Nucleotide Pairs Research Articles

Complete mitochondrial genome of the speckled dace Rhinichthys osculus, a widely distributed cyprinid minnow of western North America

The speckled dace Rhinichthys osculus (order Cypriniformes), also known as the carpita pinta, is a small cyprinid minnow native to western North America. Here, we report the sequencing of the full mitochondrial genome (mitogenome) of R. osculus from a male fish collected from the Amargosa River Canyon in eastern California, USA. The assembled mitogenome is 16 658 base pair (bp) nucleotides, and encodes 13 protein-coding genes, and includes both a 12S and a 16S rRNA, 22 tRNAs, and a 985 bp D-loop control region. Mitogenome synteny reflects that of other Ostariophysian fishes with the majority of genes and RNAs encoded on the heavy strand (H-strand) except nd6, tRNA-Gln, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser, tRNA-Glu, and tRNA-Pro. The availability of this R. osculus mitochondrial genome – the first complete mitogenome within the lineage of Rhinichthys riffle daces – provides a foundation for resolving evolutionary relationships among morphologically differentiated populations of R. osculus.

Diversity of Endonuclease V: From DNA Repair to RNA Editing.

Deamination of adenine occurs in DNA, RNA, and their precursors via a hydrolytic reaction and a nitrosative reaction. The generated deaminated products are potentially mutagenic because of their structural similarity to natural bases, which in turn leads to erroneous nucleotide pairing and subsequent disruption of cellular metabolism. Incorporation of deaminated precursors into the nucleic acid strand occurs during nucleotide synthesis by DNA and RNA polymerases or base modification by DNA- and/or RNA-editing enzymes during cellular functions. In such cases, removal of deaminated products from DNA and RNA by a nuclease might be required depending on the cellular function. One such enzyme, endonuclease V, recognizes deoxyinosine and cleaves 3' end of the damaged base in double-stranded DNA through an alternative excision repair mechanism in Escherichia coli, whereas in Homo sapiens, it recognizes and cleaves inosine in single-stranded RNA. However, to explore the role of endonuclease V in vivo, a detailed analysis of cell biology is required. Based on recent reports and developments on endonuclease V, we discuss the potential functions of endonuclease V in DNA repair and RNA metabolism.

The unusual and dynamic character of PX-DNA.

PX-DNA is a four-stranded DNA structure that has been implicated in the recognition of homology, either continuously, or in an every-other-half-turn fashion. Some of the structural features of the molecule have been noted previously, but the structure requires further characterization. Here, we report atomic force microscopic characterization of PX molecules that contain periodically placed biotin groups, enabling the molecule to be labeled by streptavidin molecules at these sites. In comparison with conventional double stranded DNA and with antiparallel DNA double crossover molecules, it is clear that PX-DNA is a more dynamic structure. Furthermore, the spacing between the nucleotide pairs along the helix axis is shorter, suggesting a mixed B/A structure. Circular dichroism spectroscopy indicates unusual features in the PX molecule that are absent in both the molecules to which it is compared.

A family of DNA aptamers with varied duplex region length that forms complexes with thrombin and prothrombin

Structural properties determine binding affinities of DNA aptamers specific to thrombin. Our paper is the first to focus on a family of eight G-quadruplex-based aptamers with varied duplex region length (from two to eight base pairs). We have shown that the duplex, which is not the main binding domain, greatly influences the interaction with thrombin and prothrombin. Furthermore, the affinity of an aptamer to thrombin and prothrombin increases (respectively from 2.7×10−8M to 5.6×10−10M and from 1.8×10−5M to 7.1×10−9M) with an increase in the number of nucleotide pairs in the duplex region.

