Charge transport in a DNA model with solvent interaction

The charge transport in the modified DNA model is studied by taking into account the factor of solvent and the effect of coupling motions of nucleotides. We report on the presence of the modulational instability (MI) of a plane wave for charge migration in DNA and the generation of soliton-like excitations in DNA nucleotides. By applying the continuum approximation, we show that the original differential-difference equation for the DNA dynamics can be reduced to a set of three coupled nonlinear equations. The linear stability analysis of wave solutions of the coupled systems is performed and the growth rate of instability is found numerically. We also investigate the impact of solvent interaction. The solvent factor introduces a new behavior to the wave patterns, modifying also the intrinsic properties of localized structures. In the numerical simulations, we show that the solitons exists when taking into account the effect of solvent and confirms an highest propagation of localized structures in the systems. The effect of solvent forces introduces a robustness behavior to the formed patterns, reinforcing the idea that the information in the DNA model is confined and concentrated to specific regions for efficiency. We also show that the localized structures can be disappeared with the highest value of solvent factor and thereafter the information within the molecule is not perceptible or not transmitted to another sites.     

Polymorphisms in the 5′-UTR of <Emphasis Type="Italic">PTEN</Emphasis> and other gene polymorphisms in normal Japanese individuals

Polymorphisms are distributed differently in populations, including those of regions, ethnic groups, and diseased patients. In order to investigate variation in nucleotide sequences in normal individuals, we isolated genomic DNA from the blood of healthy Japanese individuals and sequenced the 5′-untranslated region (5′-UTR) of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene and the gene promoter, intron, and exon nucleotides of p53, p14 ^ARF , murine double minute 2 (MDM2), and the β₂- and β₃-adrenoceptor (?AR). We found polymorphisms in these regions, including a deletion at positions ?465 to ?463 and a substitution at position ?404 in PTEN and a substitution at position ?4924 in p14 ^ARF , in normal individuals. Individuals with or without the PTEN polymorphism harbored a different distribution of polymorphisms, including simultaneous alterations in nucleotides of p53, MDM2, and β₃-AR, and also harbored some polymorphic nucleotides located in the same set of associatively altered nucleotides. Our results show that multiple nucleotides, including the PTEN nucleotides, are altered in normal Japanese individuals and provide useful information for genotyping studies in individuals and populations.     

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

Background

Transfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene. To characterize the features of the DNA-repair mechanisms involved, we examined the maximal distance for the simultaneous exchange of two nucleotides by a single-stranded oligonucleotide. The chosen experimental system was the correction of a hprt-point mutation in a hamster cell line, the generation of an additional nucleotide exchange at a variable distance from the first exchange position and the investigation of the rate of simultaneous nucleotide exchanges.

Results

The smaller the distance between the two exchange positions, the higher was the probability of a simultaneous exchange. The detected simultaneous nucleotide exchanges were found to cluster in a region of about fourteen nucleotides upstream and downstream from the first exchange position.

Conclusion

We suggest that the mechanism involved in the repair of the targeted DNA strand utilizes only a short sequence of the single-stranded oligonucleotide, which may be physically incorporated into the DNA or be used as a matrix for a repair process.     

Approximating the dynamics of communicating cells in a diffusive medium by ODEs—homogenization with localization

Bacteria may change their behavior depending on the population density. Here we study a dynamical model in which cells of radius $R$ within a diffusive medium communicate with each other via diffusion of a signalling substance produced by the cells. The model consists of an initial boundary value problem for a parabolic PDE describing the exterior concentration $u$ of the signalling substance, coupled with $N$ ODEs for the masses $a_i$ of the substance within each cell. We show that for small $R$ the model can be approximated by a hierarchy of models, namely first a system of $N$ coupled delay ODEs, and in a second step by $N$ coupled ODEs. We give some illustrations of the dynamics of the approximate model.     

