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See detailOrigin and evolution of metal P-type ATPases in Plantae (Archaeplastida)
Hanikenne, Marc ULg; Baurain, Denis ULg

in Frontiers in Plant Science (2014), 4

Metal ATPases are a subfamily of P-type ATPases involved in the transport of metal cations across biological membranes. They all share an architecture featuring eight transmembrane domains in pairs of two ... [more ▼]

Metal ATPases are a subfamily of P-type ATPases involved in the transport of metal cations across biological membranes. They all share an architecture featuring eight transmembrane domains in pairs of two and are found in prokaryotes as well as in a variety of Eukaryotes. In Arabidopsis thaliana, eight metal P-type ATPases have been described, four being specific to copper transport and four displaying a broader metal specificity, including zinc, cadmium and possibly copper and calcium. So far, few efforts have been devoted to elucidating the origin and evolution of these proteins in Eukaryotes. In this work, we use large-scale phylogenetics to show that metal P-type ATPases form a homogenous group among P-type ATPases and that their specialisation into either monovalent (Cu) or divalent (Zn, Cd…) metal transport stems from a gene duplication that took place early in the evolution of Life. Then, we demonstrate that the four subgroups of plant metal ATPases all have a different evolutionary origin and a specific taxonomic distribution, only one tracing back to the cyanobacterial progenitor of the chloroplast. Finally, we examine the subsequent evolution of these proteins in green plants and conclude that the genes thoroughly characterised in model organisms are often the result of lineage-specific gene duplications, which calls for caution when attempting to infer function from sequence similarity alone in non-model organisms. [less ▲]

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See detailChromera velia, Endosymbioses and the Rhodoplex Hypothesis - Plastid Evolution in Cryptophytes, Alveolates, Stramenopiles and Haptophytes (CASH Lineages)
Petersen, Jörn; Ludewig, Ann-Kathrin; Michael, Victoria et al

in Genome Biology and Evolution (2014)

The discovery of Chromera velia, a free-living photosynthetic relative of apicomplexan pathogens, has provided an unexpected opportunity to study the algal ancestry of malaria parasites. In this work we ... [more ▼]

The discovery of Chromera velia, a free-living photosynthetic relative of apicomplexan pathogens, has provided an unexpected opportunity to study the algal ancestry of malaria parasites. In this work we compared the molecular footprints of a eukaryote-to-eukaryote endosymbiosis in C. velia to their equivalents in peridinin-containing dinoflagellates (PCD) to re- evaluate recent claims in favor of a common ancestry of their plastids. To this end, we established the draft genome and a set of full-length cDNA sequences from C. velia via next- generation sequencing. We documented the presence of a single coxI gene in the mitochondrial genome, which thus represents the genetically most reduced aerobic organelle identified so far, but focused our analyses on five “lucky genes” of the Calvin cycle. These were selected because of their known support for a common origin of complex plastids from cryptophytes, alveolates (represented by PCDs), stramenopiles and haptophytes (CASH) via a single secondary endosymbiosis with a red alga. As expected, our broadly sampled phylogenies of the nuclear- encoded Calvin cycle markers support a rhodophycean origin for the complex plastid of Chromera. However, they also suggest an independent origin of apicomplexan and dinophycean (PCD) plastids via two eukaryote-to-eukaryote endosymbioses. Although at odds with the current view of a common photosynthetic ancestry for alveolates, this conclusion is nonetheless in line with the deviant plastome architecture in dinoflagellates and the morphological paradox of four versus three plastid membranes in the respective lineages. Further support for independent endosymbioses is provided by analysis of five additional markers, four of them involved in the plastid protein import machinery. Finally, we introduce the “rhodoplex hypothesis” as a convenient way to designate evolutionary scenarios where CASH plastids are ultimately the product of a single secondary endosymbiosis with a red alga, but were subsequently horizontally spread via higher-order eukaryote-to-eukaryote endosymbioses. [less ▲]

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See detailThe mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae.
Perez, Emilie ULg; Lapaille, Marie; Degand, Herve et al

in Mitochondrion (2014)

