The practical and pedagogical advantages of an ambigraphic nucleic acid notation

The universally applied IUPAC notation for nucleic acids was adopted primarily to facilitate the mental association of G, A, T, C, and the related ambiguity characters with the bases they represent. However it is possible to create a notation that offers greater support for the basic manipulations and analyses to which genetic sequences frequently are subjected. By designing a nucleic acid notation around ambigrams, it is possible to simplify the frequently applied process of reverse complementation and aid the visualization of palindromes. The ambigraphic notation presented here also uses common orthographic features such as stems and loops to highlight guanine and cytosine rich regions, support the derivation of ambiguity characters, and aid educators in teaching the fundamentals of molecular genetics.     

Simplicity, function, and legibility in an enhanced ambigraphic nucleic acid notation

We previously showed that an ambigraphic nucleic acid notation, based on symmetrical lowercase Roman characters, permits users to complement DNA by physically rotating the sequence text 180 degrees . This article describes an enhanced ambigraphic notation, which uses concept-related symbol design, rather than the arbitrary set of symbols that constitute the Roman alphabet, to logically encode the four DNA bases and 11 ambiguity characters. As ambigrams, the symbols continue to permit the rapid derivation of complementary sequences and visualization of palindromic DNA. In addition, the new AmbiScript notation uses legibility principles to support the identification of sequence polymorphism and improves writing efficiency by requiring fewer strokes per character than the International Union of Pure and Applied Chemistry (IUPAC) notation.     

PathVisio-Validator: a rule-based validation plugin for graphical pathway notations

PURPOSE: The PathVisio-Validator plugin aims to simplify the task of producing biological pathway diagrams that follow graphical standardized notations, such as Molecular Interaction Maps or the Systems Biology Graphical Notation. This plugin assists in the creation of pathway diagrams by ensuring correct usage of a notation, and thereby reducing ambiguity when diagrams are shared among biologists. Rulesets, needed in the validation process, can be generated for any graphical notation that a developer desires, using either Schematron or Groovy. The plugin also provides support for filtering validation results, validating on a subset of rules, and distinguishing errors and warnings.     

A comprehensive classification of nucleic acid structural families based on strand direction and base pairing.

We propose a classification of DNA structures formed from 1 to 4 strands, based only on relative strand directions, base to strand orientation and base pairing geometries. This classification and its associated notation enable all nucleic acids to be grouped into structural families and bring to light possible structures which have not yet been observed experimentally. It also helps in understanding transitions between families and can assist in the design of multistrand structures.     

Recovery of Viral RNA and Infectious Foot-and-Mouth Disease Virus from Positive Lateral-Flow Devices

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.     

A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome

BACKGROUND: This study addresses the general problem of dividing a density map of a nucleic-acid-protein complex obtained by cryo-electron microscopy (cryo-EM) or X-ray crystallography into its two components. When the resolution of the density map approaches approximately 3 A it is generally possible to interpret its shape (i. e., the envelope obtained for a standard choice of threshold) in terms of molecular structure, and assign protein and nucleic acid elements on the basis of their known sequences. The interpretation of low-resolution maps in terms of proteins and nucleic acid elements of known structure is of increasing importance in the study of large macromolecular complexes, but such analyses are difficult. RESULTS: Here we show that it is possible to separate proteins from nucleic acids in a cryo-EM density map, even at 11.5 A resolution. This is achieved by analysing the (continuous-valued) densities using the difference in scattering density between protein and nucleic acids, the contiguity constraints that the image of any nucleic acid molecule must obey, and the knowledge of the molecular volumes of all proteins. CONCLUSIONS: The new method, when applied to an 11.5 A cryo-EM map of the Escherichia coli 70S ribosome, reproduces boundary assignments between rRNA and proteins made from higher-resolution X-ray maps of the ribosomal subunits with a high degree of accuracy. Plausible predictions for the positions of as yet unassigned proteins and RNA components are also possible. One of the conclusions derived from this separation is that 23S rRNA is solely responsible for the catalysis of peptide bond formation. Application of the separation method to any nucleoprotein complex appears feasible.     

Nanopores and nucleic acids: prospects for ultrarapid sequencing

DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.     