Resonant Infrared Multiple Photon Dissociation Spectroscopy of Anionic Nucleotide Monophosphate Clusters

We report mid-infrared spectra and potential energy surfaces of four anionic, 2'-deoxynucleotide-5'-monophosphates (dNMPs) and the ionic DNA pairs [dGMP-dCMP-H](1-), [dAMP-dTMP-H](1-) with a total charge of the complex equal to -1. We recorded IR action spectra by resonant IR multiple-photon dissociation (IRMPD) using the FELIX free electron laser. The potential energy surface study employed an on-the-fly molecular dynamics quenching method (MD/Q), using a semiempirical AM1 method, followed by an optimization of the most stable structures using density functional theory. By employing infrared multiple-photon dissociation (IRMPD) spectroscopy in combination with high-level computational methods, we aim at a better understanding of the hydrogen bonding competition between the phosphate moieties and the nucleobases. We find that, unlike in multimer double stranded DNA structures, the hydrogen bonds in these isolated nucleotide pairs are predominantly formed between the phosphate groups. This intermolecular interaction appears to exceed the stabilization energy resulting from base pairing and directs the overall cluster structure and alignment.

The development of the PCR test system for identification of RNA-2 of BNYVV for environmental assessment of phytosanitary state

The aim of research was to develop diagnostic system of BNYVV based on polymerase chain reaction for the identification of protein P75 of RNA-2. The bioinformative analysis of the nucleotide sequences of RNA-2 virus isolates of beet necrotic yellow vein (BNYVV) was made. The conserved sequences of a gene that encodes a protein 75 have been shown. Primers design for identification of RNA-2 has been made. It was synthesized pair primers: Forward 5’ – CTTTGGCAGGATTAGGCTCG-3’, Reverse 5’ – CACTCGGGACTATCACCAGG-3’ with amplification product fragments of size – 490 nucleotides pairs. The developed system was tested and confirmed the effectiveness of its use in Ukrainian model cDNA of BNYVV isolate. The annealing temperature of primer was optimized and we have found that a more optimal temperature for identification of BNYVV is in the range of 50–62°C. In further studies we plan to determine the nucleotide sequences of RNA-2 of Р75 protein and establish phylogenetic relationship to world′s isolates.

Genome sequencing and analysis of an Escherichia coli phage vB_EcoM-ep3 with a novel lysin, Lysep3.

Bacteriophages represent one prospect for preventing and treating multi-drug-resistant Escherichia coli. In this study, we have isolated a novel E. coli-specific bacteriophage and characterised its biological properties. vB_EcoM-ep3 has a broad host range and was able to lyse 9 out of 15 clinical isolates of multi-drug-resistant pathogenic E. coli from chickens. The optimal multiplicity of infection for vB_EcoM-ep3 in host bacteria was 0.01. vB_EcoM-ep3 was thermostable at temperatures below 50°C for up to 60min. Electron microscopy demonstrated that vB_EcoM-ep3 belongs to Myoviridae. The vB_EcoM-ep3 genome contained 42,351 pairs of nucleotides with a GC content of 53.35%. There were 52 predicted open reading frames that appeared to overlap and have a modular structure. Phylogenetic analysis indicates that the closest evolutionary relative to vB_EcoM-ep3 is the previously reported E. coli phage vB_EcoM_ECO1230-10. However, there was no homology between reported E. coli phage lysins and the vB_EcoM-ep3 lysin gene. Lysep3 was 58% similar to the Pseudomonas phage PPpW-3 lysin despite showing no similarities at the gene sequence level. And Lysep3 has good lysis activity.

Sex identification of the rainbow trout Oncorhynchus mykiss by polymerase chain reaction

A polymerase chain reaction-based test for rapid sex identification of the rainbow trout Oncorhynchus mykiss has been elaborated. Using the data deposited with the NCBI genome bank, nucleotide sequences of the specific sex-linked locus of salmonid species have been analyzed and the oligonucleotide primers have been selected. The length of the amplification products is 800 nucleotide pairs. Amplification specificity has been tested by nucleotide analysis of the amplicon sequences. All PCR products match the Y chromosome region housing the corresponding specific locus. A comparative analysis of the nucleotide sequences of the fragments carrying the rainbow trout (and other salmonid species) sex-linked marker demonstrates a high degree of identity, amounting to 95-99%. The maximal identity between the sequences (99%) is observed when analyzing representatives of the same genus, for example, Oncorhynchus. Thus, the sex is detectable by a simple PCR test, thereby allowing the male revertants emerging via hormonal sex reversion to be identified. This test is a rapid diagnostic method, since it requires only 1 day for testing and informing the fish farm on its results. Results of abdomen opening of rainbow trout revertants and examination of their hormonal status are also described.

