollution. The data from wider-ranging surveys are more ambiguous: while some provide circumstantial evidence for a role of pollution, the apparent complexity of disease aetiology and the limitations of the epidemiological approach may prevent any clear demonstration of pollution as a cause over wide geographical areas. Extensive surveys are nevertheless useful for detecting long-term trends in disease prevalence and hot-spots of anomalously high prevalence, and for examining the relationship between disease and a complex of environmental variables.For the future, greater emphasis should be placed on the recording of liver lesions, on the measurement of exposure to pollution, and on experimental work.     

Increased flexibility as a strategy for cold adaptation: a comparative molecular dynamics study of cold- and warm-active uracil DNA glycosylase

Uracil DNA glycosylase (UDG) is a DNA repair enzyme in the base excision repair pathway and removes uracil from the DNA strand. Atlantic cod UDG (cUDG), which is a cold-adapted enzyme, has been found to be up to 10 times more catalytically active in the temperature range 15-37 degrees C as compared with the warm-active human counterpart. The increased catalytic activity of cold-adapted enzymes as compared with their mesophilic homologues are partly believed to be caused by an increase in the structural flexibility. However, no direct experimental evidence supports the proposal of increased flexibility of cold-adapted enzymes. We have used molecular dynamics simulations to gain insight into the structural flexibility of UDG. The results from these simulations show that an important loop involved in DNA recognition (the Leu(272) loop) is the most flexible part of the cUDG structure and that the human counterpart has much lower flexibility in the Leu(272) loop. The flexibility in this loop correlates well with the experimental k(cat)/K(m) values. Thus, the data presented here add strong support to the idea that flexibility plays a central role in adaptation to cold environments.     

Connectivity across the Caribbean Sea: DNA Barcoding and Morphology Unite an Enigmatic Fish Larva from the Florida Straits with a New Species of Sea Bass from Deep Reefs off Cura?ao

Integrative taxonomy, in which multiple disciplines are combined to address questions related to biological species diversity, is a valuable tool for identifying pelagic marine fish larvae and recognizing the existence of new fish species. Here we combine data from DNA barcoding, comparative morphology, and analysis of color patterns to identify an unusual fish larva from the Florida Straits and demonstrate that it is the pelagic larval phase of a previously undescribed species of Liopropoma sea bass from deep reefs off Curaçao, southern Caribbean. The larva is unique among larvae of the teleost family Serranidae, Tribe Liopropomini, in having seven elongate dorsal-fin spines. Adults of the new species are similar to the golden bass, Liopropoma aberrans, with which they have been confused, but they are distinct genetically and morphologically. The new species differs from all other western Atlantic liopropomins in having IX, 11 dorsal-fin rays and in having a unique color pattern–most notably the predominance of yellow pigment on the dorsal portion of the trunk, a pale to white body ventrally, and yellow spots scattered across both the dorsal and ventral portions of the trunk. Exploration of deep reefs to 300 m using a manned submersible off Curaçao is resulting in the discovery of numerous new fish species, improving our genetic databases, and greatly enhancing our understanding of deep-reef fish diversity in the southern Caribbean. Oh the mother and child reunion is only a moment away. Paul Simon.     

Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

The selectivity of DNA polymerases is crucial for many applications. For example, high discrimination between the extension of matched versus mismatched primer termini is desired for the detection of a single nucleotide variation at a particular locus within the genome. Here we describe the generation of thermostable mutants of the large fragment of Thermus aquaticus DNA polymerase (KlenTaq) with increased mismatch extension selectivity. In contrast to previously reported much less active KlenTaq mutants with mismatch discrimination abilities, many of the herein discovered mutants show conserved wild-type-like high activities. We demonstrate for one mutant containing the single amino acid exchange R660V the suitability for application in allele-specific amplifications directly from whole blood without prior sample purification. Also the suitability of the mutant for methylation specific amplification in the diagnostics of 5-methyl cytosines is demonstrated. Furthermore, the identified mutant supersedes other commercially available enzymes in human leukocyte antigen (HLA) analysis by sequence-specific primed polymerase chain reactions (PCRs).     

The use of Multiple Displacement Amplified DNA as a control for Methylation Specific PCR,Pyrosequencing, Bisulfite Sequencing and Methylation-Sensitive Restriction Enzyme PCR

Background  

Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples.     