StreAM-$$T_g$$: algorithms for analyzing coarse grained RNA dynamics based on Markov models of connectivity-graphs

Background

In this work, we present a new coarse grained representation of RNA dynamics. It is based on adjacency matrices and their interactions patterns obtained from molecular dynamics simulations. RNA molecules are well-suited for this representation due to their composition which is mainly modular and assessable by the secondary structure alone. These interactions can be represented as adjacency matrices of k nucleotides. Based on those, we define transitions between states as changes in the adjacency matrices which form Markovian dynamics. The intense computational demand for deriving the transition probability matrices prompted us to develop StreAM-\(T_g\), a stream-based algorithm for generating such Markov models of k-vertex adjacency matrices representing the RNA.

Results

We benchmark StreAM-\(T_g\) (a) for random and RNA unit sphere dynamic graphs (b) for the robustness of our method against different parameters. Moreover, we address a riboswitch design problem by applying StreAM-\(T_g\) on six long term molecular dynamics simulation of a synthetic tetracycline dependent riboswitch (500 ns) in combination with five different antibiotics.

Conclusions

The proposed algorithm performs well on large simulated as well as real world dynamic graphs. Additionally, StreAM-\(T_g\) provides insights into nucleotide based RNA dynamics in comparison to conventional metrics like the root-mean square fluctuation. In the light of experimental data our results show important design opportunities for the riboswitch.

     

Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase ��

A key set of reactions for the initiation of new DNA strands during herpes simplex virus-1 replication consists of the primase-catalyzed synthesis of short RNA primers followed by polymerase-catalyzed DNA synthesis (i.e. primase-coupled polymerase activity). Herpes primase (UL5-UL52-UL8) synthesizes products from 2 to ∼13 nucleotides long. However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at least 8 nucleotides long. Surprisingly, coupled activity was remarkably inefficient, even considering only those primers at least 8 nucleotides long, and herpes polymerase typically elongated <2% of the primase-synthesized primers. Of those primers elongated, only 4–26% of the primers were passed directly from the primase to the polymerase (UL30-UL42) without dissociating into solution. Comparing RNA primer-templates and DNA primer-templates of identical sequence showed that herpes polymerase greatly preferred to elongate the DNA primer by 650–26,000-fold, thus accounting for the extremely low efficiency with which herpes polymerase elongated primase-synthesized primers. Curiously, one of the DNA polymerases of the host cell, polymerase α (p70-p180 or p49-p58-p70-p180 complex), extended herpes primase-synthesized RNA primers much more efficiently than the viral polymerase, raising the possibility that the viral polymerase may not be the only one involved in herpes DNA replication.Herpes simplex virus 1 (HSV-1)² encodes seven proteins essential for replicating its double-stranded DNA genome; five of these encode the heterotrimeric helicase-primase (UL5-UL52-UL8 gene products) and the heterodimeric polymerase (UL30-UL42 gene products) (1, 2). The helicase-primase unwinds the DNA at the replication fork and generates single-stranded DNA for both leading and lagging strand synthesis. Primase synthesizes short RNA primers on the lagging strand that the polymerase presumably elongates using dNTPs (i.e. primase-coupled polymerase activity). These two protein complexes are thought to replicate the viral genome on both the leading and lagging strands (1, 2).Previous studies have focused on the helicase-primase and polymerase separately. The helicase-primase contains three subunits, UL5, UL52, and UL8 in a 1:1:1 ratio (3–5). The UL5 subunit has helicase-like motifs and the UL52 subunit has primase-like motifs, yet the minimal active complex that demonstrates either helicase or primase activities contains both UL5 and UL52 (6, 7). Although the UL8 subunit has no known catalytic activity, several functions have been proposed, including enhancing helicase and primase activities, enhancing primer synthesis on ICP8 (the HSV-1 single-stranded binding protein)-coated DNA strands, and facilitating formation of the replisome (8–12). Although primase will synthesize short (2–3 nucleotides long) primers on a variety of template sequences, synthesis of longer primers up to 13 nucleotides long requires the template sequence, 3′-deoxyguanidine-pyrimidine-pyrimidine-5′ (13). Primase initiates synthesis at the first pyrimidine via the polymerization of two purine NTPs (13). Even after initiation at this sequence, however, the vast majority of products are only 2–3 nucleotides long (13, 14).The herpes polymerase consists of the UL30 subunit, which has polymerase and 3′ → 5′ exonuclease activities (1, 2), and the UL42 subunit, which serves as a processivity factor (15–17). Unlike most processivity factors that encircle the DNA, the UL42 protein binds double-stranded DNA and thus directly tethers the polymerase to the DNA (18). Using pre-existing DNA primer-templates as the substrate, the heterodimeric polymerase (UL30-UL42) incorporates dNTPs at a rate of 150 s^–1, a rate much faster than primer synthesis (for primers >7 nucleotides long, 0.0002–0.01 s^–1) (19, 20).We examined primase-coupled polymerase activity by the herpes primase and polymerase complexes. Although herpes primase synthesizes RNA primers 2–13 nucleotides long, the polymerase only effectively elongates those at least 8 nucleotides long. Surprisingly, the polymerase elongated only a small fraction of the primase-synthesized primers (<1–2%), likely because of the polymerase elongating RNA primer-templates much less efficiently than DNA primer-templates. In contrast, human DNA polymerase α (pol α) elongated the herpes primase-synthesized primers very efficiently. The biological significance of these data is discussed.     