Performance of an in-house human immunodeficiency virus type 1 genotyping system for assessment of drug resistance in Cuba.

As commercial human immunodeficiency virus type 1 drug resistance assays are expensive, they are not commonly used in resource-limited settings. Hence, a more affordable in-house procedure was set up taking into account the specific epidemiological and economic circumstances of Cuba. The performance characteristics of the in-house assay were evaluated using clinical samples with various subtypes and resistance patterns. The lower limit of amplification was determined on dilutions series of 20 clinical isolates and ranged from 84 to 529 RNA copies/mL. For the assessment of trueness, 14 clinical samples were analyzed and the ViroSeq HIV-1 Genotyping System v2.0 was used as the reference standard. The mean nucleotide sequence identity between the two assays was 98.7% ± 1.0. Additionally, 99.0% of the amino acids at drug resistance positions were identical. The sensitivity and specificity in detecting drug resistance mutations was respectively 94.1% and 99.5%. Only few discordances in drug resistance interpretation patterns were observed. The repeatability and reproducibility were evaluated using 10 clinical samples with 3 replicates per sample. The in-house test was very precise as nucleotide sequence identity among paired nucleotide sequences ranged from 98.7% to 99.9%. The acceptance criteria were met by the in-house test for all performance characteristics, demonstrating a high degree of accuracy. Subsequently, the applicability in routine clinical practice was evaluated on 380 plasma samples. The amplification success rate was 91% and good quality consensus sequences encoding the entire protease and the first 335 codons in reverse transcriptase could be obtained for 99% of the successful amplicons. The reagent cost per sample using the in-house procedure was around € 80 per genotyping attempt. Overall, the in-house assay provided good results, was feasible with equipment and reagents available in Cuba and was half as expensive as commercial assays.

DNA barcoding of sunn pest adult parasitoids using cytochrome c oxidase subunit I (COI)

In this study, DNA barcoding was used in the identification of potential biological control agents of sunn pest adult parasitoid species, including Eliozeta helluo (F.), Phasia subcoleoptrata (L.), Ectophasia crassipennis (F.) and Elomyia lateralis (Meig). DNA analyses were assessed by sequencing cytochrome c oxidase subunit I (COI) gene. The obtained sequences were analyzed in terms of nucleotide composition, nucleotide pair frequency and haplotype diversity. Genetic divergence among haplotypes was estimated by constructing genetic distance matrix using DNA sequence variations, by Kimura 2-parameter model. Variable sites and average variations of the sequenced 603 base pair long DNA fragment were calculated. All COI barcodes were matched with reference sequences of expected species according to morphological identification. Neighbor-joining tree was drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. The evolutionary history inferred using the UPGMA method indicated two distinct mitochondrial haplotype lineages. The genetic variation between sunn pest adult parasitoids will be useful in sunn pest management, regulatory and environmental applications.

A Nucleobase-Centric Coarse-Grained Model for Structure Prediction of RNA Fragments

The structural characterization of RNA usually poses additional challenges when compared to other biomolecular systems. For example, there is a relatively scarce amount of structural data available, which demands the development of three-dimensional structure prediction tools. In addition, full-resolution simulations can be a hard task not only because of the complexity of the interactions involved, but also due to the limitations of the current force fields. At this point, coarse-grained simulations are a good candidate to fill the gaps in this growing research field. The use of these techniques has rapidly increased in the past decades in the study of biological systems on which the experiments require an additional interpretation or where an atomistic computational approach results difficult or unfeasible. Nevertheless, the development of coarse-grained models involves the understanding of the main structural features of the original system, which can represent a challenge by itself.In this work, we present a knowledge-base coarse-grained model for RNA structure prediction, representing each nucleotide by a single anisotropic particle. The mapping and the main interactions are designed to reproduce the geometrical distribution of the closest pairs of nucleotides, extracted from a set of ribosomal structures. The model is inspired in the ESCORE function [1], a knowledge-based scoring function that has been shown to perform better compared to fully atomistic techniques in identifying native-like structures from a set of decoys. Its minimalistic nature and successful application on a broad range of structures straightforwardly suggest a representation for a coarse-grained approach. We show the preliminary results of our simulations and discuss the role of pair interactions in the prediction of RNA structures.[1] S. Bottaro, F. Di Palma and G. Bussi, “The Role of Nucleobase Interactions in RNA Structure and Dynamics”, Nucleic Acids Res., accepted.

