Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown Clades

Inference of evolutionary relationships between nematodes is severely hampered by their conserved morphology, the high frequency of homoplasy, and the scarcity of phylum-wide molecular data. To study the origin of nematode radiation and to unravel the phylogenetic relationships between distantly related species, 339 nearly full-length small-subunit rDNA sequences were analyzed from a diverse range of nematodes. Bayesian inference revealed a backbone comprising 12 consecutive dichotomies that subdivided the phylum Nematoda into 12 clades. The most basal clade is dominated by the subclass Enoplia, and members of the order Triplonchida occupy positions most close to the common ancestor of the nematodes. Crown Clades 8-12, a group formerly indicated as "Secernentea" that includes Caenorhabditis elegans and virtually all major plant and animal parasites, show significantly higher nucleotide substitution rates than the more basal Clades 1-7. Accelerated substitution rates are associated with parasitic lifestyles (Clades 8 and 12) or short generation times (Clades 9-11). The relatively high substitution rates in the distal clades resulted in numerous autapomorphies that allow in most cases DNA barcode-based species identification. Teratocephalus, a genus comprising terrestrial bacterivores, was shown to be most close to the starting point of Secernentean radiation. Notably, fungal feeding nematodes were exclusively found basal to or as sister taxon next to the 3 groups of plant parasitic nematodes, namely, Trichodoridae, Longidoridae, and Tylenchomorpha. The exclusive common presence of fungivorous and plant parasitic nematodes supports a long-standing hypothesis that states that plant parasitic nematodes arose from fungivorous ancestors.     

Frequent appearance of novel protein-coding sequences by frameshift translation

Genomic duplication, followed by divergence, contributes to organismal evolution. Several mechanisms, such as exon shuffling and alternative splicing, are responsible for novel gene functions, but they generate homologous domains and do not usually lead to drastic innovation. Major novelties can potentially be introduced by frameshift mutations and this idea can explain the creation of novel proteins. Here, we employ a strategy using simulated protein sequences and identify 470 human and 108 mouse frameshift events that originate new gene segments. No obvious interspecies overlap was observed, suggesting high rates of acquisition of evolutionary events. This inference is supported by a deficiency of TpA dinucleotides in the protein-coding sequences, which decreases the occurrence of translational termination, even on the complementary strand. Increased usage of the TGA codon as the termination signal in newer genes also supports our inference. This suggests that tolerated frameshift changes are a prevalent mechanism for the rapid emergence of new genes and that protein-coding sequences can be derived from existing or ancestral exons rather than from events that result in noncoding sequences becoming exons.     

Effects of stimulus transformations on estimates of sensory neuron selectivity

Stimulus selectivity of sensory systems is often characterized by analyzing response-conditioned stimulus ensembles. However, in many cases these response-triggered stimulus sets have structure that is more complex than assumed. If not taken into account, when present it will bias the estimates of many simple statistics, and distort the estimated stimulus selectivity of a neural sensory system. We present an approach that mitigates these problems by modeling some of the response-conditioned stimulus structure as being generated by a set of transformations acting on a simple stimulus distribution. This approach corrects the estimates of key statistics and counters biases introduced by the transformations. In cases involving temporal spike jitter or spatial jitter of images, the main observed effects of transformations are blurring of the conditional mean and introduction of artefacts in the spectral decomposition of the conditional covariance matrix. We illustrate this approach by analyzing and correcting a set of model stimuli perturbed by temporal and spatial jitter. We apply the approach to neurophysiological data from the cricket cercal sensory system to correct the effects of temporal jitter. Action Editor: Matthew Wiener     

Industrial applications of hyperthermophilic enzymes: a review

Over the past two decades, research scientists have been involved in the investigation of thermophilic and hyperthermophilic microorganisms owing to the unique features of their enzymic systems. Such in-depth investigations are now on their way to mastering the cloning and industrial exploitation of a broad variety of genes encoding enzymes involved in starch hydrolysis, amino acid biosynthesis, protein hydrolysis, etc. In this work, we review the state of the art and future perspectives of industrial applications of enzymes from hyperthermophilic and extreme thermophilic microorganisms, special attention being paid to the biotechnological methods involved in their industrial exploitation.     

Microtubule binding and clustering of human Tau-4R and Tau-P301L proteins isolated from yeast deficient in orthologues of glycogen synthase kinase-3beta or cdk5

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3beta and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Delta mds1 and Delta pho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.     

Functional evidence for a small and rigid active site in a high fidelity DNA polymerase: probing T7 DNA polymerase with variably sized base pairs

Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 A) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 A beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 A. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity.     

Imaging and automated detection of Sitophilus oryzae (Coleoptera: Curculionidae) pupae in hard red winter wheat

Computed tomography, an imaging technique commonly used for diagnosing internal human health ailments, uses multiple x-rays and sophisticated software to recreate a cross-sectional representation of a subject. The use of this technique to image hard red winter wheat, Triticum aestivm L., samples infested with pupae of Sitophilus oryzae (L.) was investigated. A software program was developed to rapidly recognize and quantify the infested kernels. Samples were imaged in a 7.6-cm (o.d.) plastic tube containing 0, 50, or 100 infested kernels per kg of wheat. Interkernel spaces were filled with corn oil so as to increase the contrast between voids inside kernels and voids among kernels. Automated image processing, using a custom C language software program, was conducted separately on each 100 g portion of the prepared samples. The average detection accuracy in the five infested kernels per 100-g samples was 94.4 +/- 7.3% (mean +/- SD, n = 10), whereas the average detection accuracy in the 10 infested kernels per 100-g sample was 87.3 +/- 7.9% (n = 10). Detection accuracy in the 10 infested kernels per 100-g samples was slightly less than the five infested kernels per 100-g samples because of some infested kernels overlapping with each other or air bubbles in the oil. A mean of 1.2 +/- 0.9 (n = 10) bubbles (per tube) was incorrectly classed as infested kernels in replicates containing no infested kernels. In light of these positive results, future studies should be conducted using additional grains, insect species, and life stages.