Analysis of the dependence of surfatron acceleration of electrons by an electromagnetic wave in space plasma on the particle momentum along the wave front

Based on the numerical solution of the nonlinear nonstationary second-order equation for the wave phase on the particle trajectory, the dynamics of surfatron acceleration of electrons by an electromagnetic wave propagating across the external magnetic field in space plasma is analyzed as a function of the electron momentum along the wave front. Numerical calculations show that, for strongly relativistic initial values of the electron momentum component along the wave front g _y (0) (the other parameters of the problem being the same), electrons are trapped into the regime of ultrarelativistic surfatron acceleration within a certain interval of the initial wave phase Ψ(0) on the particle trajectory. It is assumed in the calculations that |Ψ(0)| ≤ π. For strongly relativistic values of g _y (0), electrons are immediately trapped by the wave for 19% of the initial values of the phase Ψ(0) (favorable phases). For the rest of the values of Ψ(0), trapping does not occur even at long times. This circumstance substantially simplifies estimations of the wave damping due to particle acceleration in subsequent calculations. The dynamics of the relativistic factor and the components of the electron velocity and momentum under surfatron acceleration is also analyzed. The obtained results are of interest for the development of modern concepts of possible mechanisms of generation of ultrarelativistic particle fluxes in relatively calm space plasma, as well as for correct interpretation of observational data on the fluxes of such particles and explanation of possible reasons for the deviation of ultrarelativistic particle spectra detected in the heliosphere from the standard power-law scalings and the relation of these variations to space weather and large-scale atmospheric processes similar to tropical cyclones.     

Human Triallelic Sites: Evidence for a New Mutational Mechanism?