Loss of Wild-Type Kras is Associated with Enhanced Fitness and Increased Mitogen-Activated Protein Kinase Pathway Addiction in K-RasG12D Acute Myeloid Leukemia

Functional analysis of therapeutic outlier responses in cancer therapy can identify unexpected synthetic lethal interactions and uncover biomarkers that predict enhanced sensitivity to targeted agents. We performed preclinical trials of the MEK inhibitor PD0325901 (PD901) in primary murine acute myeloid leukemias (AMLs) generated using retroviral insertional mutagenesis in KrasG12D “knock-in” mice. Similar to previous observations Nf1 and Nras mutant leukemias, treatment with PD901 prolonged survival in six independent leukemias harboring diverse retroviral integrations. Intriguingly, one outlier leukemia, clone Kras.101, demonstrated a greater than 4-fold improvement in median overall survival when treated with the MEK inhibitor. Importantly, this AML demonstrated a novel retroviral integration at relapse, indicating clonal evolution during treatment. The relapsed clone displayed phenotypic resistance upon transplantation into secondary recipients and retreatment with PD901. We performed a detailed analysis of the paired PD901-sensitive/resistant Kras.101 clones to identify the mediators of mitogen-activated protein kinase pathway (MAPK) addiction. Functional studies demonstrated that the novel dominant retroviral integration in the PD901 resistant clone did not directly alter drug response. However, genome-wide analysis of copy number alterations revealed a gain of chromosome 6 involving the Kras locus in the resistant clone, which was confirmed by spectral karyotyping and fluorescence in situ hybridization. Surprisingly, the PD901-sensitive leukemia harbored two copies of oncogenic KrasG12D due to somatic uniparental disomy, while the resistant clone harbored two mutant and one wild-type Kras alleles. This observation and paired single nucleotide polymorphism analysis indicated that the PD901-resistant leukemia was evolutionarily ancestral to the drug sensitive clone. Consistent with this hypothesis, competitive repopulation experiments with fluorescently labeled leukemia cells demonstrated increased fitness of the drug sensitive leukemia in the absence of the MEK inhibitor and over-expression of wild type K-Ras in the drug-sensitive leukemia reduced fitness in vivo. Finally, utilizing a tetracycline-inducible system, we found that wild-type Kras expression enhances the fitness of the Kras.101 clone only in the presence of the MEK inhibitor. Together, these data suggest that loss of wild-type Kras imparts a fitness advantage at the expense of increased MAPK pathway addiction, which results in enhanced sensitivity to MEK inhibition in leukemias driven by oncogenic K-Ras. DisclosuresNo relevant conflicts of interest to declare.

Graph-distance distribution of the Boltzmann ensemble of RNA secondary structures.

BackgroundLarge RNA molecules are often composed of multiple functional domains whose spatial arrangement strongly influences their function. Pre-mRNA splicing, for instance, relies on the spatial proximity of the splice junctions that can be separated by very long introns. Similar effects appear in the processing of RNA virus genomes. Albeit a crude measure, the distribution of spatial distances in thermodynamic equilibrium harbors useful information on the shape of the molecule that in turn can give insights into the interplay of its functional domains.ResultSpatial distance can be approximated by the graph-distance in RNA secondary structure. We show here that the equilibrium distribution of graph-distances between a fixed pair of nucleotides can be computed in polynomial time by means of dynamic programming. While a naïve implementation would yield recursions with a very high time complexity of O(n6D5) for sequence length n and D distinct distance values, it is possible to reduce this to O(n4) for practical applications in which predominantly small distances are of of interest. Further reductions, however, seem to be difficult. Therefore, we introduced sampling approaches that are much easier to implement. They are also theoretically favorable for several real-life applications, in particular since these primarily concern long-range interactions in very large RNA molecules.ConclusionsThe graph-distance distribution can be computed using a dynamic programming approach. Although a crude approximation of reality, our initial results indicate that the graph-distance can be related to the smFRET data. The additional file and the software of our paper are available from http://www.rna.uni-jena.de/RNAgraphdist.html.