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential ... [more ▼]

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologs to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native- and SDS-PAGE to both confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa. [less ▲]

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See detailIdentifying losses and expansions of selected gene families in incomplete genomic datasets
Di Franco, Arnaud ULg; Hanikenne, Marc ULg; Baurain, Denis ULg

Poster (2013, December 09)

Plantae (Archaeplastida) are a natural group of organisms with plastids of primary endosymbiotic origin. Within this group, members of the red algae show evidence of a reduction of their genomic content ... [more ▼]

Plantae (Archaeplastida) are a natural group of organisms with plastids of primary endosymbiotic origin. Within this group, members of the red algae show evidence of a reduction of their genomic content. In this work, we designed a bioinformatics approach to investigate the few, sometimes incomplete, genomic datasets available for red algae, with the purpose of pointing out possible gene family losses and expansions. Our pipeline first populates a relational database with precomputed orthology relationships between green plant genomes and red algal datasets and then efficiently queries the database for computing statistics of losses and expansions for a series of gene families of interest. [less ▲]

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See detailAssessment of bioinformatic pipelines for the analysis of 454 pyrosequencing data using artificial cyanobacterial communities
Stelmach Pessi, Igor ULg; De Carvalho Maalouf, Pedro; Laughinghouse IV, Haywood ULg et al

Poster (2013, November 27)

The field of microbial ecology has undergone a revolution with the advent of next-generation sequencing (NGS) technologies, which revealed a higher microbial diversity than what was previously observed ... [more ▼]

The field of microbial ecology has undergone a revolution with the advent of next-generation sequencing (NGS) technologies, which revealed a higher microbial diversity than what was previously observed. The possibility of analyzing tens to hundreds of thousands sequences in a single sequencing run has provided information on rare taxa that could constitute an important fraction of microbial communities. However, this comes with the cost of relatively high error rates for individual reads, which can lead to overestimation of diversity due to the generation of spurious Operational Taxonomic Units (OTUs) consisting of erroneous sequences. Therefore, a correct assessment of microbial diversity using NGS relies on robust bioinformatic tools in order to correct for PCR and sequencing biases. Here we report on community structures recovered for two artificial cyanobacterial communities applying three commonly used and two recently published bioinformatic pipelines. This pilot study was made in the frame of the BELSPO project CCAMBIO. Artificial communities were constructed using DNA isolated from 22 cyanobacterial strains from the BCCM/ULC Polar Cyanobacteria Collection (http://bccm.belspo.be/about/ulc.php). DNA was extracted from individual cultures and pooled at equal (community Art1) or tiered (community Art2) concentrations. The V3-V4 hypervariable region of the 16S rRNA gene was amplified by PCR using primers 359F and 781Ra/781Rb [Nübel et al (1997) Appl Environ Microbiol 63: 3327-3332], and amplicons were sequenced on a 454 GS FLX Titanium platform. Data was demultiplexed and submitted to five bioinformatic pipelines for quality control of reads, removal of chimeric sequences and Operational Taxonomic Units (OTUs) clustering: (I) “shhh.flows (450 flows)”, (II) “shhh.flows (360-720 flows)” and (III) “Sliding Window (Q35, 50 bp)”, using MOTHUR according to Schloss et al (2011) [Schloss et al (2011) PLOS One 6: e27310]; (IV) “fastq_maxee” and (V) “fastq_truncqual”, using UPARSE according to Edgar (2013) [Edgar (2013) Nat Methods 10: 996-998]. Average sequence length varied considerably among pipelines, with pipeline I generating shorter reads (220 bp in average) and pipelines IV and V longer reads (370 bp). The number of OTUs obtained in each pipeline also varied significantly. While 22 and 21 OTUs were obtained for pipelines IV and V, respectively, pipelines I-III generated a surprisingly high number of OTUs (199, 317 and 289, respectively). Despite differences in the number of OTUs, the relative abundance of each reference strain did not differ significantly between pipelines. Overall, community structures observed using the UPARSE protocol (pipelines IV and V) were the most consistent with the expected results. These findings show the importance of assessing the performance of different bioinformatic pipelines using artificial communities, in order to reduce the effects of PCR and sequencing errors, which can lead to distorted community structures estimates. [less ▲]