Misleading results of likelihood‐based phylogenetic analyses in the presence of missing data

The amount of missing data in many contemporary phylogenetic analyses has substantially increased relative to previous norms, particularly in supermatrix studies that compile characters from multiple previous analyses. In such cases the missing data are non‐randomly distributed and usually present in all partitions (i.e. groups of characters) sampled. Parametric methods often provide greater resolution and support than parsimony in such cases, yet this may be caused by extrapolation of branch lengths from one partition to another. In this study I use contrived and simulated examples to demonstrate that likelihood, even when applied to simple matrices with little or no homoplasy, homogeneous evolution across groups of characters, perfect model fit, and hundreds or thousands of variable characters, can provide strong support for incorrect topologies when the matrices have non‐random distributions of missing data distributed across all partitions. I do so using a systematic exploration of alternative seven‐taxon tree topologies and distributions of missing data in two partitions to demonstrate that these likelihood‐based artefacts may occur frequently and are not shared by parsimony. I also demonstrate that Bayesian Markov chain Monte Carlo analysis is more robust to these artefacts than is likelihood. © The Willi Hennig Society 2011.     

Phylogenetic analysis of the Hsp70 sequences reveals the monophyly of Metazoa and specific phylogenetic relationships between animals and fungi

To understand the early evolution of the Metazoa, it is necessary to determine the correct phylogenetic status of diploblastic animals. Despite cladistic studies of morphological characters and recent molecular phylogenetic studies, it remains uncertain whether diploblasts are monophyletic or paraphyletic, and how the phyla of diploblasts are phylogenetically related. The heat shock protein 70 (Hsp70) sequences, because of their ubiquity and high degree of conservation, could provide a useful model for phylogenetic analysis. We have sequenced almost the entire nucleic acid sequence of cytoplasmic Hsp70 from eight diploblastic species. Our data support the monophyly of diploblastic animals. However, the phylogenetic relationships of the diploblast groups were not significantly resolved. Our phylogenetic trees also support the monophyly of Metazoa with high bootstrap values, indicating that animals form an extremely robust clade.      

Paleobotany in cladistics and cladistics in paleobotany: enlightenment and uncertainty

Data on fossil taxa can, and should, be incorporated into cladistic analyses. Potential problems with such analyses include large amounts of missing data, and uncertainty about homology of parts that are present. Ambiguity of character data may also occur with extant taxa, but rarely to the extent that it occurs in fossil data. Such ambiguity reduces the strength of the test of character congruence among taxa, in effect relaxing the criterion of parsimony. In order to minimize such effects, composite fossil taxa should be avoided when possible, and polymorphisms reduced by breaking terminals into monomorphic subunits. When results including fossils differ radically from those that exclude fossils, such differences should be approached with caution, keeping in mind the reduced strength of the parsimony analysis when large numbers of cells in a matrix are scored as ambiguous. At this point, there is no simple way to compare the “strength” of parsimony between two data sets that have different numbers of characters and/or taxa in relation to missing data. However, methods under development may provide ways to incorporate the effect of missing values into relative measures of group support such as Bremer support, character removal, and the bootstrap.     

Classification of RNA structures based on hydrogen bond and base-base stacking patterns: application for NMR structures

A computational system, CSNA, for classifying RNA structures according to structural characters was developed. CSNA lists up all the hydrogen bonds and base-base stackings in the structures, and classifies the structures into sub-groups based on their patterns as the first step grouping. The frequency of each hydrogen bond or base-base stacking is calculated, the frequency score being defined as the sum of the frequency of existing hydrogen bonds or base-base stackings for each sub-group. Finally, the sub-groups are further classified into groups based on the frequency score defined in this study and the difference between the patterns. According to the frequency score, CSNA suggests a group that shares most frequently appearing hydrogen bonds and base-base stackings. CSNA was applied to the classification of the results of two individual simulated annealing calculations based on NMR information. It was found that CSNA could extract structures with lower energy without checking any energy term and could provide well converged groups as the lowest energy structures. Thus, CSNA could be a new tool for structural determination of nucleic acids.     

Stereochemistry of nucleic acids and their constituents. IV. Allowed and preferred conformations of nucleosides,nucleoside mono-, di-, tri-, tetraphosphates,nucleic acids and polynucleotides