Nucleic acid-based differential diagnostic assays for feline coronavirus

Feline coronavirus (FCoV) is a pleomorphic, enveloped, positive-sense single-stranded RNA virus. Owing to the differences in its genotype, FCoV belongs to a separate clade along with other viruses, such as transmissible gastroenteritis virus (TGEV) and canine coronavirus (CCoV), which can be isolated from cats. In this study, a PCR assay was developed to differentiate these coronaviruses concurrently. Multiplex differential RT-PCR was performed with primers based on the highly conserved coronavirus membrane protein. Three primer sets were designed: a primer pair (S1 and S2) that can bind to conserved sequences in all target coronaviruses, a CCoV-specific primer (S3), and a TGEV-specific primer (S4). Because of the high sequence homology among FCoV, CCoV, and TGEV, a nucleotide preceding the last pair of dissimilar nucleotides in S3 and S4 was substituted with an inosine to allow primer binding. This assay could detect and differentiate FCoV (n=7), CCoV (n=4), and TGEV (n=8) precisely and did not show any cross-reactivity with other pathogens. These results suggest that this molecular approach provides a rapid and reliable way to detect FCoV, especially in feline clinical specimens.

RNA structural constraints in the evolution of the influenza A virus genome NP segment

Conserved RNA secondary structures were predicted in the nucleoprotein (NP) segment of the influenza A virus genome using comparative sequence and structure analysis. A number of structural elements exhibiting nucleotide covariations were identified over the whole segment length, including protein-coding regions. Calculations of mutual information values at the paired nucleotide positions demonstrate that these structures impose considerable constraints on the virus genome evolution. Functional importance of a pseudoknot structure, predicted in the NP packaging signal region, was confirmed by plaque assays of the mutant viruses with disrupted structure and those with restored folding using compensatory substitutions. Possible functions of the conserved RNA folding patterns in the influenza A virus genome are discussed.

Influence of the sequence on the ab initio band structures of single and double stranded DNA models

Abstract The solid state physical approach is widely used for the characterization of electronic properties of DNA. In the simplest case the helical symmetry is explicitly utilized with a repeat unit containing only a single nucleotide or nucleotide pair. This model provides a band structure that is easily interpretable and reflects the main characteristic features of the single nucleotide or a nucleotide pair chain, respectively. The chemical variability of the different DNA chains is, however, almost completely neglected in this way. In the present work we have investigated the effect of the different sequences on the band structure of periodic DNA models. For this purpose we have applied the Hartree–Fock crystal orbital method for single and double stranded DNA chains with two different subsequent nucleotides in the repeat unit of former and two different nucleotide pairs in the latter case, respectively. These results are compared to simple helical models with uniform sequences. The valence and conduction bands related to the stacked nucleotide bases of single stranded DNA built up only from guanidine as well as of double stranded DNA built up only from guanidine–cytidine pairs showed special properties different from the other cases. Namely, they had higher conduction and lower valence band positions and this way larger band gaps and smaller widths of these bands. With the introduction of non-uniform guanidine containing sequences band structures became more similar to each other and to the band structures of other sequences without guanidine. The maximal bandwidths of the non-uniform sequences are considerably smaller than in the case of uniform sequences implying smaller charge carrier mobilities both in the conduction and valence bands.

Diversity and molecular phylogeny of mitochondrial DNA of rhesus macaques (Macaca mulatta) in Bangladesh.