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See detailComputers As Genomic Time Machines for Meeting our Ancestors
Baurain, Denis ULg

Scientific conference (2013, April 18)

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See detailImpact of Missing Data on Phylogenies Inferred from Empirical Phylogenomic Data Sets
Roure, Béatrice; Baurain, Denis ULg; Philippe, Hervé

in Molecular Biology and Evolution (2013), 30(1), 197-214

Progress in sequencing technology allows researchers to assemble ever-larger supermatrices for phylogenomic inference. However, current phylogenomic studies often rest on patchy data sets, with some ... [more ▼]

Progress in sequencing technology allows researchers to assemble ever-larger supermatrices for phylogenomic inference. However, current phylogenomic studies often rest on patchy data sets, with some having 80% missing (or ambiguous) data or more. Though early simulations had suggested that missing data per se do not harm phylogenetic inference when using sufficiently large data sets, Lemmon et al. (Lemmon AR, Brown JM, Stanger-Hall K, Lemmon EM. 2009. The effect of ambiguous data on phylogenetic estimates obtained by maximum likelihood and Bayesian inference. Syst Biol. 58:130-145.) have recently cast doubt on this consensus in a study based on the introduction of parsimony-uninformative incomplete characters. In this work, we empirically reassess the issue of missing data in phylogenomics while exploring possible interactions with the model of sequence evolution. First, we note that parsimony-uninformative incomplete characters are actually informative in a probabilistic framework. A reanalysis of Lemmon's data set with this in mind gives a very different interpretation of their results and shows that some of their conclusions may be unfounded. Second, we investigate the effect of the progressive introduction of missing data in a complete supermatrix (126 genes × 39 species) capable of resolving animal relationships. These analyses demonstrate that missing data perturb phylogenetic inference slightly beyond the expected decrease in resolving power. In particular, they exacerbate systematic errors by reducing the number of species effectively available for the detection of multiple substitutions. Consequently, large sparse supermatrices are more sensitive to phylogenetic artifacts than smaller but less incomplete data sets, which argue for experimental designs aimed at collecting a modest number (∼50) of highly covered genes. Our results further confirm that including incomplete yet short-branch taxa (i.e., slowly evolving species or close outgroups) can help to eschew artifacts, as predicted by simulations. Finally, it appears that selecting an adequate model of sequence evolution (e.g., the site-heterogeneous CAT model instead of the site-homogeneous WAG model) is more beneficial to phylogenetic accuracy than reducing the level of missing data. [less ▲]

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See detailThe unusual Gasteromycetes Lycogalopsis solmsii belongs to the gomphoid-phalloid group
Demoulin, Vincent ULg; Cornet, Luc ULg; Delbruyère, Emilie et al

in Acta Mycologica (2013), 48(1), 13-20

The rare tropical Gasteromycetes Lycogalopsis solmsii has been found twice at thirty years interval in the Singapore Botanic Gardens. From the most recent find a culture could be isolated, which allowed ... [more ▼]

The rare tropical Gasteromycetes Lycogalopsis solmsii has been found twice at thirty years interval in the Singapore Botanic Gardens. From the most recent find a culture could be isolated, which allowed DNA extraction and sequencing of about 2000 bp from the nuclear ribosomal DNA. Comparison to a large sample of Basidiomycetes was only possible for a part of the large ribosomal subunit, but clearly indicated affiliation to the gomphoid-phalloid group, without any relationship to Lycoperdales or Agaricales, as stated in the Dictionary of the Fungi. [less ▲]

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See detailThe African coelacanth genome provides insights into tetrapod evolution.
Amemiya, Chris T.; Alfoldi, Jessica; Lee, Alison P. et al

in Nature (2013), 496(7445), 311-6

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably ... [more ▼]