Most SNPs in the human genome are biallelic; however, there are some sites that are triallelic. We show here that there are approximately twice as many triallelic sites as we would expect by chance. This excess does not appear to be caused by natural selection or mutational hotspots. Instead we propose that a new mutation can induce another mutation either within the same individual or subsequently during recombination. We provide evidence for this model by showing that the rarer two alleles at triallelic sites tend to cluster on phylogenetic trees of human haplotypes. However, we find no association between the density of triallelic sites and the rate of recombination, which leads us to suggest that triallelic sites might be generated by the simultaneous production of two new mutations within the same individual on the same genetic background. Under this model we estimate that simultaneous mutation contributes ∼3% of all distinct SNPs. We also show that there is a twofold excess of adjacent SNPs. Approximately half of these seem to be generated simultaneously since they have identical minor allele frequencies. We estimate that the mutation of adjacent nucleotides accounts for a little less than 1% of all SNPs.ALTHOUGH the density of biallelic SNPs in the human genome is reasonably low, there are some sites that have three (triallelic sites) or even four nucleotides segregating in the human population. We show here that there are approximately twice as many triallelic sites as we would expect by chance. There are at least three mutational mechanisms that could potentially generate such an excess of triallelic sites. First, some sites may be hypermutable, and if the mutation rate of at least two pathways (e.g., C → T and C → A) is elevated at such sites, then there will be an excess of triallelic sites. The mutation rate of a site is known to depend upon the adjacent nucleotides, the best known example being the CpG dinucleotide (Coulondre et al. 1978; Bird 1980) at which the frequency of both transition and transversion mutations is elevated. However, other adjacent nucleotides also influence the mutation rate (Blake et al. 1992; Zhao et al. 2003; Hwang and Green 2004). Furthermore, we have recently shown that there is variation in the mutation rate that does not depend upon the identity of the adjacent nucleotides or any specific context (Hodgkinson et al. 2009).Second, it is possible that two of the alleles at a triallelic site are generated simultaneously within a single individual. Point mutations are generally assumed to involve the production of a single new allele per mutation event at a rate that is governed by the effects mentioned above. However, it is not difficult to imagine mechanisms that might induce mutations on both strands of the DNA duplex; for example, the presence of a base mismatch may itself be unstable, so we might go from a G-C base pair to a G-A, which then may mutate to C-A; if DNA replication reads through this mismatch, the G allele will have mutated to both C and T. Alternatively, the mutation may occur across both strands of the duplex at the same time, possibly as a result of a chemical or radiation event. Third, in a similar manner, we might imagine a single SNP inducing subsequent mutations if base mismatches are formed during recombination in heteroduplex DNA.Here we attempt to identify the cause of the excess of triallelic sites by analyzing sequence data around triallelic sites.     

The Structure and Assembly Dynamics of Plasmid Actin AlfA Imply a Novel Mechanism of DNA Segregation