A uniform notation and convention is suggested to describe the torsional angles in nucleic acids and their derivatives. The torsional angle χ, relating the stereochemistry of the base with respect to the sugar, shows more variation for the β-purine glycosides than for the β-pyrimidine glycosides. This variation is attributed to the fact that the β-purine derivatives may form intramolecular O(5′)-H…N(3) hydrogen bonding. The χ values for the α-purine and α-pyrimidine glycosides show preference for the –syn-clinal (or anti) conformation. The mode of puckering of the sugar also influences the χ value. The various possible conformations for the furanose ring are described by the torsional angles τ₀ τ₁, τ₂, τ₃, τ₄, about the five ring bonds. From an analysis of the torsional angles (ω, ?, ψ, ψ′, ?′, ω′) about the sugar phosphate bonds in the x-ray structures of the known nucleosides, nucleotides, phosphodiesters, nucleic acids, and related compounds, and from a consideration of molecular models, it is found that the possible conformations for the backbone of helical nucleic acids is strikingly limited. Most importantly, the preferred conformation of the nucleotide unit in poly nucleotides and nucleic acids turns out to be the same as that found for the nucleotide in the crystal structure. It is observed that base “stacking” is a consequence of the restricted backbone conformation. The torsional angles are illustrated in the form of conformational “wheels”. Interrelation between the torsion angles about successive pairs of sugar-phosphate bonds are presented in the form of conformational maps: ω,?; ?,ψ; ψ.ψ′; ψ′,?′; ?′,ω′; ω′,ω. The ω′,ω map shows the perferred conformations about the inter-nucleotide bonds of right- and left-handed helices and the possible conformations of phosphodiesters. The preferred conformation of the pyrophosphate and triphosphate is that in which the phosphate oxygens display a staggered arrangement when viewed along the P–P axis. A plausible structure and conformation for the ATPM^2? backbound complex is presented. This structure differs from that proposed by SzentGyorgi in that the metal (only transition metals are considered here) is not bound to the NH₂ nitrogen of adenine, but rather is simultaneously bound to N(7) of the ring and three phosphates (α, β, γ), or N(7) of the ring and two phosphates (β, γ). The remaining metal coordination may be satisfied by solvent–metal or enzyme–metal bonds.     

On the generation of information as motive power for molecular evolution

Molecular evolution can be described as a learning process during which previously inanimate matter developed the ability to organize all the reaction pathways that establish a living system. Common to all natural self-organizing procedures is the ability of matter to store, process and evaluate the information achieved by learning. Genetic information which is stored in RNA or DNA is the object of natural evolution. With the recognition of nature's concepts, evolutionary optimization was applied to biopolymers that are not optimally adapted for particular technical or medical purposes. Information can also be stored in molecules with structures and chemical properties that are completely different from nucleic acids. Therefore, optimization processes that mimic the natural evolutionary strategies can also be applied to small organic molecules. Much effort has been made theoretically and practically to find a certain optimized species within the (hyper)astronomical number of possible sequence alternatives. From a series of computer experiments it can be concluded that it is not necessary to search the entire sequence space in order to find a particular structure; this is advantageous because the diversity of mutant libraries that can realistically be achieved in the laboratory never extends to the number of theoretically possible sequences. Molecular mutant libraries that serve as starting populations for in vitro selection have been constructed for nucleic acids, proteins, peptides and small organic molecules.     

Action of inhibition by fluorodeoxyuridine and its reversal by thymidine on the nucleic acid fractions of the overground parts of photoperiodically inducedChenopodium rubrum plants

The nucleic acid fractions obtained by chromatography on MAK columns were compared in 4 variants ofChenopodium rubrum plants treated in different ways during floral induction. The first variant was normally induced to flowering. The second one was inhibited by application of FUDR to the apical bud on the third day of induction. In the third variant the inhibitory effect of FUDR was reversed by application of THY 24 h after FUDR treatment. In the fourth variant THY was applied 24 h after the termination of induction at a time when it was no longer able to reverse the inhibition of flowering. In plants treated with FUDR, a decrease in DNA and RNA synthesis was observed. After reversal of the inhibitory effect of FUDR by THY, DNA synthesis remained somewhat lower than in the control variant but RNA synthesis reached the same level as in the controls or even surpassed it. In plants to which THY was applied at a time when it was no longer possible to reverse flowering, the results obtained from different experiments were not identical. In some experiments nucleic acid synthesis remained lower than in the variant in which THY restituted flowering. In other experiments nucleic acid synthesis was fully restituted and reached the same level as in the control variant. This phenomenon is explained by the different size of the experimental plants at the beginning of the different experiments. The results are discussed with respect to the relation of the dynamics of nucleic acids to growth and development of the plants.     