Activation of DNA primer for DNA polymerase as an ultrasensitive assay for a subgroup of enzymes with deoxyribonuclease activity

Pretreatment of native DNA with nucleases which hydrolyze phosphodiester bonds with liberation of 3′-hydroxyl terminal groups is known to increase the rate of incorporation of nucleotides into DNA by E. coli DNA polymerase I and similar enzymes. Concentration ranges and conditions for this reaction have been established which allow specific detection and quantitative assay of such nucleases. The method permits detection of as little as 0.1 pg of pancreatic deoxyribonuclease, is simple, and requires only commercially available components. It has been utilized to monitor purification of a nuclease from HeLa cell nuclei at concentrations which could not be detected by other methods.     

Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics

The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp-5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high-LET alpha particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9+/-0.5 h and a slow component with a half-life of 16+/-9 h. For alpha particles, a fast component with a half-life of 0.7+/-0.4 h and a slow component with a half-life of 12+/-5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37+/-0.12) relative to low-LET radiation (0.22+/-0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites.     

Production of a Monoclonal Antibody Targeting the M Protein of MERS-CoV for Detection of MERS-CoV Using a Synthetic Peptide Epitope Formulated with a CpG–DNA–Liposome Complex

The Middle East respiratory syndrome-related coronavirus (MERS-CoV) contains four major structural proteins, the spike glycoprotein, nucleocapsid phosphoprotein, membrane (M) glycoprotein and small envelope glycoprotein. The M protein of MERS-CoV has a role in the morphogenesis or assembly of the virus and inhibits type I interferon expression in infected cells. Here, we produced a monoclonal antibody specific against the M protein of MERS-CoV by injecting BALB/c mice with a complex containing the epitope peptide and CpG–DNA encapsulated with a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS). The monoclonal antibody was reactive to the epitope peptide of the M protein of MERS-CoV which was confirmed by western blotting and immunoprecipitations. Indirect immunofluorescence assay and confocal image analysis showed that the monoclonal antibody binds specifically to the M protein of MERS-CoV in the virus-infected cells. Further studies using this monoclonal antibody may provide important information on the function of the M protein and its future application in diagnostics.

     

Sea Urchin Spines as a Model-System for Permeable, Light-Weight Ceramics with Graceful Failure Behavior. Part I. Mechanical Behavior of Sea Urchin Spines under Compression

The spines of pencil and lance urchins Heterocentrotus mammillatus and Phyllacanthus imperialis were studied as a modelof light-weight material with high impact resistance.The complex and variable skeleton construction ("stereom") of body andspines of sea urchins consists of highly porous Mg-bearing calcium carbonate.This basically brittle material with pronouncedsingle-crystal cleavage does not fracture by spontaneous catastrophic device failure but by graceful failure over the range of tensof millimeter of bulk compression instead.This was observed in bulk compression tests and blunt indentation experiments onregular,infiltrated and latex coated sea urchin spine segments.Microstructural characterization was carried out using X-raycomputer tomography,optical and scanning electron microscopy.The behavior is interpreted to result from the hierarchicstructure of sea urchin spines from the rnacroscale down to the nanoscale.Guidelines derived from this study see ceramics withlayered porosity as a possible biomimetic construction for appropriate applications.     

DNA hybridization with hardjobovis-specific recombinant probes as a method for type discrimination of Leptospira interrogans serovar hardjo

Restriction endonuclease analysis of DNA of Leptospira interrogans, serovar hardjo, showed two distinct types within this serovar. These two types, hardjoprajitno and hardjobovis, cannot be differentiated by monoclonal antibodies. Application of 32P- or biotin-labelled total DNA probes in dot-blot or in situ hybridization assays showed a high sensitivity of the assays but also considerable cross-hybridization. Therefore, a genomic library of hardjobovis was constructed and a number of hardjobovis-specific recombinant clones were isolated. Finally, four clones were selected on the basis of a strong hybridization signal and a high specificity for hardjobovis as compared to hardjoprajitno. In a dot-blot assay as well as in in situ hybridization experiments all four clones gave strong signals, and no cross-hybridization with hardjoprajitno was observed in either type of assay. Our results indicate that specific recombinant DNA probes might provide tools for routine diagnosis and classification in cases of hardjo infections.