Bacterial cytoskeletal proteins participate in a variety of processes, including cell division and DNA segregation. Polymerization of one plasmid-encoded, actin-like protein, ParM, segregates DNA by pushing two plasmids in opposite directions and forms the current paradigm for understanding active plasmid segregation. An essential feature of ParM assembly is its dynamically instability, the stochastic switching between growth and disassembly. It is unclear whether dynamic instability is an essential feature of all actin-like protein-based segregation mechanisms or whether bacterial filaments can segregate plasmids by different mechanisms. We expressed and purified AlfA, a plasmid-segregating actin-like protein from Bacillus subtilis, and found that it forms filaments with a unique structure and biochemistry; AlfA nucleates rapidly, polymerizes in the presence of ATP or GTP, and forms highly twisted, ribbon-like, helical filaments with a left-handed pitch and protomer nucleotide binding pockets rotated away from the filament axis. Intriguingly, AlfA filaments spontaneously associate to form uniformly sized, mixed-polarity bundles. Most surprisingly, our biochemical characterization revealed that AlfA does not display dynamic instability and is relatively stable in the presence of diphosphate nucleotides. These results (i) show that there is remarkable structural diversity among bacterial actin filaments and (ii) indicate that AlfA filaments partition DNA by a novel mechanism.Bacteria contain multiple filament-forming proteins related to eukaryotic actin (6). These actin-like proteins have multiple cellular roles, including determination of cell shape (18), arrangement of organelles (20), and segregation of DNA (36). Little is known about the assembly dynamics of most of these proteins or about the identities and activities of the factors that regulate them. The widely expressed actin-like protein MreB, for example, has been purified and studied in vitro, but its assembly appears to be strongly inhibited by physiological concentrations of monovalent cations, suggesting that its assembly in vivo is facilitated by as-yet-unknown factors (23). At present, the best-understood actin-like protein is ParM, a plasmid-encoded protein that constructs a bipolar spindle capable of pushing plasmids to opposite poles of rod-shaped cells (5, 25). In contrast to the eukaryotic actin cytoskeleton, whose assembly and architecture are regulated by a variety of accessory factors, ParM dynamics are regulated by a single factor, a complex composed of multiple copies of the repressor protein ParR bound to a DNA locus, parC (17). The ParR/parC complex binds the ends of ParM filaments and is pushed through the cytoplasm by filament elongation (5, 14, 25). The ability of ParM to function with such minimal regulation appears to be due to its unique assembly dynamics, which are dramatically different from those of eukaryotic actins. One of the most important differences is that ParM filaments are dynamically unstable (13). That is, similar to eukaryotic microtubules, they can exist in one of two states: stably growing or rapidly (catastrophically) shrinking. This property is required for the ability of ParM to segregate DNA in vivo and appears to solve several fundamental problems associated with DNA segregation. First, spontaneous disassembly of the polymer overcomes the need for an accessory factor to take filaments apart. Second, because filaments bound to ParR/parC complexes are selectively stabilized, the catastrophic disassembly of unattached filaments provides excess monomers that can preferentially elongate them. This is significant because, if the stabilities of attached and unattached filaments were similar, the concentration of free ParM monomers would equilibrate at a level not capable of promoting DNA segregation. And finally, pairs of plasmids appear to find each other via a search-and-capture mechanism (5, 14) that is dramatically enhanced by the continual growth and shortening of filaments attached to single plasmids (16).Because we have little information on the dynamics of other actin-like proteins, it is unclear to what extent ParM''s behavior reflects general properties of bacterial actins rather than specific adaptations to its role in DNA segregation. Furthermore, it is unclear whether all plasmid-segregating actins employ the same dynamic instability-based strategy to find and transport DNA molecules. To better understand the structural and functional diversity of bacterial actins, we studied a second, recently discovered plasmid-segregating actin-like protein, AlfA (1). The AlfA gene is part of an operon (alf) that is located close to the origin of replication of a ∼70-kb, low-copy-number plasmid, pLS32. This plasmid was initially isolated from a natto strain of Bacillus subtilis used in soybean fermentation (33), but a similar plasmid with an identical alf operon is also present in a colony-forming laboratory strain of B. subtilis, strain NCIB 3610 (8, 32). The function of these plasmids is cryptic. They are present at levels of only two or three copies per chromosome equivalent (33), and maintenance of their derivatives requires both AlfA and a downstream gene, alfB (1). Becker and coworkers (1) identified AlfA as a member of the actin superfamily based on the presence of a conserved nucleotide binding fold (4), although the sequence of AlfA is as different from the sequences of ParM and MreB as all three are from the sequence of conventional eukaryotic actin (∼20% identity). These authors also showed that fluorescent derivatives of AlfA form a single filamentous structure running along the long axis of the cell. Photobleached filaments recover from both ends in approximately 1 min, indicating that the structures are composed of multiple, dynamic filaments (1). By analogy with the ParR/parC complex, AlfB might be a DNA binding protein that couples AlfA assembly to plasmid movement. To date, no centromeric sequences involved in segregation have been identified in this plasmid.We expressed and purified AlfA and characterized its assembly dynamics by using light scattering, high-speed pelleting, and fluorescence microscopy, and we determined the structure of AlfA polymers by high-resolution electron microscopy (EM). We found that in the presence of ATP and GTP, AlfA forms two-strand helical filaments and filament bundles. Like ParM filaments, AlfA filaments are left-handed two-start helices, but otherwise their filament architecture is quite different. AlfA filaments appear to be more tightly twisted and ribbon-like, and AlfA subunits have a significantly different orientation with respect to the filament axis. Unlike other actin-like proteins described thus far, AlfA spontaneously forms regularly sized, mixed-polarity filament bundles driven by electrostatic interactions between filaments, even in the absence of molecular crowding. Finally, AlfA shows no evidence of the dynamic instability crucial to the function of ParM. Thus, AlfA assembles into a unique structure with a unique set of biochemical and structural properties, suggesting a novel mechanism for DNA segregation.     