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution. [less ▲]

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See detailL'évolution des Eucaryotes
Baurain, Denis ULg

Scientific conference (2012, September 20)

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See detailEvolutionary analysis of a complex multigene family by semi-automated mining of public sequence databases
Baurain, Denis ULg

Conference (2012, June 19)

Eukaryotic genes have a mosaic structure of exons and introns. After transcription, precursor mRNAs have to be spliced to yield mature mRNAs suitable for protein synthesis. This process is termed splicing ... [more ▼]

Eukaryotic genes have a mosaic structure of exons and introns. After transcription, precursor mRNAs have to be spliced to yield mature mRNAs suitable for protein synthesis. This process is termed splicing and is carried out by a complex known as the spliceosome. Serine/arginine-rich (SR) proteins play essential roles in splicing. They have a modular organization featuring at least one RNA recognition motif (RRM) domain and a carboxyl-terminal RS region enriched in arginine/serine dipeptides. However, their architectures are quite diverse, which has so far complicated their evolutionary analysis. To investigate the origin and evolution of SR splicing factors, we inferred phylogenies for more than 12,000 RRM domains representing more than 200 broadly sampled organisms. Our results show that all SR proteins share a single ancient origin. Based on refined analyses, we propose a scenario for their diversification into four natural families and a dozen subfamilies. Altogether, this work confirms the homogeneity and antiquity of SR splicing factors while establishing robust phylogenetic relationships between animal and plant proteins. In this talk, I will focus on the bioinformatics approaches required to carry out such a large-scale phylogenetic analysis. [less ▲]

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See detailMitochondrial Proteomics of a Secondary Green Alga
Perez, Emilie ULg; Degand, Hervé; Morsomme, Pierre et al

Poster (2012, June)

Euglena gracilis is an alga that derives from a secondary endosymbiosis with a green alga. Our general objective is to study the interactions established between the chloroplast and the mitochondrion ... [more ▼]

Euglena gracilis is an alga that derives from a secondary endosymbiosis with a green alga. Our general objective is to study the interactions established between the chloroplast and the mitochondrion during the endosymbiosic event and to determine the phylogenetic origin of the genes encoding the proteins involved in these interactions. As a first step, we performed a high-throughput analysis of the mitochondrial proteome of Euglena gracilis. Our MS/MS experiments mostly recover mitochondrial proteins representing 15 mitochondrial pathways, which indicates that our mitochondrial extracts are relatively pure, but the phylogenetic origins of the corresponding genes are surprisingly diverse. [less ▲]

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See detailA Single Ancient Origin for Prototypical Serine/Arginine-Rich Splicing Factors1[W][OA]
Califice, Sophie; Baurain, Denis ULg; Hanikenne, Marc ULg et al

in Plant Physiology (2012), 158(2), 546-560

Eukaryotic pre-mRNA splicing is a process involving a very complex RNA-protein edifice. Serine/arginine-rich (SR) proteins play essential roles in pre-mRNA constitutive and alternative splicing, and have ... [more ▼]

Eukaryotic pre-mRNA splicing is a process involving a very complex RNA-protein edifice. Serine/arginine-rich (SR) proteins play essential roles in pre-mRNA constitutive and alternative splicing, and have been suggested to be crucial in plant-specific forms of developmental regulation and environmental adaptation. Despite their functional importance, little is known about their origin and evolutionary history. SR splicing factors have a modular organization featuring at least one RRM domain and a C-terminal region enriched in Ser/Arg dipeptides. To investigate the evolution of SR proteins, we infer phylogenies for >12,000 RRM domains representing >200 broadly sampled organisms. Our analyses reveal that the RRM domain is not restricted to eukaryotes and that all prototypical SR proteins share a single ancient origin, including the plant-specific SR45 protein. Based on these findings, we propose a scenario for their diversification into four natural families, each corresponding to a main SR architecture, and a dozen subfamilies, of which we profile both sequence conservation and composition. Finally, using operational criteria for computational discovery and classification, we catalogue SR proteins in 20 model organisms, with a focus on green algae and land plants. Altogether, our study confirms the homogeneity and antiquity of SR splicing factors, while establishing robust phylogenetic relationships between animal and plant proteins, which should enable functional analyses of lesser characterized SR family members, especially in green plants. [less ▲]

