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Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications.

Marine macroalgae, especially the Rhodophyta, can be notoriously difficult to identify owing to their relatively simple morphology and anatomy, convergence, rampant phenotypic plasticity, and alternation of heteromorphic generations. It is thus not surprising that algal systematists have come to rely heavily on genetic tools for molecular assisted alpha taxonomy. Unfortunately the number of suitable marker systems in the three available genomes is enormous and, although most workers have settled on one of three or four models, the lack of an accepted standard hinders the comparison of results between laboratories. The advantages of a standard system are obvious for practical purposes of species discovery and identification; as well, compliance with a universal marker, such as cox1 being developed under the label 'DNA barcode', would allow algal systematists to benefit from the rapidly emerging technologies. Novel primers were developed for red algae to PCR amplify and sequence the 5' cox1 'barcode' region and were used to assess three known species-complex questions: (i) Mazzaella species in the Northeast Pacific; (ii) species of the genera Dilsea and Neodilsea in the Northeast Pacific; and (iii) Asteromenia peltata from three oceans. These models were selected because they have all caused confusion with regards to species number, distribution, and identification in the field, and because they have all been studied with molecular tools. In all cases the DNA barcode resolved accurately and unequivocally species identities and, with the enhanced sampling here, turned up a variety of novel observations in need of further taxonomic investigation.

Base Sequence↗

Defining operational taxonomic units using DNA barcode data.

The scale of diversity of life on this planet is a significant challenge for any scientific programme hoping to produce a complete catalogue, whatever means is used. For DNA barcoding studies, this difficulty is compounded by the realization that any chosen barcode sequence is not the gene 'for' speciation and that taxa have evolutionary histories. How are we to disentangle the confounding effects of reticulate population genetic processes? Using the DNA barcode data from meiofaunal surveys, here we discuss the benefits of treating the taxa defined by barcodes without reference to their correspondence to 'species', and suggest that using this non-idealist approach facilitates access to taxon groups that are not accessible to other methods of enumeration and classification. Major issues remain, in particular the methodologies for taxon discrimination in DNA barcode data.

Animals↗

TaxI: a software tool for DNA barcoding using distance methods.

DNA barcoding is a promising approach to the diagnosis of biological diversity in which DNA sequences serve as the primary key for information retrieval. Most existing software for evolutionary analysis of DNA sequences was designed for phylogenetic analyses and, hence, those algorithms do not offer appropriate solutions for the rapid, but precise analyses needed for DNA barcoding, and are also unable to process the often large comparative datasets. We developed a flexible software tool for DNA taxonomy, named TaxI. This program calculates sequence divergences between a query sequence (taxon to be barcoded) and each sequence of a dataset of reference sequences defined by the user. Because the analysis is based on separate pairwise alignments this software is also able to work with sequences characterized by multiple insertions and deletions that are difficult to align in large sequence sets (i.e. thousands of sequences) by multiple alignment algorithms because of computational restrictions. Here, we demonstrate the utility of this approach with two datasets of fish larvae and juveniles from Lake Constance and juvenile land snails under different models of sequence evolution. Sets of ribosomal 16S rRNA sequences, characterized by multiple indels, performed as good as or better than cox1 sequence sets in assigning sequences to species, demonstrating the suitability of rRNA genes for DNA barcoding.

Animals↗

Absolute configuration of the hydroxyfarnesylethyl group of haem A, determined by X-ray structural analysis of bovine heart cytochrome c oxidase using methods applicable at 2.8 Angstrom resolution.

The absolute configuration of haem A, the prosthetic group of cytochrome c oxidase, was determined to be S by analysis of the bond angles surrounding the chiral centre of haem A after refinement with X-PLOR starting from respective initial structures with R and S configurations under absolute configuration constraints at 1.8 Angstrom resolution. The same result was obtained by refinement at 1.8 Angstrom resolution without the absolute configuration constraints. Both of these methods were applicable down to a resolution of about 2.8 Angstrom. The constrained refinement converges more quickly than the unconstrained refinement.

Animals↗

HUMAN MYOPATHY WITH GIANT ABNORMAL MITOCHONDRIA.

The study of children with symmetrical nonprogressive muscle weakness and hypotonia has previously led to the discovery of two new disorders of striated muscle. A third myopathy in a child with hypotonia and proximal weakness is reported. Cytochemical and electron-microscopic studies reveal large, bizarre, interfibrillary and subsarcolemmal mitochondria in muscle obtained by biopsy. Smooth muscle of the vascular wall did not show such abnormalities nor did leucocytes or intramuscular nerves with cytochemical techniques. The basal metabolic rate of the patient was within normal limits.