Microsatellite Instability in Arabidopsis Increases with Plant Development

Plant development consists of the initial phase of intensive cell division followed by continuous genome endoreduplication, cell growth, and elongation. The maintenance of genome stability under these conditions is the main task performed by DNA repair and genome surveillance mechanisms. Our previous work showed that the rate of homologous recombination repair in older plants decreases. We hypothesized that this age-dependent decrease in the recombination rate is paralleled with other changes in DNA repair capacity. Here, we analyzed microsatellite stability using transgenic Arabidopsis (Arabidopsis thaliana) plants that carry the nonfunctional β-glucuronidase gene disrupted by microsatellite repeats. We found that microsatellite instability increased dramatically with plant age. We analyzed the contribution of various mechanisms to microsatellite instability, including replication errors and mistakes of DNA repair mechanisms such as mismatch repair, excision repair, and strand break repair. Analysis of total DNA polymerase activity using partially purified protein extracts showed an age-dependent decrease in activity and an increase in fidelity. Analysis of the steady-state RNA level of DNA replicative polymerases α, δ, Pol I-like A, and Pol I-like B and the expression of mutS homolog 2 (Msh2) and Msh6 showed an age-dependent decrease. An in vitro repair assay showed lower efficiency of nonhomologous end joining in older plants, paralleled by an increase in Ku70 gene expression. Thus, we assume that the more frequent involvement of nonhomologous end joining in strand break repair and the less efficient end-joining repair together with lower levels of mismatch repair activities may be the main contributors to the observed age-dependent increase in microsatellite instability.The genome of Arabidopsis (Arabidopsis thaliana) is extensively repetitive, which leads many to believe that Arabidopsis is subject to ancient autoploid events with many subsequent rearrangements and alterations (Meinke et al., 1998; Arabidopsis Genome Initiative, 2000; Blanc et al., 2000). Despite the highly reduplicated genome with the potential for a high degree of genetic redundancy, maintaining a consistent level of genome stability is critical. This is especially important when considering that plants do not have a predetermined germ line and that gametes are produced from meristematic cells that are products of many somatic cell divisions (Hays, 2002). Furthermore, as plants are sessile organisms, they are continuously exposed to various genotoxic elements such as heavy metals, reactive oxygen species, and UV irradiation. This constant exposure to harsh environmental conditions imposes a need for precise and efficient genome maintenance pathways, as the persistence of DNA damage and mutagenesis can decrease the fitness of current and future generations (Britt, 1996).DNA mutagenesis cannot solely be attributed to environmentally induced genotoxic stress, as DNA is prone to spontaneous or replication-induced mutagenesis. For example, transitions of 5-methylcytosine to thymine are common spontaneous mutations (Britt, 1996), while DNA replication and repair infidelity can induce numerous errors (Sia et al., 1997; Tuteja et al., 2001). Hundreds of mutations are introduced upon each genome replication due to DNA polymerase infidelity. Repetitive elements are particularly prone to this type of mutation due to replication slippage, which refers to DNA polymerase dissociation during the replication of short repetitive sequences followed by the separation and subsequent reassociation of the daughter strand in a different but identical repeat (Viguera et al., 2001). Polymerase reloading and the resumption of DNA synthesis can result in addition or subtraction of the repeated sequence. Microsatellites, the simple tandem repeats of one to six nucleotides (Viguera et al., 2001), are highly susceptible to replication slippage.The frequency at which these and other polymerase-derived errors persist depends largely on the DNA polymerase proofreading activity and the precision and fidelity of core DNA repair enzymes. Since many repair pathways involve DNA polymerase activity, many of them can potentially contribute to an increase in microsatellite instability. Mismatch repair (MMR) is a repair mechanism involved in the correction of replication errors. It is essential for the maintenance of repeated sequences, as mutations in MMR genes are associated with a substantial destabilization of microsatellites (Karran, 1996), and in humans, microsatellite instability increases with aging (Ben Yehuda et al., 2000; Krichevsky et al., 2004; Neri et al., 2005).The fidelity of different repair pathways can vary largely in the same or similar types of lesions. For example, single- and double-strand breaks can be repaired via homologous recombination (HR) or nonhomologous end joining (NHEJ) pathways (Britt, 1996; Tuteja et al., 2001; Kovalchuk et al., 2004; Boyko et al., 2006a). Of these pathways, HR is believed to be precise and largely error free, while NHEJ can induce numerous mutations ranging from single- to thousand-nucleotide insertions or deletions (Pelczar et al., 2003; Boyko et al., 2006b). It is unclear how either of these pathways is chosen for repair, but recent evidence from our laboratory suggests that the HR pathway is developmentally regulated, whereby NHEJ is up-regulated and HR is down-regulated with plant development (Boyko et al., 2006b). Currently, there is no information on whether other DNA repair pathways in plants are developmentally regulated.Previous publications suggest that aging human cells have a higher frequency of mutations in microsatellites (Ben Yehuda et al., 2000; Krichevsky et al., 2004; Neri et al., 2005). No such data exist for plants. Here, we investigated microsatellite stability during the development of Arabidopsis using the uidA (GUS) reporter gene inactivated by an artificially incorporated microsatellite (Azaiez et al., 2006). We found a strong increase in instability with plant maturity. We tested the contributions of various repair pathways to age-dependent microsatellite instability and suggest that these changes are primarily due to more frequent involvement of the NHEJ pathway in DNA repair.     