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See detailVariants modulating the expression of a chromosome domain encompassing PLAG1 influence bovine stature.
Karim, Latifa ULg; Takeda, Haruko ULg; Lin, Li et al

in Nature Genetics (2011), 43(5), 405-13

We report mapping of a quantitative trait locus (QTL) with a major effect on bovine stature to a approximately 780-kb interval using a Hidden Markov Model-based approach that simultaneously exploits ... [more ▼]

We report mapping of a quantitative trait locus (QTL) with a major effect on bovine stature to a approximately 780-kb interval using a Hidden Markov Model-based approach that simultaneously exploits linkage and linkage disequilibrium. We re-sequenced the interval in six sires with known QTL genotype and identified 13 clustered candidate quantitative trait nucleotides (QTNs) out of >9,572 discovered variants. We eliminated five candidate QTNs by studying the phenotypic effect of a recombinant haplotype identified in a breed diversity panel. We show that the QTL influences fetal expression of seven of the nine genes mapping to the approximately 780-kb interval. We further show that two of the eight candidate QTNs, mapping to the PLAG1-CHCHD7 intergenic region, influence bidirectional promoter strength and affect binding of nuclear factors. By performing expression QTL analyses, we identified a splice site variant in CHCHD7 and exploited this naturally occurring null allele to exclude CHCHD7 as single causative gene. [less ▲]

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See detailResolving difficult phylogenetic questions: why more sequences are not enough.
Philippe, Hervé; Brinkmann, Henner; Lavrov, Dennis V et al

in PLoS Biology (2011), 9(3), 1000602

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See detailA phylogenomic falsification of the chromalveolate hypothesis
Baurain, Denis ULg; Brinkmann, Henner; Petersen, Jorn et al

Poster (2010, July)

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See detailPhylogenomic evidence for separate acquisition of plastids in cryptophytes, haptophytes, and stramenopiles
Baurain, Denis ULg; Brinkmann, Henner; Petersen, Jorn et al

in Molecular Biology and Evolution (2010), 27(7), 1698-709

According to the chromalveolate hypothesis (Cavalier-Smith T. 1999. Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and sporozoan plastid origins and the ... [more ▼]

According to the chromalveolate hypothesis (Cavalier-Smith T. 1999. Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and sporozoan plastid origins and the eukaryote family tree. J Eukaryot Microbiol 46:347-366), the four eukaryotic groups with chlorophyll c-containing plastids originate from a single photosynthetic ancestor, which acquired its plastids by secondary endosymbiosis with a red alga. So far, molecular phylogenies have failed to either support or disprove this view. Here, we devise a phylogenomic falsification of the chromalveolate hypothesis that estimates signal strength across the three genomic compartments: If the four chlorophyll c-containing lineages indeed derive from a single photosynthetic ancestor, then similar amounts of plastid, mitochondrial, and nuclear sequences should allow to recover their monophyly. Our results refute this prediction, with statistical support levels too different to be explained by evolutionary rate variation, phylogenetic artifacts, or endosymbiotic gene transfer. Therefore, we reject the chromalveolate hypothesis as falsified in favor of more complex evolutionary scenarios involving multiple higher order eukaryote-eukaryote endosymbioses. [less ▲]

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See detailAtypical subunit composition of the chlorophycean mitochondrial F1FO ATP synthase and role of Asa7 protein in stability and oligomycin resistance of the enzyme.
Lapaille, Marie; Escobar-Ramirez, Adelma; Degand, Hervé et al

in Molecular Biology and Evolution (2010), 27(7), 1630-1644

Background. In yeast, mammals, and land plants, mitochondrial F(1)F(O) ATP synthase (complex V) is a remarkable enzymatic machinery which comprises about 15 conserved subunits. Peculiar among eukaryotes ... [more ▼]