Zur formalen Genetik von Isoenzymen,dargestellt am Beispiel der 6-PGD (EC: 1.1.1.44)

Summary The results obtained in all animals examined for 6-PGD so far, indicate that this enzyme is a dimer molecule. In lower vertebrates, dimer molecules consist of identical subunits (=only a single locus). The majority of vertebrates possess 6-PGD isoenzymes composed of non identical subunits; thus, at least two loci are presumed. In homozygotes, three electrophoretic bands are seen when random association occurs. In individuals heterozygous for one locus six different electrophoretic bands are to be expected of which up to six have been identified. Thus, it is suggested that the formal notation should be for homozygous phenotypes: 6-PGD AB; for phenotypes heterozygous for one locus e.g.: 6-PGD AA¹B. A corresponding notation for other isoenzyme systems is possible on the presumption that the quarternary structure and formal genetics is known.     

Some formal consequences of a specific association between double stranded nucleic acid molecules—Properties of multistranded structures

Some possible consequences of a suggested specific association of double stranded nucleic acid to form multistranded, in particular, four stranded structure are discussed in a formal way. A considerable range of isomeric structures and of interactions and transformations between structures seem possible. For example it is possible for one circular nucleic acid chain to exist in several single, double, and quadruple stranded forms, and to form polymers. The suggestion is made that single stranded circles might be involved in some chromosomal material.     

Redescription of the Central American electric fish Gymnotus cylindricus (Ostariophysi: Gymnotiformes: Gymnotidae), with comments on character ambiguity within the ostariophysan clade

Gymnotus cylindricus LaMonte, a poorly studied Central American electric fish primarily observed in the Rio Motagua basin (Guatemala) and north-western Honduras, is redescribed in detail. A clear diagnosis of this species is provided, with new information on overall morphology, osteology, and colour pattern. In a separate section, comments on character ambiguity within the Ostariophysi are made, which led to a reinterpretation of seven homoplastic characters proposed by Fink & Fink (1981) in their work on interrelationships among five major lineages of the superorder. Although the topology of their cladogram remains unchanged, the present view is that alternative explanations regarding evolution of those characters are possible, and the new ones here offered agree with initial hypotheses of primary homologies.     

Molecular interaction maps of bioregulatory networks: a general rubric for systems biology

A standard for bioregulatory network diagrams is urgently needed in the same way that circuit diagrams are needed in electronics. Several graphical notations have been proposed, but none has become standard. We have prepared many detailed bioregulatory network diagrams using the molecular interaction map (MIM) notation, and we now feel confident that it is suitable as a standard. Here, we describe the MIM notation formally and discuss its merits relative to alternative proposals. We show by simple examples how to denote all of the molecular interactions commonly found in bioregulatory networks. There are two forms of MIM diagrams. "Heuristic" MIMs present the repertoire of interactions possible for molecules that are colocalized in time and place. "Explicit" MIMs define particular models (derived from heuristic MIMs) for computer simulation. We show also how pathways or processes can be highlighted on a canonical heuristic MIM. Drawing a MIM diagram, adhering to the rules of notation, imposes a logical discipline that sharpens one's understanding of the structure and function of a network.     

Linguistics of biomolecules and the protein-first hypothesis for the origins of cells

In a protein-first model for cellular origins, genes are intermediates in a pathway of substitution of coded proteins for the geochemical ones. The ambiguity detected in genetic coding suggests that metabolism and coding systems started evolving before the settling of genetic functions. Models are the anucleated erythrocytes, as proteic metabolic systems, then reticulocytes, as nucleoproteic translation systems. Self-organization mechanisms prevailed in early evolution, later adding the genetic (replication, coding) ones. The genecentric (nucleic acid-first) top-down model starts with a poorly functional molecule to build a functional systemde novo. It shortens the span of early self-organization, kept mainly for the generation of the first nucleic acids, starting genetic mechanisms right away. This is also considered less likely due to the difficulty in obtaining nucleotides and nucleic acids under abiotic conditions, where aminoacids and proteins are abundantly produced.     

Application of multivariate resolution methods to the study of biochemical and biophysical processes

Multivariate resolution methods make up a set of mathematical tools that may be applied to the analysis and interpretation of spectroscopic data recorded when monitoring a physical or chemical process with multichannel detectors. The goal of resolution methods is the recovery of chemical and/or physical information from the experimental data. Such data include, for example, the number of intermediates present in a reaction, the rate or equilibrium constants, and the spectra for each one of those intermediates. Multivariate resolution methods have been shown to be useful for the study of biophysical and biochemical processes such as folding/unfolding of proteins or nucleic acids. The present article reviews the most frequently used resolution methods, the limitations on their use, and their latest applications in protein and nucleic acid research.