A new efficient method for analyzing fungi species using correlations between nucleotides

Background

In recent years, DNA barcoding has become an important tool for biologists to identify species and understand their natural biodiversity. The complexity of barcode data makes it difficult to analyze quickly and effectively. Manual classification of this data cannot keep up to the rate of increase of available data.

Results

In this study, we propose a new method for DNA barcode classification based on the distribution of nucleotides within the sequence. By adding the covariance of nucleotides to the original natural vector, this augmented 18-dimensional natural vector makes good use of the available information in the DNA sequence. The accurate classification results we obtained demonstrate that this new 18-dimensional natural vector method, together with the random forest classifier algorthm, can serve as a computationally efficient identification tool for DNA barcodes. We performed phylogenetic analysis on the genus Megacollybia to validate our method. We also studied how effective our method was in determining the genetic distance within and between species in our barcoding dataset.

Conclusions

The classification performs well on the fungi barcode dataset with high and robust accuracy. The reasonable phylogenetic trees we obtained further validate our methods. This method is alignment-free and does not depend on any model assumption, and it will become a powerful tool for classification and evolutionary analysis.

     

GaussianCpG: a Gaussian model for detection of CpG island in human genome sequences

Background

As crucial markers in identifying biological elements and processes in mammalian genomes, CpG islands (CGI) play important roles in DNA methylation, gene regulation, epigenetic inheritance, gene mutation, chromosome inactivation and nuclesome retention. The generally accepted criteria of CGI rely on: (a) %G+C content is ≥ 50%, (b) the ratio of the observed CpG content and the expected CpG content is ≥ 0.6, and (c) the general length of CGI is greater than 200 nucleotides. Most existing computational methods for the prediction of CpG island are programmed on these rules. However, many experimentally verified CpG islands deviate from these artificial criteria. Experiments indicate that in many cases %G+C is < 50%, CpG _obs /CpG _exp varies, and the length of CGI ranges from eight nucleotides to a few thousand of nucleotides. It implies that CGI detection is not just a straightly statistical task and some unrevealed rules probably are hidden.