Background. In yeast, mammals, and land plants, mitochondrial F(1)F(O) ATP synthase (complex V) is a remarkable enzymatic machinery which comprises about 15 conserved subunits. Peculiar among eukaryotes, complex V from Chlamydomonadales algae (order of chlorophycean class) has an atypical subunit composition of its peripheral stator and dimerization module, with 9 subunits of unknown evolutionary origin (Asa subunits). In vitro, this enzyme exhibits an increased stability of its dimeric form, and in vivo, Chlamydomonas reinhardtii cells are insensitive to oligomycins, which are potent inhibitors of proton translocation through the F(O) moiety. Methodology/Principal Findings. In this work, we showed that the atypical features of the Chlamydomonadales complex V enzyme are shared by the other chlorophycean orders. By biochemical and in silico analyses, we detected several atypical Asa subunits in Scenedesmus obliquus (Sphaeropleales) and Chlorococcum ellipsoideum (Chlorococcales). In contrast, Complex V has a canonical subunit composition in other classes of Chlorophytes (Trebouxiophyceae, Prasinophyceae, and Ulvophyceae) as well as in Streptophytes (land plants) and in Rhodophytes (red algae). Growth, respiration and ATP levels in Chlorophyceae were also barely affected by oligomycin concentrations that affect representatives of the other classes of Chlorophytes. We finally studied the function of the Asa7 atypical subunit by using RNA interference in C. reinhardtii. Although the loss of Asa7 subunit has no impact on cell bioenergetics or mitochondrial structures, it destabilizes in vitro the enzyme dimeric form and renders growth, respiration and ATP level sensitive to oligomycins. Conclusions/Significance. Altogether, our results suggest that the loss of canonical components of the Complex V stator happened at the root of chlorophycean lineage and was accompanied by the recruitment of novel polypeptides. Such a massive modification of Complex V stator features might have conferred novel properties, including the stabilization of the enzyme dimeric form and the shielding of the proton channel. In these respects, we discuss an evolutionary scenario for F(1)F(O) ATP synthase in the whole green lineage (i.e. Chlorophyta and Streptophyta). [less ▲]

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See detailAssessing the effect of the CLPG mutation on the microRNA catalog of skeletal muscle using high-throughput sequencing.
Caiment, Florian ULg; Charlier, Carole ULg; Hadfield, Tracy et al

in Genome Research (2010), 20(12), 1651-62

The callipyge phenotype is a monogenic muscular hypertrophy that is only expressed in heterozygous sheep receiving the CLPG mutation from their sire. The wild-type phenotype of CLPG/CLPG animals is ... [more ▼]

The callipyge phenotype is a monogenic muscular hypertrophy that is only expressed in heterozygous sheep receiving the CLPG mutation from their sire. The wild-type phenotype of CLPG/CLPG animals is thought to result from translational inhibition of paternally expressed DLK1 transcripts by maternally expressed miRNAs. To identify the miRNA responsible for this trans effect, we used high-throughput sequencing to exhaustively catalog miRNAs expressed in skeletal muscle of sheep of the four CLPG genotypes. We have identified 747 miRNA species of which 110 map to the DLK1-GTL2 or callipyge domain. We demonstrate that the latter are imprinted and preferentially expressed from the maternal allele. We show that the CLPG mutation affects their level of expression in cis ( approximately 3.2-fold increase) as well as in trans ( approximately 1.8-fold increase). In CLPG/CLPG animals, miRNAs from the DLK1-GTL2 domain account for approximately 20% of miRNAs in skeletal muscle. We show that the CLPG genotype affects the levels of A-to-I editing of at least five pri-miRNAs of the DLK1-GTL2 domain, but that levels of editing of mature miRNAs are always minor. We present suggestive evidence that the miRNAs from the domain target the ORF of DLK1, thereby causing the trans inhibition underlying polar overdominance. We highlight the limitations of high-throughput sequencing for digital gene expression profiling as a result of biased and inconsistent amplification of specific miRNAs. [less ▲]

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