While studies of rhesus macaques (Macaca mulatta) in the eastern (e.g., China) and western (e.g., India) parts of their geographic range have revealed major genetic differences that warrant the recognition of two different subspecies, little is known about genetic characteristics of rhesus macaques in the transitional zone extending from eastern India and Bangladesh through the northern part of Indo-China, the probable original homeland of the species. We analyzed genetic variation of 762 base pairs of mitochondrial DNA from 86 fecal swab samples and 19 blood samples from 25 local populations of rhesus macaque in Bangladesh collected from January 2010 to August 2012. These sequences were compared with those of rhesus macaques from India, China, and Myanmar. Forty-six haplotypes defined by 200 (26%) polymorphic nucleotide sites were detected. Estimates of gene diversity, expected heterozygosity, and nucleotide diversity for the total population were 0.9599 ± 0.0097, 0.0193 ± 0.0582, and 0.0196 ± 0.0098, respectively. A mismatch distribution of paired nucleotide differences yielded a statistically significantly negative value of Tajima's D, reflecting a population that rapidly expanded after the terminal Pleistocene. Most haplotypes throughout regions of Bangladesh, including an isolated region in the southwestern area (Sundarbans), clustered with haplotypes assigned to the minor haplogroup Ind-2 from India reflecting an east to west dispersal of rhesus macaques to India. Haplotypes from the southeast region of Bangladesh formed a cluster with those from Myanmar, and represent the oldest rhesus macaque haplotypes of Bangladesh. These results are consistent with the hypothesis that rhesus macaques first entered Bangladesh from the southeast, probably from Indo-China, then dispersed westward throughout eastern and central India.

A new type of localized fast moving electronic excitations in molecular chains

It is shown that in a Holstein molecular chain placed in a strong longitudinal electric field some new types of excitations can arise. These excitations can transfer a charge over large distance (more than 1000 nucleotide pairs) along the chain retaining approximately their shapes. Excitations are formed only when a strong electric field either exists or quickly arises under especially preassigned conditions. These excitations transfer a charge even in the case when Holstein polarons are practically immobile. The results obtained are applied to synthetic homogeneous PolyG/PolyC DNA duplexes. They can also be provide the basis for explanation of famous H.W. Fink and C. Schönenberger experiment on long-range charge transfer in DNA.

Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins, Ago and TRBP, in mammalian cells

Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5′ proximal nucleotides, which include seed sequence (positions 2–8 from the 5′ end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable with cognate deoxyribonucleotides without reducing RNAi activity. However, RNA sequences at positions 9-12 and 15-18 were essential for effective gene silencing, and these two double-stranded RNA cores are required for binding of the trans-activation response RNA-binding protein (TRBP). The terminal RNA (positions 19–21) provided Argonaute protein binding sites. Argonaute binding was enhanced by the presence of RNAs at positions 15–18. Knockdown experiments showed that, unlike Argonaute and TRBP, Dicer was dispensable for RNAi. Based on these observations, we discuss possible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.

Mechanisms of cellular proliferation stimulation under the effect of L-amino acids in the tissue culture of young and old rats

The statistical method of rank correlation was used to analyze the correlation of individual physicochemical characteristics of L-amino acids, as well as of dipole moments of the first pair of nucleotides of their codons, to the biological activity of amino acids, i.e. the stimulation of cellular proliferation. The experimental material was obtained from the organotypic tissues of ecto- and mesodermal origin in young and old rats. It was demonstrated that each of the stimulating amino acids acts 1.5–6.0 times less often in explants from old rats as compared to the explants from young rats. A weak correlation was detected between the stimulation of cellular proliferation and physicochemical characteristics (hydrophobicity, dipole moment and isoelectric point of the amino acids) in many tissues, but high and moderate correlation was detected in the brain subcortical explants, cerebellum and skin. However, a moderately close correlation was detected between the dipole moment of the first pair of nucleotides of their codons and the stimulating activity for explants of the brain subcortical structures (young and old rats), cerebellum and skin (young rats), and Spearman’s correlation coefficient (r s = 0.52; 0.45; 0.60; and 0.71, respectively). A moderate correlation was also detected for explants of the testis and cartilage. It can be suggested that some physicochemical and biochemical characteristics of amino acids correlate to their biological activity, which is especially remarkable in tissues derived from ectoderm.