Results

A novel Gaussian model, GaussianCpG, is developed for detection of CpG islands on human genome. We analyze the energy distribution over genomic primary structure for each CpG site and adopt the parameters from statistics of Human genome. The evaluation results show that the new model can predict CpG islands efficiently by balancing both sensitivity and specificity over known human CGI data sets. Compared with other models, GaussianCpG can achieve better performance in CGI detection.

Conclusions

Our Gaussian model aims to simplify the complex interaction between nucleotides. The model is computed not by the linear statistical method but by the Gaussian energy distribution and accumulation. The parameters of Gaussian function are not arbitrarily designated but deliberately chosen by optimizing the biological statistics. By using the pseudopotential analysis on CpG islands, the novel model is validated on both the real and artificial data sets.

     

Molecular dynamics simulation of the opposite-base preference and interactions in the active site of formamidopyrimidine-DNA glycosylase

Background

Formamidopyrimidine-DNA glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1′ of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg–DNA complexes have been solved, yet no structure with A opposite the lesion is available.

Results

Here we use molecular dynamic simulation to model interactions in the pre-catalytic complex of Lactococcus lactis Fpg with DNA containing oxoG opposite C or A, the latter in either syn or anti conformation. The catalytic dyad, Pro1–Glu2, was modeled in all four possible protonation states. Only one transition was observed in the experimental reaction rate pH dependence plots, and Glu2 kept the same set of interactions regardless of its protonation state, suggesting that it does not limit the reaction rate. The adenine base opposite oxoG was highly distorting for the adjacent nucleotides: in the more stable syn models it formed non-canonical bonds with out-of-register nucleotides in both the damaged and the complementary strand, whereas in the anti models the adenine either formed non-canonical bonds or was expelled into the major groove. The side chains of Arg109 and Phe111 that Fpg inserts into DNA to maintain its kinked conformation tended to withdraw from their positions if A was opposite to the lesion. The region showing the largest differences in the dynamics between oxoG:C and oxoG:A substrates was unexpectedly remote from the active site, located near the linker joining the two domains of Fpg. This region was also highly conserved among 124 analyzed Fpg sequences. Three sites trapping water molecules through multiple bonds were identified on the protein–DNA interface, apparently helping to maintain enzyme-induced DNA distortion and participating in oxoG recognition.

Conclusion

Overall, the discrimination against A opposite to the lesion seems to be due to incorrect DNA distortion around the lesion-containing base pair and, possibly, to gross movement of protein domains connected by the linker.

     

Quantification of microtubule stutters: dynamic instability behaviors that are strongly associated with catastrophe

Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting DI mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA) that identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term “stutters.” During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our in vitro experiments and dimer-scale MT simulations, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anticatastrophe factor CLASP2γ works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared with previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.     

Measuring DNA content in live cells by fluorescence microscopy

Background

Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell’s integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique.

Results

Presented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm’s accurate assessment of DNA content was validated by parallel flow cytometric studies.

Conclusions

This method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.

     

The cell ratchet: Interplay between efficient protrusions and adhesion determines cell motion

Many physiological and pathological processes involve directed cell motion. In general, migrating cells are represented with a polarized morphology with extending and retracting protrusions at the leading edge. However, cell motion is a more complex phenomenon. Cells show heterogeneous morphologies and high protrusive dynamics is not always related to cell shape. This prevents the quantitative prediction of cell motion and the identification of cellular mechanisms setting directionality. Here we discuss the importance of protrusion fluctuations in directed cell motion. We show how their spatiotemporal distribution and dynamics determine the fluctuations and directions of cell motion for NIH3T3 fibroblasts plated on micro-patterned adhesive ratchets.¹ We introduce efficient protrusions and direction index which capture short-term cell motility over hours: these new read-outs allow the prediction of parameters characteristic for the long-term motion of cells over days. The results may have important implications for the study of biological phenomena where directed cell migration is involved, in morphogenesis and in cancer.