Acid Phosphatase↗

A functional model related to cytochrome c oxidase and its electrocatalytic four-electron reduction of O2.

A cytochrome c oxidase model that consists of a cobalt(II) porphyrin with a copper(I) triazacyclononane macrocycle fastened on the distal face and an imidazole covalently attached to the proximal face has been synthesized and characterized. Redox titrations with molecular oxygen (O2) and cobaltocene were carried out, and O2 was found to bind irreversibly in a 1:1 ratio to the model compound. This O2 adduct (a bridged peroxide) can be fully reduced to the deoxygenated form with four equivalents of cobaltocene. The model compound was adsorbed on an edge-plane graphite electrode, and rotating ring-disk voltammetry was used to monitor the electrocatalytic reduction of O2. Four-electron reduction of O2 was observed at physiological pH.

Binding Sites↗

ISOLATION AND PROPERTIES OF INTACT MITOCHONDRIA FROM SPHEROPLASTS OF YEAST.

Duell, E. A. (Western Reserve University, Cleveland, Ohio), Sakae Inoue, and Merton F. Utter. Isolation and properties of intact mitochondria from spheroplasts of yeast. J. Bacteriol. 88:1762-1773. 1964.-Functionally intact mitochondria can be obtained in good yields by osmotic disruption of spheroplasts formed from yeast by treatment with an enzyme mixture from the snail digestive tract. The useful range of this method is extended greatly by pretreatment of the yeast cells with 2-mercaptoethylamine and ethylene-diaminetetraacetate. The concentration of the yeast suspension can be increased greatly, the incubation period can be shortened considerably, and the requirement for log-phase cells is obviated. Mitochondria prepared by this method have been compared with those obtained by mechanical disruption in terms of respiratory control, specific activity on a wide range of oxidizable substrates, heterogeneity during centrifugation, and structures observed by electron microscopy. In all cases, the mitochondria obtained from spheroplasts appear to be much less damaged by the preparative procedures.

Centrifugation↗

Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system.

Anomalies both kinetic and equilibrium in nature are described for the inhibition of cytochrome c oxidase activity by sulphide in the isolated enzyme and in submitochondrial particles. These anomalies are related to the involvement of more than 1 mol of sulphide in the blockage of one cytochrome aa3 centre. Sulphide reduces resting cytochrome a3, a reaction that results in oxygen uptake and the loss of a sulphide molecule. Sulphide can also reduce cytochromes c and a; in the former case, a part of the one-equivalent oxidation product, presumed to be the SH radical, reacts with oxygen. Such oxygen uptake is also seen under aerobic conditions when ferricyanide reacts with sulphide. Three phases are identified in the inhibitory interaction of sulphide with the cytochrome c oxidase enzyme itself: an initial rapid reaction involving sulphide oxidation, oxygen uptake, and conversion of cytochrome aa3 into the low-spin "oxyferri" form; a subsequent step in which sulphide reduces cytochrome a; and the final inhibitory step in which a third molecule of sulphide binds the a3 iron centre in the cytochrome a2+ a3 3+ (oxy) species to give cytochrome a2+ a3 3+ H2S. the initial events parallel some of the events in the interaction of the cytochrome c-cytochrome aa3 system with monothiols; the final inhibitory event resembles that with cyanide.

Animals↗

Identification of Birds through DNA Barcodes.

Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10x average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens.

Animals↗

Coupled proton and electron transfer reactions in cytochrome oxidase.

Cytochrome oxidase catalyzes the four-electron reduction of O2 to water and conserves the substantial free energy of the reaction in the form of a protonmotive force. For each electron, two full charges are translocated across the membrane, resulting in a voltage. One of the mechanisms to generate the charge separation in cytochrome oxidase is via a proton pump. A single reaction cycle can be monitored over the course of about 1 msec using absorption spectroscopy, revealing distinct intermediates. Thus, the reaction cycle can be studied as a series of steps. Each of the reaction steps in the catalytic cycle involves a sequence of coupled electron and proton transfer reaction, where protons are either consumed in the chemistry of water formation or pumped across the membrane. The pumping mechanism requires consideration of both the thermodynamics of the various species but also the favored kinetic pathways that assure proton pumping is unidirectional. Hence, a knowledge of transition states and transiently, poorly populated intermediates is likely to be important to understand the mechanism of the pump.

Biological Transport↗