The All-Data-Based Evolutionary Hypothesis of Ciliated Protists with a Revised Classification of the Phylum Ciliophora (Eukaryota, Alveolata)

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Differentially expressed genes during the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum.

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Evaluating and Correcting Inherent Bias of microRNA Expression in Illumina Sequencing Analysis

International audiencemicroRNA (miRNA) expression profiles based on the highly powerful Illumina sequencing technology rely on the construction of cDNA libraries in which adaptor ligation is known to deeply favor some miRNAs over others. This introduces erroneous measurements of the miRNA abundances and relative miRNA quantities in biological samples. Here, by using the commercial miRXplore Universal Reference that contains an equimolar mixture of 963 animal miRNAs and TruSeq or bulged adaptors, we describe a method for correcting ligation biases in expression profiles obtained with standard protocols of cDNA library construction and provide data for quantifying the true miRNA abundances in biological samples. Ligation biases were evaluated at three ratios of miRNA to 3'-adaptor and four numbers of polymerase chain reaction amplification cycles by calculating efficiency captures/correcting factors for each miRNA. We show that ligation biases lead to over- or under-expression covering a 105 amplitude range. We also show that, at each miRNA:3'-adaptor ratio, coefficients of variation (CVs) of efficiency captures calculated over the four number of amplification cycles using sliding windows of 10 values ranged from 0.1 for the miRNAs of high expression to 0.6 for the miRNAs of low expression. Efficiency captures of miRNAs of high and low expression in profiles are therefore differently impacted by the number of amplification cycles. Importantly, we observed that at a given number of amplification cycles, CVs of efficiency captures calculated over the three miRNA:3'-adaptor ratios displayed a steady value of 0.3 +/- 0.05 STD for miRNAs of high and low expression. This allows, at a given number of amplification cycles, accurate comparison of miRNA expression between biological samples over a substantial expression range. Finally we provide tables of correcting factors that allow to measure the abundances of 963 miRNAs in biological samples from TruSeq-based expression profiles and, an example of their use by characterizing miRNAs of the let-7, miR-26, miR-29, and miR-30 families as the more abundant miRNAs of the rat adult cerebellum

A broad molecular phylogeny of ciliates: identification of major evolutionary trends and radiations within the phylum.

The cellular architecture of ciliates is one of the most complex known within eukaryotes. Detailed systematic schemes have thus been constructed through extensive comparative morphological and ultrastructural analysis of the ciliature and of its internal cytoskeletal derivatives (the infraciliature), as well as of the architecture of the oral apparatus. In recent years, a consensus was reached in which the phylum was divided in eight classes as defined by Lynn and Corliss [Lynn, D. H. & Corliss, J. O. (1991) in Microscopic Anatomy of Invertebrates: Protozoa (Wiley-Liss, New York), Vol. 1, pp. 333-467]. By comparing partial sequences of the large subunit rRNA molecule, and by using both distance-matrix and maximum-parsimony-tree construction methods (checked by boot-strapping), we examine the phylogenetic relationships of 22 species belonging to seven of these eight classes. At low taxonomic levels, the traditional grouping of the species is generally confirmed. At higher taxonomic levels, the branching pattern of these seven classes is resolved in several deeply separated major branches. Surprisingly, the first emerging one contains the heterotrichs and is strongly associated with a karyorelictid but deeply separated from hypotrichs. The litostomes, the oligohymenophorans, and the hypotrichs separate later in a bush-like topology hindering the resolution of their order of diversification. These results show a much more ancient origin of heterotrichs than was classically assumed, indicating that asymmetric, abundantly ciliated oral apparatuses do not correspond to "highly evolved" traits as previously thought. They also suggest the occurrence of a major radiative explosion in the evolutionary history of the ciliates, yielding five of the eight classes of the phylum. These classes appear to differ essentially according to the cytoskeletal architecture used to shape and sustain the cellular cortex (a process of essential adaptative and morphogenetic importance in ciliates)

Proposed Function of the Accumulation of Plasma Membrane-Type Ca(2+)-ATPase mRNA in Resting Cysts of the Ciliate Sterkiella histriomuscorum

From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca(2+)-ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca(2+)-ATPase family identified in Sterkiella. The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii. Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca(2+)-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined

Holistic and Affordable Analyses of MicroRNA Expression Profiles Using Tagged cDNA Libraries and a Multiplex Sequencing Strategy

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